@article{ghaffari_kashani_do_weninger_riehn_2023, title={A nanophotonic interferometer}, volume={34}, ISSN={["1361-6528"]}, url={https://doi.org/10.1088/1361-6528/acb443}, DOI={10.1088/1361-6528/acb443}, abstractNote={Abstract}, number={18}, journal={NANOTECHNOLOGY}, author={Ghaffari, Abbas and Kashani, Somayeh and Do, Kevin and Weninger, Keith and Riehn, Robert}, year={2023}, month={Apr} }
 @article{himebaugh_robertson_weninger_gilger_ekesten_oh_2023, title={Ex vivo analysis of ultraviolet radiation transmission through ocular media and retina in select species}, volume={233}, ISSN={0014-4835}, url={http://dx.doi.org/10.1016/j.exer.2023.109550}, DOI={10.1016/j.exer.2023.109550}, abstractNote={The aim of this study was to assess the transmission of the ultraviolet (UV) radiation (200–400 nm) through intact enucleated globes of different species (dogs, cats, pigs, rabbits, horses, and humans) using spectrophotometry. Globes of cats (n = 6), dogs (n = 18), pigs (n = 10), rabbits (n = 6), horses (n = 10), and humans (n = 4) were analyzed. A 5–10 mm circular area of sclera and choroid from the posterior aspect of the globe was removed under a surgical microscope, leaving the retina intact in all species except the horse. Glass coverslips were added in horses and rabbits due to retinal and globe fragility. The %T of wavelengths from 200 to 800 nm were measured through the ocular media (cornea, aqueous humor, lens, and vitreous humor) and retina, and compared between species. The globes of cats and dogs allowed the most amount of UV radiation transmission, while those of pigs and humans allowed the least amount of UV radiation transmission. A small amount of UV radiation transmission through the ocular media was detected in the rabbit and horse. Results from this study will support further vision research that may be used to train companion, working, and service animals.}, journal={Experimental Eye Research}, publisher={Elsevier BV}, author={Himebaugh, Nicole E. and Robertson, James B. and Weninger, Keith and Gilger, Brian C. and Ekesten, Bjorn and Oh, Annie}, year={2023}, month={Aug}, pages={109550} }
 @article{xu_zhang_pan_mahn_roland_sagui_weninger_2023, title={Frustration Between Preferred States of Complementary Trinucleotide Repeat DNA Hairpins Anticorrelates with Expansion Disease Propensity}, volume={435}, ISSN={["1089-8638"]}, DOI={10.1016/j.jmb.2023.168086}, abstractNote={DNA trinucleotide repeat (TRs) expansion beyond a threshold often results in human neurodegenerative diseases. The mechanisms causing expansions remain unknown, although the tendency of TR ssDNA to self-associate into hairpins that slip along their length is widely presumed related. Here we apply single molecule FRET (smFRET) experiments and molecular dynamics simulations to determine conformational stabilities and slipping dynamics for CAG, CTG, GAC and GTC hairpins. Tetraloops are favored in CAG (89%), CTG (89%) and GTC (69%) while GAC favors triloops. We also determined that TTG interrupts near the loop in the CTG hairpin stabilize the hairpin against slipping. The different loop stabilities have implications for intermediate structures that may form when TR-containing duplex DNA opens. Opposing hairpins in the (CAG) ∙ (CTG) duplex would have matched stability whereas opposing hairpins in a (GAC) ∙ (GTC) duplex would have unmatched stability, introducing frustration in the (GAC) ∙ (GTC) opposing hairpins that could encourage their resolution to duplex DNA more rapidly than in (CAG) ∙ (CTG) structures. Given that the CAG and CTG TR can undergo large, disease-related expansion whereas the GAC and GTC sequences do not, these stability differences can inform and constrain models of expansion mechanisms of TR regions.}, number={10}, journal={JOURNAL OF MOLECULAR BIOLOGY}, author={Xu, Pengning and Zhang, Jiahui and Pan, Feng and Mahn, Chelsea and Roland, Christopher and Sagui, Celeste and Weninger, Keith}, year={2023}, month={May} }
 @misc{evans_ramisetty_kulkarni_weninger_2023, title={Illuminating Intrinsically Disordered Proteins with Integrative Structural Biology}, volume={13}, ISSN={["2218-273X"]}, DOI={10.3390/biom13010124}, abstractNote={Intense study of intrinsically disordered proteins (IDPs) did not begin in earnest until the late 1990s when a few groups, working independently, convinced the community that these ‘weird’ proteins could have important functions. Over the past two decades, it has become clear that IDPs play critical roles in a multitude of biological phenomena with prominent examples including coordination in signaling hubs, enabling gene regulation, and regulating ion channels, just to name a few. One contributing factor that delayed appreciation of IDP functional significance is the experimental difficulty in characterizing their dynamic conformations. The combined application of multiple methods, termed integrative structural biology, has emerged as an essential approach to understanding IDP phenomena. Here, we review some of the recent applications of the integrative structural biology philosophy to study IDPs.}, number={1}, journal={BIOMOLECULES}, author={Evans, Rachel and Ramisetty, Sravani and Kulkarni, Prakash and Weninger, Keith}, year={2023}, month={Jan} }
 @article{ghaffari_do_kashani_weninger_riehn_2022, title={A Nanophotonic Interferometer for small particle detection}, volume={12223}, ISBN={["978-1-5106-5430-3"]}, ISSN={["1996-756X"]}, DOI={10.1117/12.2634318}, abstractNote={The transmission of light through sub-wavelength apertures (zero-mode waveguides, ZMW) in metal films is wellexplored. It introduces both an amplitude modulation as well as a phase shift to the transmitted oscillating electromagnetic field. We propose a nanophotonic interferometer by bringing two zero-mode waveguides in proximity and monitoring the distribution of light in the back-focal plane of the collecting microscope objective. We demonstrate that both an asymmetry induced by the binding of a quantum dot in one of the two ZMW, as well as a asymmetry in ZMW diameter yield qualitatively similar deflection patterns. Using ZMW pairs with diameter asymmetries, we find that the complex pattern of the transmitted light can be quantified through a scalar measure of asymmetry along the symmetry axis of the aperture pair. We find that this scalar asymmetry is a monotonous function of the diameter difference of the two apertures.}, journal={INTERFEROMETRY XXI}, author={Ghaffari, A. and Do, K. and Kashani, S. and Weninger, K. and Riehn, Robert}, year={2022} }
 @article{goetz_barth_bohr_boerner_chen_cordes_erie_gebhardt_hadzic_hamilton_et al._2022, title={A blind benchmark of analysis tools to infer kinetic rate constants from single-molecule FRET trajectories}, volume={13}, ISSN={["2041-1723"]}, DOI={10.1038/s41467-022-33023-3}, abstractNote={Abstract}, number={1}, journal={NATURE COMMUNICATIONS}, author={Goetz, Markus and Barth, Anders and Bohr, Soren S-R and Boerner, Richard and Chen, Jixin and Cordes, Thorben and Erie, Dorothy A. and Gebhardt, Christian and Hadzic, Melodie C. A. S. and Hamilton, George L. and et al.}, year={2022}, month={Sep} }
 @article{li_xie_godec_weninger_liu_smith_hong_2022, title={Non-ergodicity of a globular protein extending beyond its functional timescale}, volume={8}, ISSN={["2041-6539"]}, DOI={10.1039/d2sc03069a}, abstractNote={Internal motions of folded proteins have been assumed to be ergodic, i.e., that the dynamics of a single protein molecule averaged over a very long time resembles that of an ensemble.}, journal={CHEMICAL SCIENCE}, author={Li, Jun and Xie, JingFei and Godec, Aljaz and Weninger, Keith R. and Liu, Cong and Smith, Jeremy C. and Hong, Liang}, year={2022}, month={Aug} }
 @article{saikia_yanez-orozco_qiu_hao_milikisiyants_ou_hamilton_weninger_smirnova_sanabria_et al._2021, title={Integrative structural dynamics probing of the conformational heterogeneity in synaptosomal-associated protein 25}, volume={2}, ISSN={["2666-3864"]}, DOI={10.1016/j.xcrp.2021.100616}, abstractNote={SNAP-25 (synaptosomal-associated protein of 25 kDa) is a prototypical intrinsically disordered protein (IDP) that is unstructured by itself but forms coiled-coil helices in the SNARE complex. With high conformational heterogeneity, detailed structural dynamics of unbound SNAP-25 remain elusive. Here, we report an integrative method to probe the structural dynamics of SNAP-25 by combining replica-exchange discrete molecular dynamics (rxDMD) simulations and label-based experiments at ensemble and single-molecule levels. The rxDMD simulations systematically characterize the coil-to-molten globular transition and reconstruct structural ensemble consistent with prior ensemble experiments. Label-based experiments using Förster resonance energy transfer and double electron-electron resonance further probe the conformational dynamics of SNAP-25. Agreements between simulations and experiments under both ensemble and single-molecule conditions allow us to assign specific helix-coil transitions in SNAP-25 that occur in submillisecond timescales and potentially play a vital role in forming the SNARE complex. We expect that this integrative approach may help further our understanding of IDPs.}, number={11}, journal={CELL REPORTS PHYSICAL SCIENCE}, author={Saikia, Nabanita and Yanez-Orozco, Inna S. and Qiu, Ruoyi and Hao, Pengyu and Milikisiyants, Sergey and Ou, Erkang and Hamilton, George L. and Weninger, Keith R. and Smirnova, Tatyana I and Sanabria, Hugo and et al.}, year={2021}, month={Nov} }
 @misc{pan_zhang_xu_man_roland_weninger_sagui_2021, title={Molecular conformations and dynamics of nucleotide repeats associated with neurodegenerative diseases: double helices and CAG hairpin loops}, volume={19}, ISSN={["2001-0370"]}, DOI={10.1016/j.csbj.2021.04.037}, abstractNote={Pathogenic DNA secondary structures have been identified as a common and causative factor for expansion in trinucleotide, hexanucleotide, and other simple sequence repeats. These expansions underlie about fifty neurological and neuromuscular disorders known as “anticipation diseases”. Cell toxicity and death have been linked to the pathogenic conformations and functional changes of the RNA transcripts, of DNA itself and, when trinucleotides are present in exons, of the translated proteins. We review some of our results for the conformations and dynamics of pathogenic structures for both RNA and DNA, which include mismatched homoduplexes formed by trinucleotide repeats CAG and GAC; CCG and CGG; CTG(CUG) and GTC(GUC); the dynamics of DNA CAG hairpins; mismatched homoduplexes formed by hexanucleotide repeats (GGGGCC) and (GGCCCC); and G-quadruplexes formed by (GGGGCC) and (GGGCCT). We also discuss the dynamics of strand slippage in DNA hairpins formed by CAG repeats as observed with single-molecule Fluorescence Resonance Energy Transfer. This review focuses on the rich behavior exhibited by the mismatches associated with these simple sequence repeat noncanonical structures.}, journal={COMPUTATIONAL AND STRUCTURAL BIOTECHNOLOGY JOURNAL}, author={Pan, Feng and Zhang, Yuan and Xu, Pengning and Man, Viet Hoang and Roland, Christopher and Weninger, Keith and Sagui, Celeste}, year={2021}, pages={2819–2832} }
 @article{pan_kaur_barnes_detwiler_sanford_liu_xu_mahn_tang_hao_et al._2021, title={Structure, dynamics, and regulation of TRF1-TIN2-mediated trans- and cis-interactions on telomeric DNA}, volume={297}, ISSN={["1083-351X"]}, DOI={10.1016/j.jbc.2021.101080}, abstractNote={TIN2 is a core component of the shelterin complex linking double-stranded telomeric DNA-binding proteins (TRF1 and TRF2) and single-strand overhang-binding proteins (TPP1-POT1). In vivo, the large majority of TRF1 and TRF2 exist in complexes containing TIN2 but lacking TPP1/POT1; however, the role of TRF1-TIN2 interactions in mediating interactions with telomeric DNA is unclear. Here, we investigated DNA molecular structures promoted by TRF1-TIN2 interaction using atomic force microscopy (AFM), total internal reflection fluorescence microscopy (TIRFM), and the DNA tightrope assay. We demonstrate that the short (TIN2S) and long (TIN2L) isoforms of TIN2 facilitate TRF1-mediated DNA compaction (cis-interactions) and DNA-DNA bridging (trans-interactions) in a telomeric sequence- and length-dependent manner. On the short telomeric DNA substrate (six TTAGGG repeats), the majority of TRF1-mediated telomeric DNA-DNA bridging events are transient with a lifetime of ~1.95 s. On longer DNA substrates (270 TTAGGG repeats), TIN2 forms multiprotein complexes with TRF1 and stabilizes TRF1-mediated DNA-DNA bridging events that last on the order of minutes. Preincubation of TRF1 with its regulator protein Tankyrase 1 and the cofactor NAD+ significantly reduced TRF1-TIN2 mediated DNA-DNA bridging, whereas TIN2 protected the disassembly of TRF1-TIN2 mediated DNA-DNA bridging upon Tankyrase 1 addition. Furthermore, we showed that TPP1 inhibits TRF1-TIN2L-mediated DNA-DNA bridging. Our study, together with previous findings, supports a molecular model in which protein assemblies at telomeres are heterogeneous with distinct subcomplexes and full shelterin complexes playing distinct roles in telomere protection and elongation. TIN2 is a core component of the shelterin complex linking double-stranded telomeric DNA-binding proteins (TRF1 and TRF2) and single-strand overhang-binding proteins (TPP1-POT1). In vivo, the large majority of TRF1 and TRF2 exist in complexes containing TIN2 but lacking TPP1/POT1; however, the role of TRF1-TIN2 interactions in mediating interactions with telomeric DNA is unclear. Here, we investigated DNA molecular structures promoted by TRF1-TIN2 interaction using atomic force microscopy (AFM), total internal reflection fluorescence microscopy (TIRFM), and the DNA tightrope assay. We demonstrate that the short (TIN2S) and long (TIN2L) isoforms of TIN2 facilitate TRF1-mediated DNA compaction (cis-interactions) and DNA-DNA bridging (trans-interactions) in a telomeric sequence- and length-dependent manner. On the short telomeric DNA substrate (six TTAGGG repeats), the majority of TRF1-mediated telomeric DNA-DNA bridging events are transient with a lifetime of ~1.95 s. On longer DNA substrates (270 TTAGGG repeats), TIN2 forms multiprotein complexes with TRF1 and stabilizes TRF1-mediated DNA-DNA bridging events that last on the order of minutes. Preincubation of TRF1 with its regulator protein Tankyrase 1 and the cofactor NAD+ significantly reduced TRF1-TIN2 mediated DNA-DNA bridging, whereas TIN2 protected the disassembly of TRF1-TIN2 mediated DNA-DNA bridging upon Tankyrase 1 addition. Furthermore, we showed that TPP1 inhibits TRF1-TIN2L-mediated DNA-DNA bridging. Our study, together with previous findings, supports a molecular model in which protein assemblies at telomeres are heterogeneous with distinct subcomplexes and full shelterin complexes playing distinct roles in telomere protection and elongation. Telomeres are nucleoprotein structures that prevent the degradation or fusion of the ends of linear chromosomes, which are threatened by at least seven distinct DNA damage response (DDR) pathways (1Palm W. de Lange T. How shelterin protects mammalian telomeres.Annu. Rev. Genet. 2008; 42: 301-334Crossref PubMed Scopus (1344) Google Scholar, 2Muraki K. Nyhan K. Han L. Murnane J.P. Mechanisms of telomere loss and their consequences for chromosome instability.Front. Oncol. 2012; 2: 135Crossref PubMed Google Scholar, 3de Lange T. Shelterin-mediated telomere protection.Annu. Rev. Genet. 2018; 52: 223-247Crossref PubMed Scopus (280) Google Scholar). Human telomeres contain ~2–20 kb of TTAGGG repeats and a G-rich 3′ overhang of ~50–400 nt in length (1Palm W. de Lange T. How shelterin protects mammalian telomeres.Annu. Rev. Genet. 2008; 42: 301-334Crossref PubMed Scopus (1344) Google Scholar, 4Wright W.E. Tesmer V.M. Huffman K.E. Levene S.D. Shay J.W. Normal human chromosomes have long G-rich telomeric overhangs at one end.Genes Dev. 1997; 11: 2801-2809Crossref PubMed Scopus (572) Google Scholar). In humans, a specialized six-protein shelterin complex consisting of TRF1, TRF2, RAP1, TIN2, TPP1, and POT1 binds specifically to the unique sequence and structure at telomeres to protect chromosome ends. Prevention of telomeres from being falsely recognized as double-strand DNA breaks and regulation of DNA repair protein access depend on the biochemical activities of shelterin proteins and their collaborative actions with other proteins involved in the genome maintenance pathways (5Cech T.R. Beginning to understand the end of the chromosome.Cell. 2004; 116: 273-279Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar, 6Songyang Z. Liu D. Inside the mammalian telomere interactome: Regulation and regulatory activities of telomeres.Crit. Rev. Eukaryot. Gene Expr. 2006; 16: 103-118Crossref PubMed Scopus (38) Google Scholar, 7Verdun R.E. Karlseder J. Replication and protection of telomeres.Nature. 2007; 447: 924-931Crossref PubMed Scopus (370) Google Scholar, 8Canudas S. Houghtaling B.R. Kim J.Y. Dynek J.N. Chang W.G. Smith S. Protein requirements for sister telomere association in human cells.EMBO J. 2007; 26: 4867-4878Crossref PubMed Scopus (80) Google Scholar, 9Giraud-Panis M.J. Pisano S. Poulet A. Le Du M.H. Gilson E. Structural identity of telomeric complexes.FEBS Lett. 2010; 584: 3785-3799Crossref PubMed Scopus (36) Google Scholar). Extensive telomere shortening or dramatic telomere loss due to DNA damage causes telomere deprotection, which triggers cell senescence and aging-related pathologies (10d'Adda di Fagagna F. Reaper P.M. Clay-Farrace L. Fiegler H. Carr P. Von Zglinicki T. Saretzki G. Carter N.P. Jackson S.P. A DNA damage checkpoint response in telomere-initiated senescence.Nature. 2003; 426: 194-198Crossref PubMed Scopus (1979) Google Scholar, 11Jaskelioff M. Muller F.L. Paik J.H. Thomas E. Jiang S. Adams A.C. Sahin E. Kost-Alimova M. Protopopov A. Cadinanos J. Horner J.W. Maratos-Flier E. Depinho R.A. Telomerase reactivation reverses tissue degeneration in aged telomerase-deficient mice.Nature. 2011; 469: 102-106Crossref PubMed Scopus (556) Google Scholar). The main protein–protein and protein–DNA interactions at telomeres have been investigated using crystallography, biochemical assays, yeast two-hybrid systems, coimmunoprecipitation, as well as visualization of shelterin subcomponents in vitro and in vivo using fluorescence imaging (3de Lange T. Shelterin-mediated telomere protection.Annu. Rev. Genet. 2018; 52: 223-247Crossref PubMed Scopus (280) Google Scholar). Among shelterin components, both TRF1 and TRF2 specifically recognize double-stranded telomeric DNA through the Myb/SANT domain facilitated by homodimerization through the TRFH domain (12Broccoli D. Chong L. Oelmann S. Fernald A.A. Marziliano N. van Steensel B. Kipling D. Le Beau M.M. de Lange T. Comparison of the human and mouse genes encoding the telomeric protein, TRF1: Chromosomal localization, expression and conserved protein domains.Hum. Mol. Genet. 1997; 6: 69-76Crossref PubMed Scopus (80) Google Scholar, 13Broccoli D. Smogorzewska A. Chong L. de Lange T. Human telomeres contain two distinct Myb-related proteins, TRF1 and TRF2.Nat. Genet. 1997; 17: 231-235Crossref PubMed Scopus (741) Google Scholar). However, TRF1 and TRF2 display distinct DNA-binding properties and functions. TRF1 and TRF2 contain an acidic and a basic domain, respectively, at their N-termini (14Poulet A. Pisano S. Faivre-Moskalenko C. Pei B. Tauran Y. Haftek-Terreau Z. Brunet F. Le Bihan Y.V. Ledu M.H. Montel F. Hugo N. Amiard S. Argoul F. Chaboud A. Gilson E. et al.The N-terminal domains of TRF1 and TRF2 regulate their ability to condense telomeric DNA.Nucleic Acids Res. 2012; 40: 2566-2576Crossref PubMed Scopus (55) Google Scholar). TRF2 prevents Mre11/Rad50/Nbs1-dependent ATM kinase signaling, classical nonhomologous end-joining (NHEJ), as well as alternative nonhomologous end-joining (alt-NHEJ) pathways at telomeres. These distinct functions of TRF2 are believed to be mediated through its activities in promoting and stabilizing T-loops, in which the 3′ single-stranded overhang invades the upstream double-stranded telomeric region (15Griffith J.D. Comeau L. Rosenfield S. Stansel R.M. Bianchi A. Moss H. de Lange T. Mammalian telomeres end in a large duplex loop.Cell. 1999; 97: 503-514Abstract Full Text Full Text PDF PubMed Scopus (1886) Google Scholar, 16Doksani Y. Wu J.Y. de Lange T. Zhuang X. Super-resolution fluorescence imaging of telomeres reveals TRF2-dependent T-loop formation.Cell. 2013; 155: 345-356Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar, 17Benarroch-Popivker D. Pisano S. Mendez-Bermudez A. Lototska L. Kaur P. Bauwens S. Djerbi N. Latrick C.M. Fraisier V. Pei B. Gay A. Jaune E. Foucher K. Cherfils-Vicini J. Aeby E. et al.TRF2-Mediated control of telomere DNA topology as a mechanism for chromosome-end protection.Mol. Cell. 2016; 61: 274-286Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 18Van Ly D. Low R.R.J. Frolich S. Bartolec T.K. Kafer G.R. Pickett H.A. Gaus K. Cesare A.J. Telomere loop dynamics in chromosome end protection.Mol. Cell. 2018; 71: 510-525.e516Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). In comparison, TRF1 represses telomere fragility by preventing DNA replication fork stalling at telomeres (19Sfeir A. Kosiyatrakul S.T. Hockemeyer D. MacRae S.L. Karlseder J. Schildkraut C.L. de Lange T. Mammalian telomeres resemble fragile sites and require TRF1 for efficient replication.Cell. 2009; 138: 90-103Abstract Full Text Full Text PDF PubMed Scopus (685) Google Scholar) and promotes parallel pairing of telomeric DNA tracts (20Griffith J. Bianchi A. de Lange T. TRF1 promotes parallel pairing of telomeric tracts in vitro.J. Mol. Biol. 1998; 278: 79-88Crossref PubMed Scopus (123) Google Scholar, 21Lin J. Countryman P. Buncher N. Kaur P. E L. Zhang Y. Gibson G. You C. Watkins S.C. Piehler J. Opresko P.L. Kad N.M. Wang H. TRF1 and TRF2 use different mechanisms to find telomeric DNA but share a novel mechanism to search for protein partners at telomeres.Nucleic Acids Res. 2014; 42: 2493-2504Crossref PubMed Scopus (44) Google Scholar). A flexible domain in TRF1 enables the two Myb domains in the TRF1 dimer to interact with DNA independently and to mediate looping of telomeric DNA (22Bianchi A. Stansel R.M. Fairall L. Griffith J.D. Rhodes D. de Lange T. TRF1 binds a bipartite telomeric site with extreme spatial flexibility.EMBO J. 1999; 18: 5735-5744Crossref PubMed Scopus (163) Google Scholar). TIN2 itself does not have binding affinity to either double-stranded or single-stranded DNA (23Kim S.H. Kaminker P. Campisi J. TIN2, a new regulator of telomere length in human cells.Nat. Genet. 1999; 23: 405-412Crossref PubMed Scopus (418) Google Scholar). However, it is a core shelterin component that bridges double-stranded (TRF1 and TRF2) and single-stranded telomeric DNA-binding proteins (TPP1-POT1) (24Kim S.H. Beausejour C. Davalos A.R. Kaminker P. Heo S.J. Campisi J. TIN2 mediates functions of TRF2 at human telomeres.J. Biol. Chem. 2004; 279: 43799-43804Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 25Houghtaling B.R. Cuttonaro L. Chang W. Smith S. A dynamic molecular link between the telomere length regulator TRF1 and the chromosome end protector TRF2.Curr. Biol. 2004; 14: 1621-1631Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 26Kim S.H. Davalos A.R. Heo S.J. Rodier F. Zou Y. Beausejour C. Kaminker P. Yannone S.M. Campisi J. Telomere dysfunction and cell survival: Roles for distinct TIN2-containing complexes.J. Cell Biol. 2008; 181: 447-460Crossref PubMed Scopus (44) Google Scholar, 27O'Connor M.S. Safari A. Xin H. Liu D. Songyang Z. A critical role for TPP1 and TIN2 interaction in high-order telomeric complex assembly.Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 11874-11879Crossref PubMed Scopus (181) Google Scholar, 28Ye J.Z. Hockemeyer D. Krutchinsky A.N. Loayza D. Hooper S.M. Chait B.T. de Lange T. POT1-interacting protein PIP1: A telomere length regulator that recruits POT1 to the TIN2/TRF1 complex.Genes Dev. 2004; 18: 1649-1654Crossref PubMed Scopus (340) Google Scholar). Crystal structures revealed the interfaces between TRF1-TIN2, TRF2-TIN2, and TPP1-TIN2 (29Hu C. Rai R. Huang C. Broton C. Long J. Xu Y. Xue J. Lei M. Chang S. Chen Y. Structural and functional analyses of the mammalian TIN2-TPP1-TRF2 telomeric complex.Cell Res. 2017; 27: 1485-1502Crossref PubMed Scopus (50) Google Scholar, 30Chen Y. Yang Y. van Overbeek M. Donigian J.R. Baciu P. de Lange T. Lei M. A shared docking motif in TRF1 and TRF2 used for differential recruitment of telomeric proteins.Science. 2008; 319: 1092-1096Crossref PubMed Scopus (187) Google Scholar). TIN2 stabilizes both TRF1 and TRF2 at telomeres (31Kim S.H. Han S. You Y.H. Chen D.J. Campisi J. The human telomere-associated protein TIN2 stimulates interactions between telomeric DNA tracts in vitro.EMBO Rep. 2003; 4: 685-691Crossref PubMed Scopus (42) Google Scholar, 32Ye J.Z. Donigian J.R. van Overbeek M. Loayza D. Luo Y. Krutchinsky A.N. Chait B.T. de Lange T. TIN2 binds TRF1 and TRF2 simultaneously and stabilizes the TRF2 complex on telomeres.J. Biol. Chem. 2004; 279: 47264-47271Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). The loss of the TRF1 or TRF2-binding domains in TIN2 triggers a DNA damage response (33Takai K.K. Kibe T. Donigian J.R. Frescas D. de Lange T. Telomere protection by TPP1/POT1 requires tethering to TIN2.Mol. Cell. 2011; 44: 647-659Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Binding of TPP1 to TIN2 is required for POT1-mediated telomere protection (34Frescas D. de Lange T. Binding of TPP1 protein to TIN2 protein is required for POT1a,b protein-mediated telomere protection.J. Biol. Chem. 2014; 289: 24180-24187Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). As an integral component of the “TIN2/TPP1/POT1 processivity complex,” TIN2 functions together with TPP1/POT1 to stimulate telomerase processivity (35Pike A.M. Strong M.A. Ouyang J.P.T. Greider C.W. TIN2 functions with TPP1/POT1 to stimulate telomerase processivity.Mol. Cell. Biol. 2019; 39e00593-18Crossref PubMed Scopus (22) Google Scholar). Furthermore, TIN2 directly interacts with the cohesin subunit SA1 and plays a key role in a distinct SA1-TRF1-TIN2-mediated sister telomere cohesion pathway that is largely independent of the cohesin ring subunits (8Canudas S. Houghtaling B.R. Kim J.Y. Dynek J.N. Chang W.G. Smith S. Protein requirements for sister telomere association in human cells.EMBO J. 2007; 26: 4867-4878Crossref PubMed Scopus (80) Google Scholar, 36Canudas S. Smith S. Differential regulation of telomere and centromere cohesion by the Scc3 homologues SA1 and SA2, respectively, in human cells.J. Cell Biol. 2009; 187: 165-173Crossref PubMed Scopus (122) Google Scholar). Binding of TRF1-TIN2 to telomeres is regulated by the poly(ADP-ribose) polymerase Tankyrase 1 (37Smith S. Giriat I. Schmitt A. de Lange T. Tankyrase, a poly(ADP-ribose) polymerase at human telomeres.Science. 1998; 282: 1484-1487Crossref PubMed Scopus (891) Google Scholar). ADP-ribosylation of TRF1 by Tankyrase 1 reduces its binding to telomeric DNA in vitro, and the depletion of Tankyrase 1 using siRNA leads to mitotic arrest and persistent telomere cohesion that can be rescued by depletion of TIN2 (8Canudas S. Houghtaling B.R. Kim J.Y. Dynek J.N. Chang W.G. Smith S. Protein requirements for sister telomere association in human cells.EMBO J. 2007; 26: 4867-4878Crossref PubMed Scopus (80) Google Scholar, 36Canudas S. Smith S. Differential regulation of telomere and centromere cohesion by the Scc3 homologues SA1 and SA2, respectively, in human cells.J. Cell Biol. 2009; 187: 165-173Crossref PubMed Scopus (122) Google Scholar, 38Dynek J.N. Smith S. Resolution of sister telomere association is required for progression through mitosis.Science. 2004; 304: 97-100Crossref PubMed Scopus (216) Google Scholar). Three distinct TIN2 isoforms have been identified in human cell lines (35Pike A.M. Strong M.A. Ouyang J.P.T. Greider C.W. TIN2 functions with TPP1/POT1 to stimulate telomerase processivity.Mol. Cell. Biol. 2019; 39e00593-18Crossref PubMed Scopus (22) Google Scholar, 39Kaminker P.G. Kim S.H. Desprez P.Y. Campisi J. A novel form of the telomere-associated protein TIN2 localizes to the nuclear matrix.Cell Cycle. 2009; 8: 931-939Crossref PubMed Scopus (35) Google Scholar, 40Smith S. The long and short of it: A new isoform of TIN2 in the nuclear matrix.Cell Cycle. 2009; 8: 797-798Crossref PubMed Scopus (2) Google Scholar) that include TIN2S (354 AAs), TIN2L (451 AAs), and TIN2M (TIN2 medium, 420 AAs). TIN2S, TIN2L, and TIN2M share the same TRF1, TRF2, and TPP1-binding domains and localize to telomeres (23Kim S.H. Kaminker P. Campisi J. TIN2, a new regulator of telomere length in human cells.Nat. Genet. 1999; 23: 405-412Crossref PubMed Scopus (418) Google Scholar, 35Pike A.M. Strong M.A. Ouyang J.P.T. Greider C.W. TIN2 functions with TPP1/POT1 to stimulate telomerase processivity.Mol. Cell. Biol. 2019; 39e00593-18Crossref PubMed Scopus (22) Google Scholar, 39Kaminker P.G. Kim S.H. Desprez P.Y. Campisi J. A novel form of the telomere-associated protein TIN2 localizes to the nuclear matrix.Cell Cycle. 2009; 8: 931-939Crossref PubMed Scopus (35) Google Scholar). Consistent with its key role in telomere maintenance, germline inactivation of TIN2 in mice is embryonic lethal (41Chiang Y.J. Kim S.H. Tessarollo L. Campisi J. Hodes R.J. Telomere-associated protein TIN2 is essential for early embryonic development through a telomerase-independent pathway.Mol. Cell. Biol. 2004; 24: 6631-6634Crossref PubMed Scopus (61) Google Scholar). Removal of TIN2 leads to the formation of telomere dysfunction-induced foci (TIFs). Importantly, clinical studies further highlight the biological significance of TIN2 in telomere protection (42Savage S.A. Giri N. Baerlocher G.M. Orr N. Lansdorp P.M. Alter B.P. TINF2, a component of the shelterin telomere protection complex, is mutated in dyskeratosis congenita.Am. J. Hum. Genet. 2008; 82: 501-509Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar, 43Walne A.J. Vulliamy T. Beswick R. Kirwan M. Dokal I. TINF2 mutations result in very short telomeres: Analysis of a large cohort of patients with dyskeratosis congenita and related bone marrow failure syndromes.Blood. 2008; 112: 3594-3600Crossref PubMed Scopus (227) Google Scholar). TINF2, which encodes TIN2, is the second most frequently mutated gene in the telomere elongation and protection disorder dyskeratosis congenita (DKC). DKC-associated TIN2 mutations are most frequently de novo and cluster at a highly conserved region near the end of its TRF1-binding domain. Two decades of research since the first discovery of TIN2 have shed light on protein-interaction networks around TIN2 and its multifaceted roles in telomere maintenance. However, since TIN2 itself does not directly bind to DNA and instead serves as a "mediator/enhancer" for shelterin and telomerase activities, defining TIN2's distinct function at the molecular level has been challenging. The bottleneck for studying TIN2 lies in the fact that results from bulk biochemical assays do not fully reveal the heterogeneity and dynamics of the protein–protein and protein–DNA interactions. Furthermore, cell-based assays only provide information on the outcomes from downstream effectors after the knocking down of TIN2 that also removes TRF1 and TRF2 from telomeres. These approaches do not allow us to investigate the molecular structures and dynamics in which TIN2 directly participates. In vivo, the amount of TIN2 is sufficient for binding every TRF1 and TRF2 molecule (44Takai K.K. Hooper S. Blackwood S. Gandhi R. de Lange T. In vivo stoichiometry of shelterin components.J. Biol. Chem. 2010; 285: 1457-1467Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar), while TPP1 and POT1 are ~10-fold less than TRF1 and TIN2. Thus, it is important to study the DNA-binding properties of TRF1-TIN2 complexes. To fill this important knowledge gap, we applied complementary single-molecule imaging platforms, including atomic force microscopy (AFM) (45Yang Y. Wang H. Erie D.A. Quantitative characterization of biomolecular assemblies and interactions using atomic force microscopy.Methods. 2003; 29: 175-187Crossref PubMed Scopus (88) Google Scholar, 46Wang H. Nora G.J. Ghodke H. Opresko P.L. Single molecule studies of physiologically relevant telomeric tails reveal POT1 mechanism for promoting G-quadruplex unfolding.J. Biol. Chem. 2011; 286: 7479-7489Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 47Kaur P. Wu D. Lin J. Countryman P. Bradford K.C. Erie D.A. Riehn R. Opresko P.L. Wang H. Enhanced electrostatic force microscopy reveals higher-order DNA looping mediated by the telomeric protein TRF2.Sci. Rep. 2016; 6: 20513Crossref PubMed Scopus (20) Google Scholar), total internal reflection fluorescence microscopy (TIRFM) (48Erie D.A. Weninger K.R. Single molecule studies of DNA mismatch repair.DNA Repair. 2014; 20: 71-81Crossref PubMed Scopus (46) Google Scholar), and the DNA tightrope assay to monitor TRF1-TIN2-mediated DNA compaction and DNA-DNA bridging (49Lin J. Countryman P. Chen H. Pan H. Fan Y. Jiang Y. Kaur P. Miao W. Gurgel G. You C. Piehler J. Kad N.M. Riehn R. Opresko P.L. Smith S. et al.Functional interplay between SA1 and TRF1 in telomeric DNA binding and DNA-DNA pairing.Nucleic Acids Res. 2016; 44: 6363-6376Crossref PubMed Scopus (18) Google Scholar, 50Countryman P. Fan Y. Gorthi A. Pan H. Strickland J. Kaur P. Wang X. Lin J. Lei X. White C. You C. Wirth N. Tessmer I. Piehler J. Riehn R. et al.Cohesin SA2 is a sequence-independent DNA-binding protein that recognizes DNA replication and repair intermediates.J. Biol. Chem. 2018; 293: 1054-1069Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 51Pan H. Jin M. Ghadiyaram A. Kaur P. Miller H.E. Ta H.M. Liu M. Fan Y. Mahn C. Gorthi A. You C. Piehler J. Riehn R. Bishop A.J.R. Tao Y.J. et al.Cohesin SA1 and SA2 are RNA binding proteins that localize to RNA containing regions on DNA.Nucleic Acids Res. 2020; 48: 5639-5655Crossref PubMed Google Scholar). Through using DNA substrates on different length scales (6 and 270 TAAGGG repeats), these imaging platforms provide complementary results demonstrating that both TIN2S and TIN2L facilitate TRF1-mediated DNA compaction (cis-interactions) and DNA-DNA bridging (trans-interactions) in a telomeric sequence- and length-dependent manner. In some cases, TRF1-TIN2 is capable of mediating the bridging of multiple copies of telomeric DNA fragments. Importantly, our results demonstrate that TIN2 protects the disassembly of TRF1-TIN2-mediated DNA-DNA bridging by Tankyrase 1. In addition, the N-terminal domain of TPP1 inhibits TRF1-TIN2-mediated DNA-DNA bridging. In summary, this study uncovered the unique biophysical function of TIN2 as a telomeric architectural protein, acting together with TRF1 to mediate interactions between distant telomeric sequences. Tankyrase 1 and TPP1 regulate TRF1-TIN2-mediated DNA-DNA bridging. Furthermore, this work establishes a unique combination of single-molecule imaging platforms for future examination of TIN2 disease variants and provides a new direction for investigating molecular mechanisms underlying diverse TIN2 functions. A previous study suggested that TIN2 modulates the bridging of telomeric DNA by TRF1 (31Kim S.H. Han S. You Y.H. Chen D.J. Campisi J. The human telomere-associated protein TIN2 stimulates interactions between telomeric DNA tracts in vitro.EMBO Rep. 2003; 4: 685-691Crossref PubMed Scopus (42) Google Scholar). However, the bulk biochemical assays using short telomeric DNA (six telomeric repeats) did not provide information regarding the structure and dynamics of the TRF1-TIN2-DNA complex. To investigate the molecular function of TIN2, we applied AFM imaging to investigate how TIN2 affects the telomeric DNA-DNA pairing mediated by TRF1 at the single-molecule level on longer telomeric DNA substrates (270 TTAGGG repeats). We purified TRF1 (Fig. S1A) and obtained TIN2S (1–354 amino acids, 39.4 kDa) and TIN2L (1–451 amino acids, 50.0 kDa) proteins purified from insect cells (Fig. 1A and Fig. S1D). Previously, we established an AFM imaging-based calibration method to investigate the oligomeric states and protein–protein interactions by correlating AFM volumes of proteins and their molecular weights (45Yang Y. Wang H. Erie D.A. Quantitative characterization of biomolecular assemblies and interactions using atomic force microscopy.Methods. 2003; 29: 175-187Crossref PubMed Scopus (88) Google Scholar, 47Kaur P. Wu D. Lin J. Countryman P. Bradford K.C. Erie D.A. Riehn R. Opresko P.L. Wang H. Enhanced electrostatic force microscopy reveals higher-order DNA looping mediated by the telomeric protein TRF2.Sci. Rep. 2016; 6: 20513Crossref PubMed Scopus (20) Google Scholar, 52Wang H. Yang Y. Erie D.A. Characterization of protein-protein interactions using atomic force microscopy.in: Schuck P. Protein Interactions Biophysical approaches for the Study of Complex Reversible Systems. Springer Science+Business Media, LLC, Berlin, Germany2007: 39-78Crossref Google Scholar). AFM volumes of TRF1 alone in solution showed two distinct peaks, which were consistent with TRF1 monomers (51 KDa) and dimers (102 KDa, Fig. S1B). In addition, based on the population of TRF1 under the monomer and dimer peaks (53Wang H. DellaVecchia M.J. Skorvaga M. Croteau D.L. Erie D.A. Van Houten B. UvrB domain 4, an autoinhibitory gate for regulation of DNA binding and ATPase activity.J. Biol. Chem. 2006; 281: 15227-15237Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), the estimated TRF1 dimer equilibrium dissociation constant (Kd) is 18.4 nM (Fig. S1C). Meanwhile, AFM volumes of purified TIN2S at 41.3 nm3 (±28.3 nm3) and TIN2L at 41.9 nm3 (±12.8 nm3) were consistent with the notion that TIN2 does not interact with itself (23Kim S.H. Kaminker P. Campisi J. TIN2, a new regulator of telomere length in human cells.Nat. Genet. 1999; 23: 405-412Crossref PubMed Scopus (418) Google Scholar), and TIN2 exists in a monomeric state in solution (Fig. S1D). Furthermore, we conducted size-exclusive chromatography using TRF1 and TIN2S and confirmed the presence of TRF1 dimers, TIN2 monomers, as well as the interaction between TRF1 and TIN2S in solution (Fig. S2). To further validate the activities of TIN2, we used electrophoresis mobility shift assays (EMSAs) to verify the interaction of TIN2 with TRF1 on a double-stranded telomeric DNA substrate (48 bp containing three TTAGGG repeats, Fig. S3, A–C). Consistent with previous studies (23Kim S.H. Kaminker P. Campisi J. TIN2, a new regulator of telomere length in human cells.Nat. Genet. 1999; 23: 405-412Crossref PubMed Scopus (418) Google Scholar), EMSA experiments showed that TIN2S and TIN2L did not directly bind to telomeric dsDNA (Fig. S3A). Both TRF1-TIN2S and TRF1-TIN2L induced a clear supershift of the telomeric DNA substrate compared with TRF1 alone (Complex III in Fig. S3, B and C), indicating the formation of stable TRF1-TIN2-telomeric DNA complexes. Next, to study TRF1-TIN2 DNA binding at the single-molecule level, we used the linear DNA substrate (5.4 kb) that contains 1.6 kb (270 TTAGGG) telomeric repeats in the middle region that is 35%–50% from DNA ends (T270 DNA, Experimental procedures, Fig. 1A) (21Lin J. Countryman P. Buncher N. Kaur P. E L. Zhang Y. Gibson G. You C. Watkins S.C. Piehler J. Opresko P.L. Kad N.M. Wang H. TRF1 and TRF2 use different mechanisms to find telomeric DNA but share a novel mechanism to search for protein partners at telomeres.Nucleic Acids Res. 2014; 42: 2493-2504Crossref PubMed Scopus (44) Google Scholar, 49Lin J. Countryman P. Chen H. Pan H. Fan Y. Jiang Y. Kaur P. Miao W. Gurgel G. You C. Piehler J. Kad N.M. Riehn R. Opresko P.L. Smith S. et al.Functional interplay between SA1 and TRF1 in telomeric DNA binding and DNA-DNA pairing.Nucleic Acids Res. 2016; 44: 6363-6376Crossref PubMed Scopus (18) Google Scholar). Previously, AFM and electron microscopy imaging–based studies established that TRF1 specifically binds to the telomeric region and mediates DNA-DNA pairing (21Lin J. Countryman P. Buncher N. Kaur P. E L. Zhang Y. Gibson G. You C. Watkins S.C. Piehler J. Opresko P.L. Kad N.M. Wang H. TRF1 and TRF2 use different mechanisms to find telomeric DNA but share a novel mechanism to search for protein partners at telomeres.Nucleic Acids Res. 2014; 42: 2493-2504Crossref PubMed Scopus (44) Google Scholar, 22Bianchi A. Stansel R.M. Fairall L. Griffith J.D. Rhodes D. de Lange T. TRF1 binds a bipartite telomeric site with extreme spatial flexibility.EMBO J. 1999; 18: 5735-5744Crossref PubMed Scopus (163) Google Scholar, 49Lin J. Countryman P. Chen H. Pan H. Fan Y. Jiang Y. Kaur P. Miao W. Gurgel G. You C. Piehler J. Kad N.M. Riehn R. Opresko P.L. Smith S. et al.Functional interplay between SA1 and TRF1 in telomeric DNA binding and DNA-DNA pairing.Nucleic Acids Res. 2016; 44: 6363-6376Crossref PubMed Scopus (18) Google Scholar). To study the function of TIN2, we preincubated TRF1 without or with TIN2 (either TIN2S or TIN2L), followed by the addition}, number={3}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Pan, Hai and Kaur, Parminder and Barnes, Ryan and Detwiler, Ariana C. and Sanford, Samantha Lynn and Liu, Ming and Xu, Pengning and Mahn, Chelsea and Tang, Qingyu and Hao, Pengyu and et al.}, year={2021}, month={Sep} }
 @article{bradford_wilkins_hao_li_wang_burke_wu_smith_spaller_du_et al._2020, title={Dynamic human MutS alpha-MutL alpha complexes compact mismatched DNA}, volume={117}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.1918519117}, abstractNote={Significance}, number={28}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Bradford, Kira C. and Wilkins, Hunter and Hao, Pengyu and Li, Zimeng M. and Wang, Bangchen and Burke, Dan and Wu, Dong and Smith, Austin E. and Spaller, Logan and Du, Chunwei and et al.}, year={2020}, month={Jul}, pages={16302–16312} }
 @article{xu_pan_roland_sagui_weninger_2020, title={Dynamics of strand slippage in DNA hairpins formed by CAG repeats: roles of sequence parity and trinucleotide interrupts}, volume={1}, url={https://doi.org/10.1093/nar/gkaa036}, DOI={10.1093/nar/gkaa036}, abstractNote={Abstract}, journal={Nucleic Acids Research}, publisher={Oxford University Press (OUP)}, author={Xu, Pengning and Pan, Feng and Roland, Christopher and Sagui, Celeste and Weninger, Keith}, year={2020}, month={Mar} }
 @article{larsen_rosholm_kennard_pedersen_munch_tkach_sakon_bjornholm_weninger_bendix_et al._2020, title={How Membrane Geometry Regulates Protein Sorting Independently of Mean Curvature}, volume={6}, ISSN={["2374-7951"]}, DOI={10.1021/acscentsci.0c00419}, abstractNote={Biological membranes have distinct geometries that confer specific functions. However, the molecular mechanisms underlying the phenomenological geometry/function correlations remain elusive. We studied the effect of membrane geometry on the localization of membrane-bound proteins. Quantitative comparative experiments between the two most abundant cellular membrane geometries, spherical and cylindrical, revealed that geometry regulates the spatial segregation of proteins. The measured geometry-driven segregation reached 50-fold for membranes of the same mean curvature, demonstrating a crucial and hitherto unaccounted contribution by Gaussian curvature. Molecular-field theory calculations elucidated the underlying physical and molecular mechanisms. Our results reveal that distinct membrane geometries have specific physicochemical properties and thus establish a ubiquitous mechanistic foundation for unravelling the conserved correlations between biological function and membrane polymorphism.}, number={7}, journal={ACS CENTRAL SCIENCE}, author={Larsen, Jannik B. and Rosholm, Kadla R. and Kennard, Celeste and Pedersen, Soren L. and Munch, Henrik K. and Tkach, Vadym and Sakon, John J. and Bjornholm, Thomas and Weninger, Keith R. and Bendix, Poul Martin and et al.}, year={2020}, month={Jul}, pages={1159–1168} }
 @article{hao_leblanc_case_elston_hingorani_erie_weninger_2020, title={Recurrent mismatch binding by MutS mobile clamps on DNA localizes repair complexes nearby}, volume={117}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.1918517117}, abstractNote={Significance}, number={30}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Hao, Pengyu and LeBlanc, Sharonda J. and Case, Brandon C. and Elston, Timothy C. and Hingorani, Manju M. and Erie, Dorothy A. and Weninger, Keith R.}, year={2020}, month={Jul}, pages={17775–17784} }
 @article{leblanc_hao_hinds_morgan_gbozah_weninger_erie_2019, title={Investigating the Function of Mutl Conformational Changes in Mismatch Repair using smFRET}, volume={116}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/J.BPJ.2018.11.311}, DOI={10.1016/J.BPJ.2018.11.311}, abstractNote={Mismatch repair (MMR) is a highly conserved enzymatic system that corrects errors made during DNA replication. MMR involves the coordinated stepwise action of about ten different proteins on a DNA mismatch. In humans, mutations in the genes that code for mismatch repair proteins have been linked to hereditary non-polyposis colorectal cancer (HNPCC), and some sporadic cancers. The connection between cancer and mutations in DNA repair proteins has been firmly established with biochemical and in vivo studies, but we currently lack a molecular understanding of precisely how these mutations lead to tissue-specific tumorigenesis. MutS recognizes a mismatch by binding to DNA, and then undergoes ATP-dependent conformational changes to interact with MutL. This MutS-MutL complex then interacts with the processivity clamp in an ATP-dependent manner that activates MutL to nick the daughter strand. This nick serves as an entry point for exonucleases and polymerase to complete repair. MutL is the key player in the middle of the pathway that directs repair, likely acting as a switch to turn molecular interactions of repair machinery on and off. Despite its importance in repair, the mechanism by which MutL carries out its functions is poorly understood. Structural, biochemical, and single molecule experiments indicate that ATP binding and hydrolysis regulate conformational changes in MutL to modulate transient interactions. We have used single molecule FRET to determine the key dynamic conformational changes of MutL during its initial interactions with the MutS-mismatch DNA recognition complex. We expect that conformational dynamics of MutL are critical for MutL to activate downstream machinery in the MMR system and that defective interactions are presumably involved in the malfunction that leads to cancer.}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={LeBlanc, Sharonda J. and Hao, Pengyu and Hinds, Malikiya A. and Morgan, Andi N. and Gbozah, Korene and Weninger, Keith R. and Erie, Dorothy A.}, year={2019}, month={Feb}, pages={49a–50a} }
 @misc{choi_sanabria_smirnova_bowen_weninger_2019, title={Spontaneous Switching among Conformational Ensembles in Intrinsically Disordered Proteins}, volume={9}, ISSN={["2218-273X"]}, DOI={10.3390/biom9030114}, abstractNote={The common conception of intrinsically disordered proteins (IDPs) is that they stochastically sample all possible configurations driven by thermal fluctuations. This is certainly true for many IDPs, which behave as swollen random coils that can be described using polymer models developed for homopolymers. However, the variability in interaction energy between different amino acid sequences provides the possibility that some configurations may be strongly preferred while others are forbidden. In compact globular IDPs, core hydration and packing density can vary between segments of the polypeptide chain leading to complex conformational dynamics. Here, we describe a growing number of proteins that appear intrinsically disordered by biochemical and bioinformatic characterization but switch between restricted regions of conformational space. In some cases, spontaneous switching between conformational ensembles was directly observed, but few methods can identify when an IDP is acting as a restricted chain. Such switching between disparate corners of conformational space could bias ligand binding and regulate the volume of IDPs acting as structural or entropic elements. Thus, mapping the accessible energy landscape and capturing dynamics across a wide range of timescales are essential to recognize when an IDP is acting as such a switch.}, number={3}, journal={BIOMOLECULES}, author={Choi, Ucheor B. and Sanabria, Hugo and Smirnova, Tatyana and Bowen, Mark E. and Weninger, Keith R.}, year={2019}, month={Mar} }
 @article{lin_kulkarni_bocci_schafer_roy_tsai_he_chen_rajagopalan_mooney_et al._2019, title={Structural and Dynamical Order of a Disordered Protein: Molecular Insights into Conformational Switching of PAGE4 at the Systems Level}, volume={9}, ISSN={2218-273X}, url={http://dx.doi.org/10.3390/biom9020077}, DOI={10.3390/biom9020077}, abstractNote={Folded proteins show a high degree of structural order and undergo (fairly constrained) collective motions related to their functions. On the other hand, intrinsically disordered proteins (IDPs), while lacking a well-defined three-dimensional structure, do exhibit some structural and dynamical ordering, but are less constrained in their motions than folded proteins. The larger structural plasticity of IDPs emphasizes the importance of entropically driven motions. Many IDPs undergo function-related disorder-to-order transitions driven by their interaction with specific binding partners. As experimental techniques become more sensitive and become better integrated with computational simulations, we are beginning to see how the modest structural ordering and large amplitude collective motions of IDPs endow them with an ability to mediate multiple interactions with different partners in the cell. To illustrate these points, here, we use Prostate-associated gene 4 (PAGE4), an IDP implicated in prostate cancer (PCa) as an example. We first review our previous efforts using molecular dynamics simulations based on atomistic AWSEM to study the conformational dynamics of PAGE4 and how its motions change in its different physiologically relevant phosphorylated forms. Our simulations quantitatively reproduced experimental observations and revealed how structural and dynamical ordering are encoded in the sequence of PAGE4 and can be modulated by different extents of phosphorylation by the kinases HIPK1 and CLK2. This ordering is reflected in changing populations of certain secondary structural elements as well as in the regularity of its collective motions. These ordered features are directly correlated with the functional interactions of WT-PAGE4, HIPK1-PAGE4 and CLK2-PAGE4 with the AP-1 signaling axis. These interactions give rise to repeated transitions between (high HIPK1-PAGE4, low CLK2-PAGE4) and (low HIPK1-PAGE4, high CLK2-PAGE4) cell phenotypes, which possess differing sensitivities to the standard PCa therapies, such as androgen deprivation therapy (ADT). We argue that, although the structural plasticity of an IDP is important in promoting promiscuous interactions, the modulation of the structural ordering is important for sculpting its interactions so as to rewire with agility biomolecular interaction networks with significant functional consequences.}, number={2}, journal={Biomolecules}, publisher={MDPI AG}, author={Lin, Xingcheng and Kulkarni, Prakash and Bocci, Federico and Schafer, Nicholas and Roy, Susmita and Tsai, Min-Yeh and He, Yanan and Chen, Yihong and Rajagopalan, Krithika and Mooney, Steven and et al.}, year={2019}, month={Feb}, pages={77} }
 @article{pan_dangi_kaur_hao_weninger_riehn_opresko_wang_2019, title={TIN2 is an Architectural Protein Stabilizing TRF1 at Telomere}, volume={116}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/J.BPJ.2018.11.1168}, DOI={10.1016/J.BPJ.2018.11.1168}, abstractNote={Telomeres, consisting of duplex TTAGGG repeats and associating with protein complexes at chromosome ends, play a crucial role in maintaining the stability of chromosomes. The protein complex - shelterin contains six subunits (TRF1, TRF2, RAP1, TIN2, TPP1 and POT1), which bind to telomeres and protect the chromosome ends from DNA repairing, and recruit telomerase when chromosome gets shortened abnormally. Among these six subunits, TIN2 has no affinity to either double-strand or single-strand DNA. However, it is a core component bridging the double-strand DNA binding proteins (TRF1 and TRF2) to single-strand DNA binding protein complex (TPP1-POT1). Loss of TRF1 or TRF2 binding domain in TIN2 can trigger DNA damage response. Moreover, TIN2 without the TPP1 binding domain is capable of fully supporting the stabilization of TRF1 and TRF2/RAP1. Despite the significance of TIN2 in telomere maintenance, the mechanism underlying TIN2 remains elusive. To understand how TIN2 affects TRF1 binding dynamics and how TIN2 influences with TRF1-mediated telomeric DNA pairing, we conducted Atomic Force Microscopy (AFM) and used nanochannels confined DNA to study the telomeric DNA conformation upon TRF1 and TIN2 binding. We observed high-order protein-DNA complexes formation indicating TIN2 facilitates TRF1 accumulation on telomeric DNA. We also carried out single molecule fluorescence technique to investigate how TIN2 affects TRF1 binding dynamics on telomeric DNA sequences. Our results show that TIN2 can accelerate DNA-DNA pairing and stabilize TRF1 on telomeric DNA.}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Pan, Hai and Dangi, Saroj and Kaur, Parminder and Hao, Pengyu and Weninger, Keith and Riehn, Robert and Opresko, Patricia and Wang, Hong}, year={2019}, month={Feb}, pages={211a–212a} }
 @article{xu_hensley_chan_weninger_2019, title={Toward Single Molecule FRET Studies of DNA Mismatch Repair in Live Bacteria}, volume={116}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/J.BPJ.2018.11.772}, DOI={10.1016/J.BPJ.2018.11.772}, abstractNote={DNA Mismatch Repair (MMR) is an important correction system that contributes to genomic fidelity during DNA replication. The protein MutS initiates DNA MMR by recognition of base mismatch or insertion/deletion loop and activation of downstream repair processes. Single molecule FRET has previously been used in purified, in vitro studies to characterize conformational transitions in MutS as it interacts with a DNA mismatch and converts to a sliding clamp. The relationship of these conformational transitions to the setting inside live cells remains to be demonstrated. Here we report on efforts to combine in vivo single molecule tracking with single molecule FRET to study the Taq MutS behavior inside live E. coli.}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Xu, Pengning and Hensley, Andrew and Chan, Edward and Weninger, Keith R.}, year={2019}, month={Feb}, pages={139a} }
 @article{hao_leblanc_erie_weninger_2019, title={Untangling DNA Mismatch Repair Complexes with smFRET and Tethered Particle Motion Analysis}, volume={116}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/J.BPJ.2018.11.457}, DOI={10.1016/J.BPJ.2018.11.457}, abstractNote={DNA is the genetic information carrier in all organisms and therefore must be replicated and propagated across generations with high fidelity. Several DNA repair pathways correct different types of lesions that arise during DNA replication or damage. Among these pathways, DNA mismatch repair proteins recognize and activate repair of incorrectly incorporated nucleotides that escape polymerase proofreading. Although intensely studied for decades, details of the mechanisms within DNA mismatch repair are still controversial. Here we present experiments using single molecule Fluorescence Resonance Energy Transfer and tethered particle motion analysis to provide access to the dynamic and transient interactions of MutS and MutL with DNA mismatched bases. Using photocleavable end blocking of DNA, we present an assay that can report the mobility of these proteins sliding on DNA. These sorts of single molecule experiments provide insight into the mechanisms that cause activation of DNA mismatch repair processes.}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Hao, Pengyu and LeBlanc, Sharonda and Erie, Dorothy A. and Weninger, Keith R.}, year={2019}, month={Feb}, pages={77a} }
 @article{leblanc_gauer_hao_case_hingorani_weninger_erie_2018, title={Coordinated protein and DNA conformational changes govern mismatch repair initiation by MutS}, volume={46}, ISSN={["1362-4962"]}, DOI={10.1093/nar/gky865}, abstractNote={Abstract MutS homologs identify base-pairing errors made in DNA during replication and initiate their repair. In the presence of adenosine triphosphate, MutS induces DNA bending upon mismatch recognition and subsequently undergoes conformational transitions that promote its interaction with MutL to signal repair. In the absence of MutL, these transitions lead to formation of a MutS mobile clamp that can move along the DNA. Previous single-molecule FRET (smFRET) studies characterized the dynamics of MutS DNA-binding domains during these transitions. Here, we use protein–DNA and DNA–DNA smFRET to monitor DNA conformational changes, and we use kinetic analyses to correlate DNA and protein conformational changes to one another and to the steps on the pathway to mobile clamp formation. The results reveal multiple sequential structural changes in both MutS and DNA, and they suggest that DNA dynamics play a critical role in the formation of the MutS mobile clamp. Taking these findings together with data from our previous studies, we propose a unified model of coordinated MutS and DNA conformational changes wherein initiation of mismatch repair is governed by a balance of DNA bending/unbending energetics and MutS conformational changes coupled to its nucleotide binding properties.}, number={20}, journal={NUCLEIC ACIDS RESEARCH}, author={LeBlanc, Sharonda J. and Gauer, Jacob W. and Hao, Pengyu and Case, Brandon C. and Hingorani, Manju M. and Weninger, Keith R. and Erie, Dorothy A.}, year={2018}, month={Nov}, pages={10782–10795} }
 @article{roushan_azad_movahed_ray_livshits_lim_weninger_riehn_2018, title={Motor-like DNA motion due to an ATP-hydrolyzing protein under nanoconfinement}, volume={8}, ISSN={2045-2322}, url={http://dx.doi.org/10.1038/S41598-018-28278-0}, DOI={10.1038/S41598-018-28278-0}, abstractNote={Abstract}, number={1}, journal={Scientific Reports}, publisher={Springer Science and Business Media LLC}, author={Roushan, Maedeh and Azad, Zubair and Movahed, Saeid and Ray, Paul D. and Livshits, Gideon I. and Lim, Shuang Fang and Weninger, Keith R. and Riehn, Robert}, year={2018}, month={Jul} }
 @article{lin_roy_jolly_bocci_schafer_tsai_chen_he_grishaev_weninger_et al._2018, title={PAGE4 and Conformational Switching: Insights from Molecular Dynamics Simulations and Implications for Prostate Cancer}, volume={430}, ISSN={0022-2836}, url={http://dx.doi.org/10.1016/J.JMB.2018.05.011}, DOI={10.1016/J.JMB.2018.05.011}, abstractNote={Prostate-associated gene 4 (PAGE4) is an intrinsically disordered protein implicated in prostate cancer. Thestress-response kinase homeodomain-interacting protein kinase 1 (HIPK1) phosphorylates two residues in PAGE4, serine 9 and threonine 51. Phosphorylation of these two residues facilitates the interaction of PAGE4 with activator protein-1 (AP-1) transcription factor complex to potentiate AP-1's activity. In contrast, hyperphosphorylation of PAGE4 by CDC-like kinase 2 (CLK2) attenuates this interaction with AP-1. Small-angleX-ray scattering and single-molecule fluorescence resonance energy transfer measurements have shown that PAGE4 expands upon hyperphosphorylation and that this expansion is localized to its N-terminal half. To understand the interactions underlying this structural transition, we performed molecular dynamics simulations using Atomistic AWSEM, a multi-scale molecular model that combines atomistic and coarse-grained simulation approaches. Our simulations show that electrostatic interactions drive transient formation of an N-terminal loop, the destabilization of which accounts for the dramatic change in size upon hyperphosphorylation. Phosphorylation also changes the preference of secondary structure formation of the PAGE4 ensemble, which leads to a transition between states that display different degrees of disorder. Finally, we construct a mechanism-based mathematical model that allows us to capture the interactions ofdifferent phosphoforms of PAGE4 with AP-1 and its downstream target, the androgen receptor (AR)—a key therapeutic target in prostate cancer. Our model predicts intracellular oscillatory dynamics of HIPK1-PAGE4, CLK2-PAGE4, and AR activity, indicating phenotypic heterogeneity in an isogenic cell population. Thus, conformational switching of PAGE4 may potentially affect the efficiency of therapeutically targeting AR activity.}, number={16}, journal={Journal of Molecular Biology}, publisher={Elsevier BV}, author={Lin, Xingcheng and Roy, Susmita and Jolly, Mohit Kumar and Bocci, Federico and Schafer, Nicholas P. and Tsai, Min-Yeh and Chen, Yihong and He, Yanan and Grishaev, Alexander and Weninger, Keith and et al.}, year={2018}, month={Aug}, pages={2422–2438} }
 @misc{jolly_kulkarni_weninger_orban_levine_2018, title={Phenotypic Plasticity, Bet-Hedging, and Androgen Independence in Prostate Cancer: Role of Non-Genetic Heterogeneity}, volume={8}, ISSN={["2234-943X"]}, DOI={10.3389/fonc.2018.00050}, abstractNote={It is well known that genetic mutations can drive drug resistance and lead to tumor relapse. Here, we focus on alternate mechanisms—those without mutations, such as phenotypic plasticity and stochastic cell-to-cell variability that can also evade drug attacks by giving rise to drug-tolerant persisters. The phenomenon of persistence has been well-studied in bacteria and has also recently garnered attention in cancer. We draw a parallel between bacterial persistence and resistance against androgen deprivation therapy in prostate cancer (PCa), the primary standard care for metastatic disease. We illustrate how phenotypic plasticity and consequent mutation-independent or non-genetic heterogeneity possibly driven by protein conformational dynamics can stochastically give rise to androgen independence in PCa, and suggest that dynamic phenotypic plasticity should be considered in devising therapeutic dosing strategies designed to treat and manage PCa.}, journal={FRONTIERS IN ONCOLOGY}, author={Jolly, Mohit Kumar and Kulkarni, Prakash and Weninger, Keith and Orban, John and Levine, Herbert}, year={2018}, month={Mar} }
 @article{hellenkamp_schmid_doroshenko_opanasyuk_kuehnemuth_adariani_ambrose_aznauryan_barth_birkedal_et al._2018, title={Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study}, volume={15}, ISSN={["1548-7105"]}, DOI={10.1038/s41592-018-0085-0}, abstractNote={Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods.}, number={9}, journal={NATURE METHODS}, author={Hellenkamp, Bjoern and Schmid, Sonja and Doroshenko, Olga and Opanasyuk, Oleg and Kuehnemuth, Ralf and Adariani, Soheila Rezaei and Ambrose, Benjamin and Aznauryan, Mikayel and Barth, Anders and Birkedal, Victoria and et al.}, year={2018}, month={Sep}, pages={669-+} }
 @misc{salgia_jolly_dorff_lau_weninger_orban_kulkarni_2018, title={Prostate-Associated Gene 4 (PAGE4): Leveraging the Conformational Dynamics of a Dancing Protein Cloud as a Therapeutic Target}, volume={7}, ISSN={["2077-0383"]}, DOI={10.3390/jcm7060156}, abstractNote={Prostate cancer (PCa) is a leading cause of mortality and morbidity globally. While genomic alterations have been identified in PCa, in contrast to some other cancers, use of such information to personalize treatment is still in its infancy. Here, we discuss how PAGE4, a protein which appears to act both as an oncogenic factor as well as a metastasis suppressor, is a novel therapeutic target for PCa. Inhibiting PAGE4 may be a viable strategy for low-risk PCa where it is highly upregulated. Conversely, PAGE4 expression is downregulated in metastatic PCa and, therefore, reinstituting its sustained expression may be a promising option to subvert or attenuate androgen-resistant PCa. Thus, fine-tuning the levels of PAGE4 may represent a novel approach for personalized medicine in PCa.}, number={6}, journal={JOURNAL OF CLINICAL MEDICINE}, author={Salgia, Ravi and Jolly, Mohit Kumar and Dorff, Tanya and Lau, Clayton and Weninger, Keith and Orban, John and Kulkarni, Prakash}, year={2018}, month={Jun} }
 @article{hellenkamp_schmid_doroshenko_opanasyuk_kühnemuth_adariani_ambrose_aznauryan_barth_birkedal_et al._2018, title={Publisher Correction: Precision and accuracy of single-molecule
FRET measurements—a multi-laboratory benchmark study}, volume={15}, ISSN={1548-7091 1548-7105}, url={http://dx.doi.org/10.1038/S41592-018-0193-X}, DOI={10.1038/S41592-018-0193-X}, abstractNote={This paper was originally published under standard Springer Nature copyright. As of the date of this correction, the Analysis is available online as an open-access paper with a CC-BY license. No other part of the paper has been changed.}, number={11}, journal={Nature Methods}, publisher={Springer Science and Business Media LLC}, author={Hellenkamp, Björn and Schmid, Sonja and Doroshenko, Olga and Opanasyuk, Oleg and Kühnemuth, Ralf and Adariani, Soheila Rezaei and Ambrose, Benjamin and Aznauryan, Mikayel and Barth, Anders and Birkedal, Victoria and et al.}, year={2018}, month={Oct}, pages={984–984} }
 @article{hao_leblanc_erie_weninger_2018, title={Single Molecule Experiments Reveal Molecular Level Details of MutS-MutL Interactions in DNA Mismatch Activated Sliding Clamp}, volume={114}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/J.BPJ.2017.11.508}, DOI={10.1016/J.BPJ.2017.11.508}, abstractNote={Maintaining the integrity of DNA is crucial for a species to preserve genomic information. Organisms have developed several DNA repair pathways to repair different types of DNA lesion. Among these pathways, the DNA mismatch repair pathway identifies and repairs erroneous insertion or deletion of nucleotide bases during DNA replication. The first two proteins involved in this DNA mismatch repair process, MutS and MutL, are studied in this work using single molecule techniques including smFRET and tethered particle motion tracking. With smFRET we have characterized complex behaviors of MutS sliding clamps on DNA containing a mismatch. We find that inclusion of MutL in the assay greatly alters the behavior of these MutS sliding clamps. With Tethered Particle Motion (TPM) experiments, we characterize the effects of MutS and MutL on the global structure of DNA. The results constrain current models of the DNA mismatch repair phenomena.}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Hao, Pengyu and LeBlanc, Sharonda and Erie, Dorothy and Weninger, Keith}, year={2018}, month={Feb}, pages={85a} }
 @article{xu_weninger_2018, title={Single Molecule FRET Observations of Folding for DNA Hairpins Containing Trinucleotide Repeats}, volume={114}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/J.BPJ.2017.11.3275}, DOI={10.1016/J.BPJ.2017.11.3275}, abstractNote={Expansions of tracks of Trinucleotide Repeats (TR) in DNA is associated with disorders including Huntington disease, fragile X syndrome and myotonic dystrophy. Unusual TR conformations may cause mutagenic DNA replication and affect downstream protein translation changes. We have used single molecule FRET (smFRET) to study the dynamics of DNA containing repeated Cytosine-Adenine-Guanine (CAG) triplet patterns that can self-assemble into loops and haripins. Thermodynamic characteristics such as transitional entropy and enthalpy acquired from studies of single molecules are compared with results from DNA melting bulk analysis. We have found complex patterns of hairpin stability and cooperativity that depend sensitively on the number of triplet repeats.}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Xu, Pengning and Weninger, Keith}, year={2018}, month={Feb}, pages={599a} }
 @misc{leblanc_kulkarni_weninger_2018, title={Single Molecule FRET: A Powerful Tool to Study Intrinsically Disordered Proteins}, volume={8}, ISSN={["2218-273X"]}, DOI={10.3390/biom8040140}, abstractNote={Intrinsically disordered proteins (IDPs) are often modeled using ideas from polymer physics that suggest they smoothly explore all corners of configuration space. Experimental verification of this random, dynamic behavior is difficult as random fluctuations of IDPs cannot be synchronized across an ensemble. Single molecule fluorescence (or Förster) resonance energy transfer (smFRET) is one of the few approaches that are sensitive to transient populations of sub-states within molecular ensembles. In some implementations, smFRET has sufficient time resolution to resolve transitions in IDP behaviors. Here we present experimental issues to consider when applying smFRET to study IDP configuration. We illustrate the power of applying smFRET to IDPs by discussing two cases in the literature of protein systems for which smFRET has successfully reported phosphorylation-induced modification (but not elimination) of the disordered properties that have been connected to impacts on the related biological function. The examples we discuss, PAGE4 and a disordered segment of the GluN2B subunit of the NMDA receptor, illustrate the great potential of smFRET to inform how IDP function can be regulated by controlling the detailed ensemble of disordered states within biological networks.}, number={4}, journal={BIOMOLECULES}, author={LeBlanc, Sharonda J. and Kulkarni, Prakash and Weninger, Keith R.}, year={2018}, month={Dec} }
 @article{leblanc_gauer_hao_walser_weninger_erie_2017, title={Dynamic Studies of MutS-MutL-DNA Complexes in Mismatch Repair}, volume={112}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/J.BPJ.2016.11.2793}, DOI={10.1016/J.BPJ.2016.11.2793}, abstractNote={DNA mismatch repair (MMR) is a post-replicative system of proteins that corrects rare mistakes in the genome of all organisms. In the human genome of 6 billion bases, there are ∼ 600 errors per round of replication, per cell. If left uncorrected, errors accumulate as permanent mutations in a genome, and can lead to a disease state in the organism. MutS and MutL homologs are tasked with recognizing a mismatch in 107 correctly paired bases, discriminating between parent and daughter strand, then initiating repair. Single amino acid mutations in MutS and MutL proteins have been linked to hereditary and sporadic colorectal cancer, the third most common cancer worldwide. Although these mutations, mostly associated with MutL, have been identified in cancer cases, it is unclear how MMR deficiencies initiate and advance the disease. Failures in the mismatch repair pathway likely initiate tumorigenesis, but we lack a fundamental understanding of the MMR process. MutS and MutL are ATPases that undergo conformational changes upon ATP binding and hydrolysis. We seek to understand how these conformations are functional in coordinating repair. On the molecular level, we know that MutS initially recognizes a DNA mismatch, and undergoes ATP-dependent conformational changes to slide along the DNA. MutL is recruited to the site, and interacts with MutS on DNA to coordinate repair with PCNA, EXO1, DNA polymerase, RFC clamp loader, RPA single strand binding protein, and DNA ligase. The formation of the ternary MutS-MutL-DNA complex is a key step in this pathway that involves multiple transient protein-protein and protein-DNA interactions. In these studies, we used in vitro single molecule fluorescence resonance energy transfer (smFRET) to investigate the dynamic molecular mechanism of MMR.}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={LeBlanc, Sharonda and Gauer, Jacob and Hao, Pengyu and Walser, T' Yasah and Weninger, Keith and Erie, Dorothy}, year={2017}, month={Feb}, pages={516a–517a} }
 @article{hao_qiu_erie_weninger_2017, title={Mismatch Repair on the Go: Extending the Mechanical Model of DNA Mismatch Repair through Single Molecule Study}, volume={112}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/J.BPJ.2016.11.2782}, DOI={10.1016/J.BPJ.2016.11.2782}, abstractNote={DNA polymerase has a small but non-zero rate of making single base mismatch errors during DNA replication. To preserve the integrity of genomic information, a cellular system comprised of DNA mismatch repair (MMR) protein machinery is present that is conserved in all organisms. Among the proteins in this system, MutS and MutL are crucial for mismatch recognition and discrimination of parent and daughter strand following replication. MutS protein initiates the MMR cascade by recognizing and binding onto the base-base mismatches on the hybrid daughter-parent dsDNA; MutL follows and creates a nick on the newly synthesized DNA strand to guide the downstream repair processes. We have used single-molecule methods to monitor the interaction between MutS/MutL and mismatched DNA in various nucleotide conditions. The dynamics of MutS and MutL interacting with mismatched DNA is reported by single molecule Fluorescence Resonance Energy Transfer (smFRET) measurements, which are sensitive to nanoscale molecular motions. In addition to FRET, experiments using the Tethered Particle Motion (TPM) technique are conducted to estimate the dynamic changes in the end-to-end length of a DNA molecule when interacting with MMR proteins. In this method, one end of the DNA is anchored on microscope slide and the other attached to a micrometer-sized bead. Imaging the excursions of the bead position as a function of time reveals the length of the DNA tether length. Our observations combine to reveal a picture of the molecular behaviors of MMR proteins interacting with mismatched DNA that supports models of the key processes underlying the MMR pathway.}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Hao, Pengyu and Qiu, Ruoyi and Erie, Dorothy and Weninger, Keith}, year={2017}, month={Feb}, pages={514a–515a} }
 @article{kulkarni_jolly_jia_mooney_bhargava_kagohara_chen_hao_he_veltri_et al._2017, title={Phosphorylation-induced conformational dynamics in an intrinsically disordered protein and potential role in phenotypic heterogeneity}, volume={114}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.1700082114}, abstractNote={Significance}, number={13}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Kulkarni, Prakash and Jolly, Mohit Kumar and Jia, Dongya and Mooney, Steven M. and Bhargava, Ajay and Kagohara, Luciane T. and Chen, Yihong and Hao, Pengyu and He, Yanan and Veltri, Robert W. and et al.}, year={2017}, month={Mar}, pages={E2644–E2653} }
 @article{erie_bradford_wilkins_bower_wang_gauer_satusky_qiu_weninger_kaur_et al._2017, title={Single Molecule Fluorescence and Atomic Force Microscopy Studies of DNA Repair}, volume={112}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/J.BPJ.2016.11.061}, DOI={10.1016/J.BPJ.2016.11.061}, abstractNote={DNA polymerases that are responsible for replication make approximately one error for every 107 bases copied, but the human genome contains ∼6 billion bases, which results in ∼600 errors per round of replication. The DNA mismatch repair (MMR) system corrects these DNA synthesis errors that occur during replication. MMR is initiated by the highly conserved MutS and MutL homologs, which are both dimers and contain DNA binding and ATPase activities that are essential for MMR in vivo. MutS homologs initiate repair by binding to a mismatch and undergoing an ATP-dependent conformational change that promotes its interaction with MutL homologs. This complex signals the initiation of excision and resynthesis of the newly synthesized DNA strand containing the incorrect nucleotide. We have been using a combination of atomic force microscopy (AFM) and single molecule fluorescence to characterize the stoichiometries and the conformational and dynamic properties of MutS and MutL homologs and their assembly on DNA containing a mismatch. We have also developed a new dual resonance frequency enhanced electrostatic force microscopy (DREEM), in which we simultaneously collect the AFM topographic image and an image of the electrostatic potential of the surface. The DREEM images reveal the path of DNA inside individual protein-DNA complexes, yielding unprecedented details about DNA conformations within simple and complicated complexes. I will discuss our studies on the assembly of MutS and MutL homologs on mismatches, with a focus on how AFM, DREEM, and single-molecule fluorescence can be powerful tools to study the stoichiometries, conformations, and dynamic assembly of multi-component complexes.}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Erie, Dorothy and Bradford, Kira and Wilkins, Hunter and Bower, Jacqueline and Wang, Zimeng and Gauer, Jacob and Satusky, Matthew and Qiu, Rouyi and Weninger, Keith and Kaur, Parminder and et al.}, year={2017}, month={Feb}, pages={7a} }
 @misc{kulkarni_dunker_weninger_orban_2016, title={Prostate-associated gene 4 (PAGE4), an intrinsically disordered cancer/testis antigen, is a novel therapeutic target for prostate cancer}, volume={18}, ISSN={["1745-7262"]}, DOI={10.4103/1008-682x.181818}, abstractNote={Prostate-associated gene 4 (PAGE4) is a remarkably prostate-specific Cancer/Testis Antigen that is highly upregulated in the human fetal prostate and its diseased states but not in the adult normal gland. PAGE4 is an intrinsically disordered protein (IDP) that functions as a stress-response protein to suppress reactive oxygen species as well as prevent DNA damage. In addition, PAGE4 is also a transcriptional regulator that potentiates transactivation by the oncogene c-Jun. c-Jun forms the AP-1 complex by heterodimerizing with members of the Fos family and plays an important role in the development and pathology of the prostate gland, underscoring the importance of the PAGE4/c-Jun interaction. HIPK1, also a component of the stress-response pathway, phosphorylates PAGE4 at T51 which is critical for its transcriptional activity. Phosphorylation induces conformational and dynamic switching in the PAGE4 ensemble leading to a new cellular function. Finally, bioinformatics evidence suggests that the PAGE4 mRNA could be alternatively spliced resulting in four potential isoforms of the polypeptide alluding to the possibility of a range of conformational ensembles with latent functions. Considered together, the data suggest that PAGE4 may represent the first molecular link between stress and prostate cancer (PCa). Thus, pharmacologically targeting PAGE4 may be a novel opportunity for treating and managing patients with PCa, especially patients with low-risk disease.}, number={5}, journal={ASIAN JOURNAL OF ANDROLOGY}, author={Kulkarni, Prakash and Dunker, A. Keith and Weninger, Keith and Orban, John}, year={2016}, month={Sep}, pages={695–703} }
 @article{gauer_leblanc_hao_qiu_case_sakato_hingorani_erie_weninger_2016, title={Single-molecule FRET to measure conformational dynamics of DNA mismatch repair proteins}, volume={581}, journal={Single-molecule enzymology: fluorescence-based and high-throughput methods}, author={Gauer, J. W. and LeBlanc, S. and Hao, P. and Qiu, R. and Case, B. C. and Sakato, M. and Hingorani, M. M. and Erie, D. A. and Weninger, K. R.}, year={2016}, pages={285–315} }
 @article{fletcher_fletcher_weninger_martin_2016, title={Venoms from Centruroides sp. Scorpions Cleave SNARE Proteins}, volume={117}, ISSN={0041-0101}, url={http://dx.doi.org/10.1016/J.TOXICON.2016.04.025}, DOI={10.1016/J.TOXICON.2016.04.025}, journal={Toxicon}, publisher={Elsevier BV}, author={Fletcher, Paul L., Jr. and Fletcher, Maryann D. and Weninger, Keith R. and Martin, Brian M.}, year={2016}, month={Jul}, pages={109} }
 @article{weninger_qiu_ou_milikisiyants_sanabria_smirnova_2016, title={smFRET and DEER Distance Measurements as Applied to Disordered and Structured Proteins}, volume={110}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/J.BPJ.2015.11.2987}, DOI={10.1016/J.BPJ.2015.11.2987}, abstractNote={FRET and DEER are two spectroscopic methods that are widely applied for biophysical studies of protein structure. Both methods are based on measuring dipolar interactions - electrical dipoles in case of FRET and magnetic dipoles in case of DEER - between specifically labeled protein sites. The experimental data are then analyzed to derive the distance between the interacting dipoles and relate this distance to the structure of biomacromolecule(s). Molecular volume of EPR labels is generally smaller vs. that of the fluorescent probes and DEER experiments can be carried out by labeling cysteines with identical molecular tags whereas FRET typically relies on orthogonal labeling with distinct donor and acceptor fluorophores. Another essential difference is that FRET can be performed under physiological conditions, but DEER typically requires cryogenic or near cryogenic temperatures because of short phase memory time for nitroxides at ambient conditions. Finally, single molecule (sm) FRET reports on conformation of individual protein molecules whereas DEER provides information on ensemble average. While the distance ranges of these two methods overlaps the direct comparison of FRET and DEER data is rarely found in the literature. Here we report on the distance measurements and conformational states using both smFRET and DEER on three protein systems. We attached probes to a unique pair of cysteines in the neuronal SNARE protein SNAP-25. SNAP-25 is highly disordered in isolation, but it folds into a stable alpha-helix bundle upon forming SNARE complex with syntaxin and synaptobrevin. We also labeled intrinsically disordered Glutamate Receptor Cytoplasmic Domain N2B. Results of smFRET and DEER distances and distance distribution are compared for disordered SNAP-25 and folded SNAP-25 within the SNARE complex and for disordered N2B.}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Weninger, Keith and Qiu, Ruoyi and Ou, Erkang and Milikisiyants, Sergey and Sanabria, Hugo and Smirnova, Tatyana I.}, year={2016}, month={Feb}, pages={559a} }
 @article{breuer_larsen_røskva rosholm_pedersenb_munch_tkach_sakon_bjørnholm_weninger_bendix_et al._2016, title={tN-Ras, Synaptotagmin1 C2Ab, Annexinb12 and Amphiphysin NBAR can Discriminate Spherical from Cylindrical Membrane Curvature}, volume={110}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/J.BPJ.2015.11.1924}, DOI={10.1016/J.BPJ.2015.11.1924}, abstractNote={Membrane shape or geometrical curvature emerged recently as a potent regulator of membrane recruitment during protein trafficking and sorting. Cellular membranes display distinct curvature geometries e.g. spherical (trafficking- and synaptic vesicles) or cylindrical (tubes in the ER and Golgi), however quantitative studies of protein recruitment by membrane curvature typically focus on a single geometry. Thus the biological implications of different curvature geometries remain largely unexplored. We recently used our single liposome assay to show that the N-Ras lipid anchor (tN-Ras) is recruited by spherical membrane curvature. Here we report the development of a novel membrane tube assay, allowing us to quantitatively compare the recruitment of tN-Ras by spherical and cylindrical membrane curvature. Furthermore we expand the study to include representatives of the four most common families of membrane-binding domains (MBDs); the lipid anchor of N-Ras (tN-Ras), the C2AB-domain of Synaptotagmin1 (Syt), AnnexinB12 (Anx) and Amphiphysin NBAR (Amph). Our data revealed an increased recruitment of all four MBDs by spherical as compared to cylindrical curvature. Molecular field theory calculations attributed this trend to the greater perturbation of lipid packing parameters by spherically curved membranes. Importantly, the four MBDs displayed markedly distinct abilities to discriminate the two curvature geometries e.g. tN-Ras had a moderate 2-fold discrimination while Amph a remarkable absolute preference for spherical membranes. This demonstrated that discrimination of curvature geometry can be modulated and likely adapted to specific biological functions. Our results reveal membrane curvature geometry as a novel regulator of protein recruitment during trafficking and sorting for a plethora of membrane-binding proteins, and thus suggest a novel functional role to the diversity of conserved organelle morphologies.}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Breuer, Artu' and Larsen, Jannik and Røskva Rosholm, Kadla and Pedersenb, Søren L. and Munch, Henrik K. and Tkach, Vadym and Sakon, John J. and Bjørnholm, Thomas and Weninger, Keith R. and Bendix, Poul M. and et al.}, year={2016}, month={Feb}, pages={357a} }
 @article{fletcher_fletcher_weninger_2016, title={“Metalloproteases in new world scorpion venoms cleave intracellular membrane fusion SNARE proteins VAMP2 and VAMP8”}, volume={117}, ISSN={0041-0101}, url={http://dx.doi.org/10.1016/J.TOXICON.2016.04.008}, DOI={10.1016/J.TOXICON.2016.04.008}, journal={Toxicon}, publisher={Elsevier BV}, author={Fletcher, Paul L., Jr. and Fletcher, Maryann D. and Weninger, Keith R.}, year={2016}, month={Jul}, pages={104} }
 @article{qiu_sakato_sacho_wilkins_zhang_modrichd_hingorani_erie_weninger_2015, title={MutL traps MutS at a DNA mismatch}, volume={112}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.1505655112}, abstractNote={Significance}, number={35}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Qiu, Ruoyi and Sakato, Miho and Sacho, Elizabeth J. and Wilkins, Hunter and Zhang, Xingdong and Modrichd, Paul and Hingorani, Manju M. and Erie, Dorothy A. and Weninger, Keith R.}, year={2015}, month={Sep}, pages={10914–10919} }
 @article{he_chen_mooney_rajagopalan_bhargava_sacho_keith_bryan_kulkarni_orban_et al._2015, title={Phosphorylation-induced Conformational Ensemble Switching in an Intrinsically Disordered Cancer/Testis Antigen}, volume={290}, ISSN={["1083-351X"]}, DOI={10.1074/jbc.m115.658583}, abstractNote={Background: PAGE4, an intrinsically disordered protein up-regulated in prostate cancer, binds to c-Jun and potentiates its transactivation. Results: The effects of phosphorylation on PAGE4 conformation, dynamics, and c-Jun binding were determined by NMR. Conclusion: Phosphorylation induces a more compact conformational ensemble, restricting access to the c-Jun binding site. Significance: This study may help to explain how phosphorylation of PAGE4 alters its binding to c-Jun. Prostate-associated gene 4 (PAGE4) is an intrinsically disordered cancer/testis antigen that is up-regulated in the fetal and diseased human prostate. Knocking down PAGE4 expression results in cell death, whereas its overexpression leads to a growth advantage of prostate cancer cells (Zeng, Y., He, Y., Yang, F., Mooney, S. M., Getzenberg, R. H., Orban, J., and Kulkarni, P. (2011) The cancer/testis antigen prostate-associated gene 4 (PAGE4) is a highly intrinsically disordered protein. J. Biol. Chem. 286, 13985–13994). Phosphorylation of PAGE4 at Thr-51 is critical for potentiating c-Jun transactivation, an important factor in controlling cell growth, apoptosis, and stress response. Using NMR spectroscopy, we show that the PAGE4 polypeptide chain has local and long-range conformational preferences that are perturbed by site-specific phosphorylation at Thr-51. The population of transient turn-like structures increases upon phosphorylation in an ∼20-residue acidic region centered on Thr-51. This central region therefore becomes more compact and more negatively charged, with increasing intramolecular contacts to basic sequence motifs near the N and C termini. Although flexibility is decreased in the central region of phospho-PAGE4, the polypeptide chain remains highly dynamic overall. PAGE4 utilizes a transient helical structure adjacent to the central acidic region to bind c-Jun with low affinity in vitro. The binding interaction is attenuated by phosphorylation at Thr-51, most likely because of masking the effects of the more compact phosphorylated state. Therefore, phosphorylation of PAGE4 leads to conformational shifts in the dynamic ensemble, with large functional consequences. The changes in the structural ensemble induced by posttranslational modifications are similar conceptually to the conformational switching events seen in some marginally stable (“metamorphic”) folded proteins in response to mutation or environmental triggers.}, number={41}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={He, Y. N. and Chen, Y. H. and Mooney, S. M. and Rajagopalan, K. and Bhargava, A. and Sacho, E. and keith and Bryan, P. N. and Kulkarni, P. and Orban, J. and et al.}, year={2015}, month={Oct}, pages={25090–25102} }
 @article{weninger_hao_yang_sacho_qiu_2015, title={Toward Adding Complexity in Single Molecule FRET Studies of DNA Mismatch Repair}, volume={108}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/J.BPJ.2014.11.414}, DOI={10.1016/J.BPJ.2014.11.414}, abstractNote={Mistakes made during DNA replication are targeted for post-replicative repair by the DNA mismatch repair system. Purified reconstitutions of DNA mismatch repair require multiple protein interactions, and in vivo studies of DNA mismatch repair function have identified layers of spatial and temporal regulation. We have previously developed single molecule FRET experiments for studies of purified in vitro studies of DNA mismatch interactions from Thermus Aquaticus MutS and MutL. In this poster we present our recent progress developing single molecule FRET assays for more complex studies of DNA repair. In particular, we highlight our work using unnatural amino acid engineering and other methods for site-specific labeling of yeast repair proteins, detailed analysis of kinetics to reveal nucleotide regulation of protein conformations, and steps toward studies in live cells.}, number={2}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={weninger, keith and Hao, Pengyu and Yang, Yue and Sacho, Elizabeth J. and Qiu, Ruoyi}, year={2015}, month={Jan}, pages={69a–70a} }
 @article{mooney_qiu_kim_sacho_rajagopalan_johng_shiraishi_kulkarni_weninger_2014, title={Cancer/Testis Antigen PAGE4, a Regulator of c-Jun Transactivation, Is Phosphorylated by Homeodomain-Interacting Protein Kinase 1, a Component of the Stress-Response Pathway}, volume={53}, ISSN={["0006-2960"]}, DOI={10.1021/bi500013w}, abstractNote={Prostate-associated gene 4 (PAGE4) is a cancer/testis antigen that is typically restricted to the testicular germ cells but is aberrantly expressed in cancer. Furthermore, PAGE4 is developmentally regulated with dynamic expression patterns in the developing prostate and is also a stress-response protein that is upregulated in response to cellular stress. PAGE4 interacts with c-Jun, which is activated by the stress-response kinase JNK1, and plays an important role in the development and pathology of the prostate gland. Here, we have identified homeodomain-interacting protein kinase 1 (HIPK1), also a component of the stress-response pathway, as a kinase that phosphorylates PAGE4 at T51. We show that phosphorylation of PAGE4 is critical for its transcriptional activity since mutating this T residue abolishes its ability to potentiate c-Jun transactivation. In vitro single molecule FRET indicates phosphorylation results in compaction of (still) intrinsically disordered PAGE4. Interestingly, however, while our previous observations indicated that the wild-type nonphosphorylated PAGE4 protein interacted with c-Jun [RajagopalanK. et al. (2014) Biochim, Biophys. Acta1842, 154−16324263171], here we show that phosphorylation of PAGE4 weakens its interaction with c-Jun in vitro. These data suggest that phosphorylation induces conformational changes in natively disordered PAGE4 resulting in its decreased affinity for c-Jun to promote interaction of c-Jun with another, unidentified, partner. Alternatively, phosphorylated PAGE4 may induce transcription of a novel partner, which then potentiates c-Jun transactivation. Regardless, the present results clearly implicate PAGE4 as a component of the stress-response pathway and uncover a novel link between components of this pathway and prostatic development and disease.}, number={10}, journal={BIOCHEMISTRY}, author={Mooney, Steven M. and Qiu, Ruoyi and Kim, John J. and Sacho, Elizabeth J. and Rajagopalan, Krithika and Johng, Dorhyun and Shiraishi, Takumi and Kulkarni, Prakash and Weninger, Keith R.}, year={2014}, month={Mar}, pages={1670–1679} }
 @article{derocco_sass_qiu_weninger_erie_2014, title={Dynamics of MutS-Mismatched DNA Complexes Are Predictive of Their Repair Phenotypes}, volume={53}, ISSN={["0006-2960"]}, DOI={10.1021/bi401429b}, abstractNote={MutS recognizes base–base mismatches and base insertions/deletions (IDLs) in newly replicated DNA. Specific interactions between MutS and these errors trigger a cascade of protein–protein interactions that ultimately lead to their repair. The inability to explain why different DNA errors are repaired with widely varying efficiencies in vivo remains an outstanding example of our limited knowledge of this process. Here, we present single-molecule Förster resonance energy transfer measurements of the DNA bending dynamics induced by Thermus aquaticus MutS and the E41A mutant of MutS, which is known to have error specific deficiencies in signaling repair. We compared three DNA mismatches/IDLs (T-bulge, GT, and CC) with repair efficiencies ranging from high to low. We identify three dominant DNA bending states [slightly bent/unbent (U), intermediately bent (I), and significantly bent (B)] and find that the kinetics of interconverting among states varies widely for different complexes. The increased stability of MutS–mismatch/IDL complexes is associated with stabilization of U and lowering of the B to U transition barrier. Destabilization of U is always accompanied by a destabilization of B, supporting the suggestion that B is a “required” precursor to U. Comparison of MutS and MutS-E41A dynamics on GT and the T-bulge suggests that hydrogen bonding to MutS facilitates the changes in base–base hydrogen bonding that are required to achieve the U state, which has been implicated in repair signaling. Taken together with repair propensities, our data suggest that the bending kinetics of MutS–mismatched DNA complexes may control the entry into functional pathways for downstream signaling of repair.}, number={12}, journal={BIOCHEMISTRY}, author={DeRocco, Vanessa C. and Sass, Lauryn E. and Qiu, Ruoyi and Weninger, Keith R. and Erie, Dorothy A.}, year={2014}, month={Apr}, pages={2043–2052} }
 @article{wessels_weninger_2014, title={Investigation of Calcein for Reporting Content Mixing During Viral Membrane Fusion Experiments}, volume={106}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/J.BPJ.2013.11.3922}, DOI={10.1016/J.BPJ.2013.11.3922}, abstractNote={Simultaneous diagnosis of lipid mixing and content mixing during fusion of enveloped virus particle with a target liposome is critical for thorough understanding of the completeness of the membrane fusion reaction. Here we have investigated calcein for fluorescence detection of content mixing in virus fusion experiments. Calcein was encapsulated into target liposomes and lipophilic fluorescent dyes were incorporated into virus samples, both at self-quenching concentrations, to allow hemifusion and full fusion to be distinguished by the relative timing of the dequenching signals. We report that osmotic balancing of the interior and exterior of the calcein-loaded vesicles is essential for these experiments to reproduce native virus fusion behaviors.}, number={2}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Wessels, Laura and Weninger, Keith}, year={2014}, month={Jan}, pages={707a} }
 @article{erie_weninger_2014, title={Single molecule studies of DNA mismatch repair}, volume={20}, ISSN={["1568-7856"]}, DOI={10.1016/j.dnarep.2014.03.007}, abstractNote={DNA mismatch repair, which involves is a widely conserved set of proteins, is essential to limit genetic drift in all organisms. The same system of proteins plays key roles in many cancer related cellular transactions in humans. Although the basic process has been reconstituted in vitro using purified components, many fundamental aspects of DNA mismatch repair remain hidden due in part to the complexity and transient nature of the interactions between the mismatch repair proteins and DNA substrates. Single molecule methods offer the capability to uncover these transient but complex interactions and allow novel insights into mechanisms that underlie DNA mismatch repair. In this review, we discuss applications of single molecule methodology including electron microscopy, atomic force microscopy, particle tracking, FRET, and optical trapping to studies of DNA mismatch repair. These studies have led to formulation of mechanistic models of how proteins identify single base mismatches in the vast background of matched DNA and signal for their repair.}, journal={DNA REPAIR}, author={Erie, Dorothy A. and Weninger, Keith R.}, year={2014}, month={Aug}, pages={71–81} }
 @article{rajagopalan_qiu_mooney_rao_shiraishi_sacho_huang_shapiro_keith_kulkarni_et al._2014, title={The Stress-response protein prostate-associated gene 4, interacts with c-Jun and potentiates its transactivation}, volume={1842}, ISSN={["0006-3002"]}, DOI={10.1016/j.bbadis.2013.11.014}, abstractNote={The Cancer/Testis Antigen (CTA), Prostate-associated Gene 4 (PAGE4), is a stress-response protein that is upregulated in prostate cancer (PCa) especially in precursor lesions that result from inflammatory stress. In cells under stress, translocation of PAGE4 to mitochondria increases while production of reactive oxygen species decreases. Furthermore, PAGE4 is also upregulated in human fetal prostate, underscoring its potential role in development. However, the proteins that interact with PAGE4 and the mechanisms underlying its pleiotropic functions in prostatic development and disease remain unknown. Here, we identified c-Jun as a PAGE4 interacting partner. We show that both PAGE4 and c-Jun are overexpressed in the human fetal prostate; and in cell-based assays, PAGE4 robustly potentiates c-Jun transactivation. Single-molecule Förster resonance energy transfer experiments indicate that upon binding to c-Jun, PAGE4 undergoes conformational changes. However, no interaction is observed in presence of BSA or unilamellar vesicles containing the mitochondrial inner membrane diphosphatidylglycerol lipid marker cardiolipin. Together, our data indicate that PAGE4 specifically interacts with c-Jun and that, conformational dynamics may account for its observed pleiotropic functions. To our knowledge, this is the first report demonstrating crosstalk between a CTA and a proto-oncogene. Disrupting PAGE4/c-Jun interactions using small molecules may represent a novel therapeutic strategy for PCa.}, number={2}, journal={BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR BASIS OF DISEASE}, author={Rajagopalan, K. and Qiu, R. Y. and Mooney, S. M. and Rao, S. and Shiraishi, T. and Sacho, E. and Huang, H. Y. and Shapiro, E. and keith and Kulkarni, P. and et al.}, year={2014}, month={Feb}, pages={154–163} }
 @article{qiu_weninger_2013, title={Dynamics of DNA Mismatch Repair Initiation Complex Revealed by Single Molecule Fluorescence}, volume={104}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/j.bpj.2012.11.2043}, DOI={10.1016/j.bpj.2012.11.2043}, abstractNote={The integrity of the genetic information is dependent on the fidelity of DNA replication and several DNA repair processes. Among these repair systems, DNA mismatch repair (MMR) is responsible for correcting base-base mismatches and small nucleotide insertion/deletion (IDL) mispairs that arise from polymerase misincorporation, elevating fidelity of replication 50-1000 fold. MMR is initiated when MutS binds to mismatched bases on dsDNA. The communication between the mismatch site and a distal strand discrimination signal is required for removing the mismatch from the newly synthesized strand. MutS-MutL-heteroduplex ternary complex is thought to play an key role in the coupling of these two sites on DNA. We used single molecule fluorescence resonance energy transfer (smFRET) to characterize conformational changes in this ternary complex through the process of mismatch recognition, MutS-MutL interaction and large MutS-MutL assembly formation on DNA. We found that the sliding clamp formation of MutS is inhibited by MutL interaction. The initial MutS-MutL complex stays at the mismatch site and recruits more MutS and MutL to form a large protein assembly. The structural information revealed by our single molecule measurements provides constraints for modeling the mechanism of MMR in the initiation stage.}, number={2}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Qiu, Ruoyi and Weninger, Keith}, year={2013}, month={Jan}, pages={368a} }
 @article{qiu_weninger_2012, title={Conformational Changes in MutS during Mismtach Repair Signaling Determined with Single Molecule FRET}, volume={102}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/j.bpj.2011.11.1564}, DOI={10.1016/j.bpj.2011.11.1564}, abstractNote={DNA mismatch repair (MMR) is required for high replication fidelity in organisms ranging from bacteria to humans. MutS protein initiates MMR by recognizing base-base mismatches and insertion-deletion mismatches in double stranded DNA. In spite of extensive study, a temporally resolved picture of MutS conformations during mismatch repair remains elusive. We used single molecule fluorescence resonance energy transfer (smFRET) to characterize conformational changes in Thermus aquaticus (Taq) MutS as it scans homoduplex DNA, recognizes mismatches, activates to a sliding clamp, and interacts with MutL. We found that DNA binding domains of MutS undergo large movements as it is converted to sliding clamp in a two step process. First, the proximate domains I, which are initially stabilized by interaction with a mismatch, partially open while MutS remains at the mismatch. The domains then move farther apart, which results in a ring conformation that allows MutS to slide on DNA. We also report interactions between MutS and MutL while bound to mismatched DNA. This information provides constraints for modeling the downstream MMR pathways.}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Qiu, Ruoyi and Weninger, Keith}, year={2012}, month={Jan}, pages={283a} }
 @article{stamou_christensen_ehrlich_tkach_sakon_choi_weninger_2012, title={Cooperative All-Or-None Recruitment of Synaptotagmin C2AB on Single Vesicles Explains Why Ca2+ Regulates the Amplitude of SNARE Mediated Vesicle Fusion}, volume={102}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/j.bpj.2011.11.1750}, DOI={10.1016/j.bpj.2011.11.1750}, abstractNote={Synaptotagmin-1 (syt) has been identified as the principle determinant of synchronous release of neurotransmitters at the synapse. The clear correlation between the Ca2+-sensitivity of release and the Ca2+-dependent binding of different syt mutants to negatively charged phospholipid membranes (in the form of small unilamellar vesicles) is in strong favor of this hypothesis. Both syt wt and C2AB have been exhibiting an enigmatic behavior in all in vitro studies reporting Ca2+regulated vesicle fusion, namely they predominantly, or in some cases exclusively, modulated fusion amplitudes and not fusion kinetics. This means that contrary to intuition syt and Ca2+ do not increase the probability of vesicle fusion. Instead they increase the total fraction of vesicles that are available for fusion through a yet unknown mechanism. Interestingly, this phenotype is reminiscent of the observation that in vivo Ca2+influx in addition to changing fusion kinetics increases the size of the RRP. Here we demonstrate that this striking phenotype originates from the highly cooperative binding of syt to membranes that results to an all-or-none recruitment at the single vesicle level1-4 which is regulated by Ca2+. References 1. Christensen, S.M., Mortensen, M.W. & Stamou, D.G. Single Vesicle Assaying of SNARE-Synaptotagmin-Driven Fusion Reveals Fast and Slow Modes of Both Docking and Fusion and Intrasample Heterogeneity. Biophysj 100, 957-967 (2011). 2. Bendix, P.M., Pedersen, M.S. & Stamou, D. Quantification of nano-scale intermembrane contact areas by using fluorescence resonance energy transfer. Proceedings of the National Academy of Sciences 106, 12341-12346 (2009). 3. Hatzakis, N.S. et al. How curved membranes recruit amphipathic helices and protein anchoring motifs. Nat. Chem. Biol. 5, 835-841 (2009). 4. Andreas H. Kunding, et al. Intermembrane docking reactions are regulated by membrane curvature. Biophysical Journal, in press.}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Stamou, Dimitrios and Christensen, Sune M. and Ehrlich, Nicky and Tkach, Vadym and Sakon, John J. and Choi, Ucheor B. and Weninger, Keith R.}, year={2012}, month={Jan}, pages={318a–319a} }
 @article{weninger_2012, title={Dynamics of DNA Mismatch Repair Revealed by Single Molecule FRET}, volume={102}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/j.bpj.2011.11.1291}, DOI={10.1016/j.bpj.2011.11.1291}, abstractNote={Single molecule FRET (smFRET) has rapidly gained popularity because it can provide unique information about biomolecular systems. In particular, smFRET provides quantitative nanoscale resolution of dynamic molecular motions and multimolecular interactions, all for unsynchronizable samples that may have multiple reaction pathways operating in parallel. As the experimental capabilities of smFRET advance, this method has been applied to increasingly complex biological systems. I will illustrate these complex system advantages of single molecule FRET by presenting measurements of DNA mismatch repair proteins MutS and MutL interacting with mismatched DNA. These proteins are the initial sentries that detect single base mismatches and insertions/deletions and activate repair cascades. We use smFRET to determine dynamic DNA bending by MutS, concomitant conformational changes within MutS itself, motion of MutS scanning along DNA, ATP binding states that commit MutS:mismatch DNA complexes to convert to sliding states used in signaling, and the modulation of these MutS behaviors by interactions with MutL.}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Weninger, Keith}, year={2012}, month={Jan}, pages={235a} }
 @article{qiu_derocco_harris_sharma_hingorani_erie_weninger_2012, title={Large conformational changes in MutS during DNA scanning, mismatch recognition and repair signalling}, volume={31}, ISSN={["1460-2075"]}, DOI={10.1038/emboj.2012.95}, abstractNote={MutS protein recognizes mispaired bases in DNA and targets them for mismatch repair. Little is known about the transient conformations of MutS as it signals initiation of repair. We have used single-molecule fluorescence resonance energy transfer (FRET) measurements to report the conformational dynamics of MutS during this process. We find that the DNA-binding domains of MutS dynamically interconvert among multiple conformations when the protein is free and while it scans homoduplex DNA. Mismatch recognition restricts MutS conformation to a single state. Steady-state measurements in the presence of nucleotides suggest that both ATP and ADP must be bound to MutS during its conversion to a sliding clamp form that signals repair. The transition from mismatch recognition to the sliding clamp occurs via two sequential conformational changes. These intermediate conformations of the MutS:DNA complex persist for seconds, providing ample opportunity for interaction with downstream proteins required for repair.}, number={11}, journal={EMBO JOURNAL}, author={Qiu, Ruoyi and DeRocco, Vanessa C. and Harris, Credle and Sharma, Anushi and Hingorani, Manju M. and Erie, Dorothy A. and Weninger, Keith R.}, year={2012}, month={May}, pages={2528–2540} }
 @inbook{brunger_strop_vrljic_bowen_chu_weninger_2012, title={Macromolecular Models by Single Molecule FRET}, ISBN={9789400749221 9789400749238}, ISSN={1874-6500 1874-6535}, url={http://dx.doi.org/10.1007/978-94-007-4923-8_1}, DOI={10.1007/978-94-007-4923-8_1}, abstractNote={Single molecule fluorescence energy transfer (FRET) experiments enable investigations of macromolecular conformation and folding by the introduction of fluorescent dyes at specific sites in the macromolecule. Multiple such experiments can be performed with different labeling site combinations in order to map complex conformational changes or interactions between multiple molecules. Distances that are derived from such experiments can be used for determination of the fluorophore positions by triangulation. When combined with a known structure of the macromolecule(s) to which the fluorophores are attached, a three-dimensional model of the system can be determined by docking calculations. Here we discuss recent applications of single molecule FRET to obtain a model of the synaptotagmin-1:SNARE complex and to study the conformation of PSD-95.}, booktitle={NATO Science for Peace and Security Series B: Physics and Biophysics}, publisher={Springer Netherlands}, author={Brunger, Axel T. and Strop, Pavel and Vrljic, Marija and Bowen, Mark and Chu, Steven and Weninger, Keith R.}, year={2012}, pages={1–19} }
 @article{khalid_kappus_weninger_putterman_2012, title={Opacity and Transport Measurements Reveal That Dilute Plasma Models of Sonoluminescence Are Not Valid}, volume={108}, ISSN={["0031-9007"]}, DOI={10.1103/physrevlett.108.104302}, abstractNote={A strong interaction between a nanosecond laser and a 70 μm radius sonoluminescing plasma is achieved. The overall response of the system results in a factor of 2 increase in temperature as determined by its spectrum. Images of the interaction reveal that light energy is absorbed and trapped in a region smaller than the sonoluminescence emitting region of the bubble for over 100 ns. We interpret this opacity and transport measurement as demonstrating that sonoluminescencing bubbles can be 1000 times more opaque than what follows from the Saha equation of statistical mechanics in the ideal plasma limit. To address this discrepancy, we suggest that the effects of strong Coulomb interactions are an essential component of a first principles theory of sonoluminescence.}, number={10}, journal={PHYSICAL REVIEW LETTERS}, author={Khalid, Shahzad and Kappus, Brian and Weninger, Keith and Putterman, Seth}, year={2012}, month={Mar} }
 @article{sacho_weninger_2012, title={Single Molecule FRET Studies of the DNA Mismatch Repair Protein MutSα using Specific Labeling with Unnatural Amino Acids}, volume={102}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/j.bpj.2011.11.1560}, DOI={10.1016/j.bpj.2011.11.1560}, abstractNote={Mutations to proteins involved in DNA mismatch repair (MMR) have been linked to hereditary colorectal cancer. Understanding the mechanism by which these proteins participate in repair (and how these mutations inactivate the repair pathway) is key in furthering our understanding of cancer. We seek to understand the mechanism behind the initiation step of mismatch repair in eukaryotes: binding of MutSα (Msh2 - Msh6) to mismatched DNA. Following mismatch recognition, MutSα then starts a signaling cascade that ultimately leads to repair. Despite knowing that mismatch recognition by MutSα is responsible for initiating repair, the mechanistic steps remain a mystery. We are using single molecule FRET to study the protein:DNA interactions involved in MutSα initiation of MMR. FRET pairs attached to the DNA reveal dynamic bending of DNA by MutSα. In other experiments we want to use single molecule FRET between dyes attached to the damaged DNA and dyes on MutSα - which requires site-specific labeling of the protein. Towards that end, we report progress using unnatural amino acid labeling approaches.}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Sacho, Elizabeth J. and Weninger, Keith R.}, year={2012}, month={Jan}, pages={282a} }
 @article{choi_mccann_weninger_bowen_2011, title={Beyond the Random Coil: Stochastic Conformational Switching in Intrinsically Disordered Proteins}, volume={19}, ISSN={["1878-4186"]}, DOI={10.1016/j.str.2011.01.011}, abstractNote={Intrinsically disordered proteins (IDPs) participate in critical cellular functions that exploit the flexibility and rapid conformational fluctuations of their native state. Limited information about the native state of IDPs can be gained by the averaging over many heterogeneous molecules that is unavoidable in ensemble approaches. We used single molecule fluorescence to characterize native state conformational dynamics in five synaptic proteins confirmed to be disordered by other techniques. For three of the proteins, SNAP-25, synaptobrevin and complexin, their conformational dynamics could be described with a simple semiflexible polymer model. Surprisingly, two proteins, neuroligin and the NMDAR-2B glutamate receptor, were observed to stochastically switch among distinct conformational states despite the fact that they appeared intrinsically disordered by other measures. The hop-like intramolecular diffusion found in these proteins is suggested to define a class of functionality previously unrecognized for IDPs.}, number={4}, journal={STRUCTURE}, author={Choi, Ucheor B. and McCann, James J. and Weninger, Keith R. and Bowen, Mark E.}, year={2011}, month={Apr}, pages={566–576} }
 @article{weninger_2011, title={Complexin arrests a neighbor}, volume={18}, ISSN={["1545-9993"]}, DOI={10.1038/nsmb.2118}, number={8}, journal={NATURE STRUCTURAL & MOLECULAR BIOLOGY}, author={Weninger, Keith R.}, year={2011}, month={Aug}, pages={861–863} }
 @article{qiu_weninger_2011, title={Single Molecule FRET Study of Nucleotide Binding Effects on Muts Proteins}, volume={100}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/j.bpj.2010.12.1431}, DOI={10.1016/j.bpj.2010.12.1431}, abstractNote={The DNA mismatch repair (MMR) system is an essential component of the cellular DNA replication process that ensures high overall fidelity. The protein MutS initiates MMR by binding specifically to base-base mismatches and insertion-deletion mismatches in double stranded DNA. The active MutS dimer includes two ATP binding sites that hydrolyze ATP in both DNA bound and free states. ATP is also hydrolyzed as part of the MMR cascade involving MutS. Biochemical characterizations have shown that distinct nucleotides produce different interactions between MutS and mismatched DNA substrates, but the details of the conformations of MutS-mismatched DNA complexes under exposure to different nucleotides are not well known. We used single molecule fluorescence resonance energy transfer (smFRET) to study MutS from Thermus aquaticus in isolation and in complex with mismatched DNA substrates when exposed to a variety of different nucleotide conditions. We report results using intramolecular FRET from MutS or the DNA substrate as well as intermolecular FRET between MutS and the DNA. Our results allow MutS-DNA conformations resulting from specific nucleotide bound states to be characterized. We suggest possible roles for ATP cycling that could regulate the function of MutS in DNA MMR.}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Qiu, Ruoyi and Weninger, Keith}, year={2011}, month={Feb}, pages={223a} }
 @article{brunger_strop_vrljic_chu_weninger_2011, title={Three-dimensional molecular modeling with single molecule FRET}, volume={173}, ISSN={["1095-8657"]}, DOI={10.1016/j.jsb.2010.09.004}, abstractNote={Single molecule fluorescence energy transfer experiments enable investigations of macromolecular conformation and folding by the introduction of fluorescent dyes at specific sites in the macromolecule. Multiple such experiments can be performed with different labeling site combinations in order to map complex conformational changes or interactions between multiple molecules. Distances that are derived from such experiments can be used for determination of the fluorophore positions by triangulation. When combined with a known structure of the macromolecule(s) to which the fluorophores are attached, a three-dimensional model of the system can be determined. However, care has to be taken to properly derive distance from fluorescence energy transfer efficiency and to recognize the systematic or random errors for this relationship. Here we review the experimental and computational methods used for three-dimensional modeling based on single molecule fluorescence resonance transfer, and describe recent progress in pushing the limits of this approach to macromolecular complexes.}, number={3}, journal={JOURNAL OF STRUCTURAL BIOLOGY}, author={Brunger, Axel T. and Strop, Pavel and Vrljic, Marija and Chu, Steven and Weninger, Keith R.}, year={2011}, month={Mar}, pages={497–505} }
 @article{sakon_weninger_2010, title={Detecting the conformation of individual proteins in live cells}, volume={7}, ISSN={["1548-7105"]}, DOI={10.1038/nmeth.1421}, abstractNote={We combined single-molecule fluorescence resonance energy transfer (smFRET) with single-particle tracking in live cells to detect the in vivo conformation of individual proteins. We site-specifically labeled recombinant SNARE proteins with a FRET donor and acceptor before microinjecting them into cultured cells. Individual proteins rapidly incorporated into folded complexes at the cell membrane, demonstrating the potential of this method to reveal dynamic interactions within cells.}, number={3}, journal={NATURE METHODS}, author={Sakon, John J. and Weninger, Keith R.}, year={2010}, month={Mar}, pages={203–U56} }
 @article{derocco_anderson_piehler_erie_weninger_2010, title={Four-color single-molecule fluorescence with noncovalent dye labeling to monitor dynamic multimolecular complexes}, volume={49}, ISSN={["1940-9818"]}, DOI={10.2144/000113551}, abstractNote={ To enable studies of conformational changes within multimolecular complexes, we present a simultaneous, four-color single molecule fluorescence methodology implemented with total internal reflection illumination and camera-based, wide-field detection. We further demonstrate labeling histidine-tagged proteins noncovalently with Tris–nitrilotriacetic acid (Tris-NTA)–conjugated dyes to achieve single molecule detection. We combine these methods to colocalize the mismatch repair protein MutSα on DNA while monitoring MutSα-induced DNA bending using Förster resonance energy transfer (FRET) and to monitor assembly of membrane-tethered SNARE protein complexes. }, number={5}, journal={BIOTECHNIQUES}, author={DeRocco, Vanessa C. and Anderson, Trevor and Piehler, Jacob and Erie, Dorothy A. and Weninger, Keith}, year={2010}, month={Nov}, pages={807-+} }
 @article{mccann_choi_zheng_weninger_bowen_2010, title={Optimizing Methods to Recover Absolute FRET Efficiency from Immobilized Single Molecules}, volume={99}, ISSN={["1542-0086"]}, DOI={10.1016/j.bpj.2010.04.063}, abstractNote={Microscopy-based fluorescence resonance energy transfer (FRET) experiments measure donor and acceptor intensities by isolating these signals with a series of optical elements. Because this filtering discards portions of the spectrum, the observed FRET efficiency is dependent on the set of filters in use. Similarly, observed FRET efficiency is also affected by differences in fluorophore quantum yield. Recovering the absolute FRET efficiency requires normalization for these effects to account for differences between the donor and acceptor fluorophores in their quantum yield and detection efficiency. Without this correction, FRET is consistent across multiple experiments only if the photophysical and instrument properties remain unchanged. Here we present what is, to our knowledge, the first systematic study of methods to recover the true FRET efficiency using DNA rulers with known fluorophore separations. We varied optical elements to purposefully alter observed FRET and examined protein samples to achieve quantum yields distinct from those in the DNA samples. Correction for calculated instrument transmission reduced FRET deviations, which can facilitate comparison of results from different instruments. Empirical normalization was more effective but required significant effort. Normalization based on single-molecule photobleaching was the most effective depending on how it is applied. Surprisingly, per-molecule gamma-normalization reduced the peak width in the DNA FRET distribution because anomalous gamma-values correspond to FRET outliers. Thus, molecule-to-molecule variation in gamma has an unrecognized effect on the FRET distribution that must be considered to extract information on sample dynamics from the distribution width.}, number={3}, journal={BIOPHYSICAL JOURNAL}, author={McCann, James J. and Choi, Ucheor B. and Zheng, Liqiang and Weninger, Keith and Bowen, Mark E.}, year={2010}, month={Aug}, pages={961–970} }
 @article{mccann_choi_zheng_weninger_bowen_2010, title={Recovering Absolute Fret Efficiency from Single Molecules: Comparing Methods of Gamma Correction}, volume={98}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/j.bpj.2009.12.998}, DOI={10.1016/j.bpj.2009.12.998}, abstractNote={Fluorescence resonance energy transfer is widely thought of as a “spectroscopic ruler.” Because biological processes and cellular assemblies occur on the nanometer scale, FRET is a popular tool for structural biology. In contrast to ensemble solution FRET measurements which record the entire emission spectrum, microscopy- based FRET experiments separate donor and acceptor intensity by passing the emission through a series of optical elements. Observed FRET efficiency, determined from the uncorrected donor and acceptor intensities, has been called a relative proximity ratio, which is internally consistent only if the photophysical properties and instrument remain unchanged. However, it is desirable to measure absolute distances using FRET, which requires that FRET efficiency be corrected for both instrument response and fluorophore properties. Thus, “gamma” correction adjusts for differences between the donor and acceptor dyes in their probability of photon emission upon excitation and the probability that emitted photons will be detected. Methods of gamma correction vary depending on the single molecule methodology. To test different methods for correcting FRET efficiency, we recorded smFRET distributions for protein and DNA on different instruments and with different filter sets which altered the observed FRET efficiency. Knowledge of filter set transmission allows for comparison of results between groups using different instruments. Applying empirically-derived corrections for instrument response and quantum yield was only slightly better than corrections based solely on filter set transmission data. We found that gamma correction based on single molecule photobleaching was the most effective particularly when gamma was determined for each sample or even each molecule. Variations in focus of the two colors and sub-pixel errors in image mapping affect both FRET and gamma. As such, per molecule correction affects distribution width because FRET outliers may also have anomalous gamma values.}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={McCann, James J. and Choi, Ucheor B. and Zheng, Liqiang and Weninger, Keith and Bowen, Mark E.}, year={2010}, month={Jan}, pages={186a–187a} }
 @article{sass_lanyi_weninger_erie_2010, title={Single-Molecule FRET TACKLE Reveals Highly Dynamic Mismatched DNA-MutS Complexes}, volume={49}, ISSN={["0006-2960"]}, DOI={10.1021/bi901871u}, abstractNote={The first step in DNA mismatch repair (MMR) is the recognition of DNA mismatches or nucleotide insertions/deletions (IDLs) by MutS and MutS homologues. To investigate the conformational properties of MutS-mismatch complexes, we used single-molecule fluorescence resonance energy transfer (smFRET) to examine the dynamics of MutS-induced DNA bending at a GT mismatch. The FRET measurements reveal that the MutS-GT mismatch recognition complex is highly dynamic, undergoing conformational transitions between many states with different degrees of DNA bending. Due to the complexity of the data, we developed an analysis approach, called FRET TACKLE, in which we combine direct analysis of FRET transitions with examination of kinetic lifetimes to identify all of the conformational states and characterize the kinetics of the binding and conformational equilibria. The data reveal that MutS-GT complexes can reside in six different conformations, which have lifetimes that differ by as much as 20-fold and exhibit rates of interconversion that vary by 2 orders of magnitude. To gain further insight into the dynamic properties of GT-MutS complexes and to bolster the validity of our analysis, we complemented our experimental data with Monte Carlo simulations. Taken together, our results suggest that the dynamics of the MutS-mismatch complex could govern the efficiency of repair of different DNA mismatches. Finally, in addition to revealing these important biological implications of MutS-DNA interactions, this FRET TACKLE method will enable the analysis of the complex dynamics of other biological systems.}, number={14}, journal={BIOCHEMISTRY}, author={Sass, Lauryn E. and Lanyi, Cherie and Weninger, Keith and Erie, Dorothy A.}, year={2010}, month={Apr}, pages={3174–3190} }
 @article{choi_strop_vrljic_chu_brunger_weninger_2010, title={Single-molecule FRET-derived model of the synaptotagmin 1-SNARE fusion complex}, volume={17}, ISSN={["1545-9985"]}, DOI={10.1038/nsmb.1763}, abstractNote={Single-molecule FRET studies have resulted in an experimentally derived model of a synaptotagmin–SNARE complex. In this complex of SNARE with synaptotagmin 1, the arrangement of the Ca2+-binding loops is similar to that of the structure of SNARE-induced Ca2+-bound synaptotagmin 3. This suggests a common molecular mechanism by which the synaptotagmin–SNARE interaction plays a role in Ca2+-triggered vesicle fusion. Synchronous neurotransmission is triggered when Ca2+ binds to synaptotagmin 1 (Syt1), a synaptic-vesicle protein that interacts with SNAREs and membranes. We used single-molecule fluorescence resonance energy transfer (FRET) between synaptotagmin's two C2 domains to determine that their conformation consists of multiple states with occasional transitions, consistent with domains in random relative motion. SNARE binding results in narrower intrasynaptotagmin FRET distributions and less frequent transitions between states. We obtained an experimentally determined model of the elusive Syt1–SNARE complex using a multibody docking approach with 34 FRET-derived distances as restraints. The Ca2+-binding loops point away from the SNARE complex, so they may interact with the same membrane. The loop arrangement is similar to that of the crystal structure of SNARE-induced Ca2+-bound Syt3, suggesting a common mechanism by which the interaction between synaptotagmins and SNAREs aids in Ca2+-triggered fusion.}, number={3}, journal={NATURE STRUCTURAL & MOLECULAR BIOLOGY}, author={Choi, Ucheor B. and Strop, Pavel and Vrljic, Marija and Chu, Steven and Brunger, Axel T. and Weninger, Keith R.}, year={2010}, month={Mar}, pages={318–U84} }
 @article{fletcher_fletcher_weninger_anderson_martin_2010, title={Vesicle-associated Membrane Protein (VAMP) Cleavage by a New Metalloprotease from the Brazilian Scorpion Tityus serrulatus}, volume={285}, ISSN={["1083-351X"]}, DOI={10.1074/jbc.m109.028365}, abstractNote={We present evidence that venom from the Brazilian scorpion Tityus serrulatus and a purified fraction selectively cleave essential SNARE proteins within exocrine pancreatic tissue. Western blotting for vesicle-associated membrane protein type v-SNARE proteins (or synaptobrevins) reveals characteristic alterations to venom-treated excised pancreatic lobules in vitro. Immunocytochemistry by electron microscopy confirms both the SNARE identity as VAMP2 and the proteolysis of VAMP2 as a marked decrease in secondary antibody-conjugated colloidal gold particles that are predominantly associated with mature zymogen granules. Studies with recombinant SNARE proteins were used to determine the specific cleavage site in VAMP2 and the susceptibility of VAMP8 (endobrevin). The VAMP2 cleavage site is between the transmembrane anchor and the SNARE motif that assembles into the ternary SNARE complex. Inclusion of divalent chelating agents (EDTA) with fraction ν, an otherwise active purified component from venom, eliminates SNARE proteolysis, suggesting the active protein is a metalloprotease. The unique cleavages of VAMP2 and VAMP8 may be linked to pancreatitis that develops following scorpion envenomation as both of these v-SNARE proteins are associated with zymogen granule membranes in pancreatic acinar cells. We have isolated antarease, a metalloprotease from fraction ν that cleaves VAMP2, and report its amino acid sequence.}, number={10}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Fletcher, Paul L., Jr. and Fletcher, Maryann D. and Weninger, Keith and Anderson, Trevor E. and Martin, Brian M.}, year={2010}, month={Mar}, pages={7405–7416} }
 @article{sakon_ribeill_garguilo_perkins_weninger_nemanich_2009, title={Fluorescence quenching effects of nanocrystalline diamond surfaces}, volume={18}, ISSN={["1879-0062"]}, DOI={10.1016/j.diamond.2008.10.028}, abstractNote={Undoped diamond has conductive properties when terminated by hydrogen and exposed to air or aqueous solution. Here, it is shown that nanocrystalline diamond, fabricated with hydrogen termination and deposited on quartz substrates using chemical vapor deposition, significantly quenched the fluorescence of adsorbed, dye-labeled fibrinogen protein in aqueous solutions at near neutral pH. Smaller levels of quenching were observed from oxygen terminated NCD surfaces. We suggest that these near-surface fluorescence quenching effects may arise from surface conductance effects in hydrogen terminated NCD. It is also shown that despite bulk quenching effects, single molecules of fibrinogen could be imaged on nanocrystalline diamond surfaces using epi-fluorescence techniques.}, number={1}, journal={DIAMOND AND RELATED MATERIALS}, author={Sakon, John J. and Ribeill, Guilhern J. and Garguilo, Jacob M. and Perkins, Jarnes and Weninger, Keith R. and Nemanich, Robert J.}, year={2009}, month={Jan}, pages={82–87} }
 @article{weninger_2009, title={How Do Adhesion Proteins Stick?}, volume={17}, ISSN={["0969-2126"]}, DOI={10.1016/j.str.2009.07.003}, abstractNote={Cadherin homodimerization mediates cell-cell adhesion, but one stabilizing structural element has inspired questions about assembly mechanisms. Combining single molecule fluorescence and force analyses allowed Sivasankar et al. to provide strong evidence for an induced fit pathway to dimerization.}, number={8}, journal={STRUCTURE}, author={Weninger, Keith}, year={2009}, month={Aug}, pages={1035–1036} }
 @article{thongthai_weninger_2009, title={Photoinactivation of Sindbis Virus Infectivity Without Inhibition of Membrane Fusion}, volume={85}, ISSN={["1751-1097"]}, DOI={10.1111/j.1751-1097.2008.00475.x}, abstractNote={Abstract}, number={3}, journal={PHOTOCHEMISTRY AND PHOTOBIOLOGY}, author={Thongthai, Wor and Weninger, Keith}, year={2009}, pages={801–806} }
 @misc{wessels_weninger_2009, title={Physical Aspects of Viral Membrane Fusion}, volume={9}, ISSN={["1537-744X"]}, DOI={10.1100/tsw.2009.76}, abstractNote={Enveloped viruses commonly employ membrane fusion during cell penetration in order to deliver their genetic material across the cell boundary. Large conformational changes in the proteins embedded in the viral membrane play a fundamental role in the membrane fusion process. Despite the tremendously wide variety of viruses that contain membranes, it appears that they all contain membrane fusion protein machinery with a remarkably conserved mechanism of action. Much of our current biochemical understanding of viral membrane fusion has been derived from high-resolution structural studies and solution-basedin vitroassays in which viruses fuse with liposomes or cells. Recently, single-particle experiments have been used to provide measurements of details not available in the bulk assays. Here we focus our discussion on the key dynamical aspects of fusion protein structure, along with some of the experimental and computational techniques presently being used to investigate viral-mediated membrane fusion.}, journal={THESCIENTIFICWORLDJOURNAL}, author={Wessels, Laura and Weninger, Keith}, year={2009}, pages={764–780} }
 @article{brunger_weninger_bowen_chu_2009, title={Single-Molecule Studies of the Neuronal SNARE Fusion Machinery}, volume={78}, ISSN={["1545-4509"]}, DOI={10.1146/annurev.biochem.77.070306.103621}, abstractNote={ SNAREs are essential components of the machinery for Ca2+-triggered fusion of synaptic vesicles with the plasma membrane, resulting in neurotransmitter release into the synaptic cleft. Although much is known about their biophysical and structural properties and their interactions with accessory proteins such as the Ca2+ sensor synaptotagmin, their precise role in membrane fusion remains an enigma. Ensemble studies of liposomes with reconstituted SNAREs have demonstrated that SNAREs and accessory proteins can trigger lipid mixing/fusion, but the inability to study individual fusion events has precluded molecular insights into the fusion process. Thus, this field is ripe for studies with single-molecule methodology. In this review, we discuss applications of single-molecule approaches to observe reconstituted SNAREs, their complexes, associated proteins, and their effect on biological membranes. Some of the findings are provocative, such as the possibility of parallel and antiparallel SNARE complexes or of vesicle docking with only syntaxin and synaptobrevin, but have been confirmed by other experiments. }, journal={ANNUAL REVIEW OF BIOCHEMISTRY}, author={Brunger, Axel T. and Weninger, Keith and Bowen, Mark and Chu, Steven}, year={2009}, pages={903–928} }
 @article{weninger_bowen_choi_chu_brunger_2008, title={Accessory proteins stabilize the acceptor complex for synaptobrevin, the 1 : 1 syntaxin/SNAP-25 complex}, volume={16}, ISSN={["1878-4186"]}, DOI={10.1016/j.str.2007.12.010}, abstractNote={Syntaxin/SNAP-25 interactions precede assembly of the ternary SNARE complex that is essential for neurotransmitter release. This binary complex has been difficult to characterize by bulk methods because of the prevalence of a 2:1 dead-end species. Here, using single-molecule fluorescence, we find the structure of the 1:1 syntaxin/SNAP-25 binary complex is variable, with states changing on the second timescale. One state corresponds to a parallel three-helix bundle, whereas other states show one of the SNAP-25 SNARE domains dissociated. Adding synaptobrevin suppresses the dissociated helix states. Remarkably, upon addition of complexin, Munc13, Munc18, or synaptotagmin, a similar effect is observed. Thus, the 1:1 binary complex is a dynamic acceptor for synaptobrevin binding, and accessory proteins stabilize this acceptor. In the cellular environment the binary complex is actively maintained in a configuration where it can rapidly interact with synaptobrevin, so formation is not likely a limiting step for neurotransmitter release.}, number={2}, journal={STRUCTURE}, author={Weninger, Keith and Bowen, Mark E. and Choi, Ucheor B. and Chu, Steven and Brunger, Axel T.}, year={2008}, month={Feb}, pages={308–320} }
 @article{wang_hernandez_keith_brown_2007, title={Infection of cells by Sindbis virus at low temperature}, volume={362}, DOI={10.1016/j.virol.2006.12.036}, abstractNote={Sindbis virus, which belongs to the family Togaviridae genus Alphavirus infects a variety of vertebrate and invertebrate cells. The initial steps of Sindbis virus infection involve attachment, penetration and uncoating. Two different pathways of infection have been proposed for Alphaviruses. One proposed mechanism involves receptor mediated virion endocytosis followed by membrane fusion triggered by endosome acidification. This virus–host membrane fusion model, well established by influenza virus, has been applied to other unrelated membrane-containing viruses including Alphaviruses. The other mechanism proposes direct penetration of the cell plasma membrane by the virus glycoproteins in the absence of membrane fusion. This alternate model is supported by both ultrastructural [Paredes, A.M., Ferreira, D., Horton, M., Saad, A., Tsuruta, H., Johnston, R., Klimstra, W., Ryman, K., Hernandez, R., Chiu, W., Brown, D.T., 2004. Conformational changes in Sindbis virions resulting from exposure to low pH and interactions with cells suggest that cell penetration may occur at the cell surface in the absence of membrane fusion. Virology 324(2), 373–386] and biochemical [Koschinski, A., Wengler, G., Wengler, G., and Repp, H., 2005. Rare earth ions block the ion pores generated by the class II fusion proteins of alphaviruses and allow analysis of the biological functions of these pores. J. Gen. Virol. 86(Pt. 12), 3311–3320] studies. We have examined the ability of Sindbis virus to infect Baby Hamster Kidney (BHK) cells at temperatures which block endocytosis. We have found that under these conditions Sindbis virus infects cells in a temperature- and time-dependent fashion.}, number={2}, journal={Virology}, author={Wang, G. B. and Hernandez, R. and keith and Brown, D. T.}, year={2007}, pages={461–467} }
 @article{li_augustine_weninger_2007, title={Kinetics of complexin binding to the SNARE complex: Correcting single molecule FRET measurements for hidden events}, volume={93}, ISSN={["0006-3495"]}, DOI={10.1529/biophysj.106.101220}, abstractNote={Virtually all measurements of biochemical kinetics have been derived from macroscopic measurements. Single-molecule methods can reveal the kinetic behavior of individual molecular complexes and thus have the potential to determine heterogeneous behaviors. Here we have used single-molecule fluorescence resonance energy transfer to determine the kinetics of binding of SNARE (soluble N-ethyl maleimide-sensitive fusion protein attachment protein receptor) complexes to complexin and to a peptide derived from the central SNARE binding region of complexin. A Markov model was developed to account for the presence of unlabeled competitor in such measurements. We find that complexin associates rapidly with SNARE complexes anchored in lipid bilayers with a rate constant of 7.0 x 10(6) M(-1) s(-1) and dissociates slowly with a rate constant of 0.3 s(-1). The complexin peptide associates with SNARE complexes at a rate slower than that of full-length complexin (1.2 x 10(6) M(-1) s(-1)), and dissociates much more rapidly (rate constant >67 s(-1)). Comparison of single-molecule fluorescence resonance energy transfer measurements made using several dye attachment sites illustrates that dye labeling of complexin can modify its rate of unbinding from SNAREs. These rate constants provide a quantitative framework for modeling of the cascade of reactions underlying exocytosis. In addition, our theoretical correction establishes a general approach for improving single-molecule measurements of intermolecular binding kinetics.}, number={6}, journal={BIOPHYSICAL JOURNAL}, author={Li, Yulong and Augustine, George J. and Weninger, Keith}, year={2007}, month={Sep}, pages={2178–2187} }
 @article{wessels_elting_scimeca_weninger_2007, title={Rapid membrane fusion of individual virus particles with supported lipid bilayers}, volume={93}, ISSN={["1542-0086"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-34447311691&partnerID=MN8TOARS}, DOI={10.1529/biophysj.106.097485}, abstractNote={Many enveloped viruses employ low-pH-triggered membrane fusion during cell penetration. Solution-based in vitro assays in which viruses fuse with liposomes have provided much of our current biochemical understanding of low-pH-triggered viral membrane fusion. Here, we extend this in vitro approach by introducing a fluorescence assay using single particle tracking to observe lipid mixing between individual virus particles (influenza or Sindbis) and supported lipid bilayers. Our single-particle experiments reproduce many of the observations of the solution assays. The single-particle approach naturally separates the processes of membrane binding and membrane fusion and therefore allows measurement of details that are not available in the bulk assays. We find that the dynamics of lipid mixing during individual Sindbis fusion events is faster than 30 ms. Although neither virus binds membranes at neutral pH, under acidic conditions, the delay between membrane binding and lipid mixing is less than half a second for nearly all virus-membrane combinations. The delay between binding and lipid mixing lengthened only for Sindbis virus at the lowest pH in a cholesterol-dependent manner, highlighting the complex interaction between lipids, virus proteins, and buffer conditions in membrane fusion.}, number={2}, journal={BIOPHYSICAL JOURNAL}, publisher={Elsevier BV}, author={Wessels, Laura and Elting, Mary Williard and Scimeca, Dominic and Weninger, Keith}, year={2007}, month={Jul}, pages={526–538} }