@article{degtyareva_placentra_gabel_klimczak_gordenin_wagner_buettner_mueller_smirnova_doetsch_2023, title={Changes in metabolic landscapes shape divergent but distinct mutational signatures and cytotoxic consequences of redox stress}, volume={4}, ISSN={["1362-4962"]}, DOI={10.1093/nar/gkad305}, abstractNote={Mutational signatures discerned in cancer genomes, in aging tissues and in cells exposed to toxic agents, reflect complex processes underlying transformation of cells from normal to dysfunctional. Due to its ubiquitous and chronic nature, redox stress contributions to cellular makeover remain equivocal. The deciphering of a new mutational signature of an environmentally-relevant oxidizing agent, potassium bromate, in yeast single strand DNA uncovered a surprising heterogeneity in the mutational signatures of oxidizing agents. NMR-based analysis of molecular outcomes of redox stress revealed profound dissimilarities in metabolic landscapes following exposure to hydrogen peroxide versus potassium bromate. The predominance of G to T substitutions in the mutational spectra distinguished potassium bromate from hydrogen peroxide and paraquat and mirrored the observed metabolic changes. We attributed these changes to the generation of uncommon oxidizing species in a reaction with thiol-containing antioxidants; a nearly total depletion of intracellular glutathione and a paradoxical augmentation of potassium bromate mutagenicity and toxicity by antioxidants. Our study provides the framework for understanding multidimensional processes triggered by agents collectively known as oxidants. Detection of increased mutational loads associated with potassium bromate-related mutational motifs in human tumors may be clinically relevant as a biomarker of this distinct type of redox stress.}, journal={NUCLEIC ACIDS RESEARCH}, author={Degtyareva, Natalya P. and Placentra, Victoria C. and Gabel, Scott A. and Klimczak, Leszek J. and Gordenin, Dmitry A. and Wagner, Brett A. and Buettner, Garry R. and Mueller, Geoffrey A. and Smirnova, Tatyana I and Doetsch, Paul W.}, year={2023}, month={Apr} }
 @article{voinov_smirnova_smirnov_2021, title={EPR Oximetry with Nitroxides: Effects of Molecular Structure, pH, and Electrolyte Concentration}, volume={11}, ISSN={["1613-7507"]}, url={https://doi.org/10.1007/s00723-021-01446-8}, DOI={10.1007/s00723-021-01446-8}, journal={APPLIED MAGNETIC RESONANCE}, author={Voinov, Maxim A. and Smirnova, Tatyana I and Smirnov, Alex I}, year={2021}, month={Nov} }
 @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} }
 @article{myers_ranieri_smirnova_hewitt_peterson_quesada_lenker_stapelmann_2021, title={Measuring plasma-generated center dot OH and O atoms in liquid using EPR spectroscopy and the non-selectivity of the HTA assay}, volume={54}, ISSN={["1361-6463"]}, url={https://doi.org/10.1088/1361-6463/abd9a6}, DOI={10.1088/1361-6463/abd9a6}, abstractNote={Abstract Plasma-generated hydroxyl radicals ( · OH) and oxygen atoms (O) produced by the COST reference plasma jet, a micro-scaled atmospheric pressure plasma jet, were investigated using a variety of experimental techniques. Several gas admixtures were studied to distinguish the contributions of the two reactive oxygen species. Large discrepancies between inferred aqueous · OH densities were noted when using a 2-hydroxyterephthalic acid (HTA) fluorescence assay and electron paramagnetic resonance (EPR) measurements with the spin trap 5,5-dimethyl-1-pyrroline N -oxide—especially when oxygen was present in the feed gas. A series of follow-up experiments including optical emission spectroscopy, H 2 O 2 quantification, and EPR measurements of atomic oxygen using the spin trap 2,2,6,6-tetramethylpiperidine, revealed that the inconsistencies between the measured aqueous · OH were likely due to the propensity of atomic oxygen to hydroxylate TA in a manner indistinguishable from · OH. This renders the HTA assay non-selective when both · OH radicals and atomic oxygen are present, which we report for all three gas admixtures in our experiments. Additionally, considerable degradation of both HTA and the spin adducts measured using EPR spectroscopy was apparent, meaning actual radical densities in the plasma-treated liquid may be considerably higher than implied. Degradation rates compared favorably to previously measured gas phase densities of atomic oxygen in the predecessor of the COST jet and reported degradation of other chemical probes. These results show the prolific role of atomic oxygen in plasma-induced liquid chemistry and caution against diagnostic techniques that are unable to account for it.}, number={14}, journal={JOURNAL OF PHYSICS D-APPLIED PHYSICS}, publisher={IOP Publishing}, author={Myers, B. and Ranieri, P. and Smirnova, T. and Hewitt, P. and Peterson, D. and Quesada, M. Herrera and Lenker, E. and Stapelmann, K.}, year={2021}, month={Apr} }
 @article{cha_milikisiyants_davidson_xue_smirnova_smirnov_guo_chang_2020, title={Alternative Reactivity of Leucine 5-Hydroxylase Using an Olefin-Containing Substrate to Construct a Substituted Piperidine Ring}, volume={59}, ISSN={["0006-2960"]}, url={https://doi.org/10.1021/acs.biochem.0c00289}, DOI={10.1021/acs.biochem.0c00289}, abstractNote={Applying enzymatic reactions to produce useful molecules is a central focus of chemical biology. Iron and 2-oxoglutarate (Fe/2OG) enzymes are found in all kingdoms of life and catalyze a broad array of oxidative transformations. Herein, we demonstrate that the activity of an Fe/2OG enzyme can be redirected when changing the targeted carbon hybridization from sp3 to sp2. During leucine 5-hydroxylase catalysis, installation of an olefin group onto the substrate redirects the Fe(IV)–oxo species reactivity from hydroxylation to asymmetric epoxidation. The resulting epoxide subsequently undergoes intramolecular cyclization to form the substituted piperidine, 2S,5S-hydroxypipecolic acid.}, number={21}, journal={BIOCHEMISTRY}, publisher={American Chemical Society (ACS)}, author={Cha, Lide and Milikisiyants, Sergey and Davidson, Madison and Xue, Shan and Smirnova, Tatyana I and Smirnov, Alex I and Guo, Yisong and Chang, Wei-Chen}, year={2020}, month={Jun}, pages={1961–1965} }
 @article{perelygin_voinov_marek_ou_krim_brenner_smirnova_smirnov_2019, title={Dielectric and Electrostatic Properties of the Silica Nanoparticle-Water Interface by EPR of pH-Sensitive Spin Probes}, volume={123}, ISSN={["1932-7455"]}, url={https://doi.org/10.1021/acs.jpcc.9b08007}, DOI={10.1021/acs.jpcc.9b08007}, abstractNote={Interfacial electrostatic properties of monodisperse silica nanoparticles (SiNPs) in aqueous suspensions as a function of bulk pH were characterized by spin labeling EPR of two ionizable nitroxides...}, number={49}, journal={JOURNAL OF PHYSICAL CHEMISTRY C}, publisher={American Chemical Society (ACS)}, author={Perelygin, Vladislav and Voinov, Maxim A. and Marek, Antonin and Ou, Erkang and Krim, Jacqueline and Brenner, Donald and Smirnova, Tatyana I and Smirnov, Alex I}, year={2019}, month={Dec}, pages={29972–29985} }
 @article{smirnova_smirnov_2019, title={EPR studies of bionanomaterials}, volume={50}, ISBN={["978-0-12-814024-6"]}, ISSN={["1079-4042"]}, DOI={10.1016/B978-0-12-814024-6.00007-8}, journal={ELECTRON MAGNETIC RESONANCE - APPLICATIONS IN PHYSICAL SCIENCES AND BIOLOGY}, author={Smirnova, Tatyana I. and Smirnov, Alex I.}, year={2019}, pages={129–159} }
 @article{ou_voinov_irving_smirnov_smirnova_2019, title={Effect of Silica Support on Electrostatics of Lipid Interfaces in Nano-Bio Hybrid Systems}, volume={116}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/J.BPJ.2018.11.479}, DOI={10.1016/J.BPJ.2018.11.479}, abstractNote={Design of new bio-nano hybrid systems calls for understanding and accounting for the influence of a nanostructured support and nanoconfinement on the structure and biophysical properties of lipid bilayer hybrid systems and membrane-protein interface. Here we report on spin-labeling EPR studies of pH-sensitive lipids and specifically labeled protein side chains to assess effects of solid inorganic interface, specifically, silica support in a form of monodispersed nanoparticles ranging from 20 to 300 nm in diameter on the surface electrostatic potential of lipid bilayers associated with the particles and effective pKa of the membrane-burred peptide ionisable sidechains. We have shown that bilayers formed from zwitterionic or mixed lipids on silica nanoparticle surfaces possess a higher negative electrostatic potential than the unsupported bilayers with the potential of mixed bilayers containing negatively charged lipids being significantly more sensitive to the silica support. Effect the silica nanoparticle size on the lipid bilayer surface electrostatic potential was also observed for particles smaller than 100 nm. pH-sensitive EPR probes were then employed to label model WALP peptide known to form an α-helix when integrated into a lipid bilayer. The silica support exerted pronounced effects on WALP dynamics and the effective pKa of the ionizable probe. It was demonstrated that the silica nanoparticles shift the effective pKa of the ionizable nitroxide probe in a membrane depth-dependent manner. Upon protonation of the membrane-burred model ionisable sidechain the silica support caused significant changes in the membrane association of WALP peptide that are not observed when WALP is integrated into unilamellar phospholipid vesicles of similar curvature. Supported by NSF 1508607.}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Ou, Erkang and Voinov, Maxim and Irving, Alex and Smirnov, Alex and Smirnova, Tatyana I.}, year={2019}, month={Feb}, pages={81a} }
 @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{yu_tang_cha_milikisiyants_smirnova_smirnov_guo_chang_2018, title={Elucidating the Reaction Pathway of Decarboxylation-Assisted Olefination Catalyzed by a Mononuclear Non-Heme Iron Enzyme}, volume={140}, ISSN={["1520-5126"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85056414780&partnerID=MN8TOARS}, DOI={10.1021/jacs.8b10077}, abstractNote={Installation of olefins into molecules is a key transformation in organic synthesis. The recently discovered decarboxylation-assisted olefination in the biosynthesis of rhabduscin by a mononuclear non-heme iron enzyme (P.IsnB) represents a novel approach in olefin construction. This method is commonly employed in natural product biosynthesis. Herein, we demonstrate that a ferryl intermediate is used for C–H activation at the benzylic position of the substrate. We further establish that P.IsnB reactivity can be switched from olefination to hydroxylation using electron-withdrawing groups appended on the phenyl moiety of the analogues. These experimental observations imply that a pathway involving an initial C–H activation followed by a benzylic carbocation species or by electron transfer coupled β-scission is likely utilized to complete C═C bond formation.}, number={45}, journal={JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, author={Yu, Cheng-Ping and Tang, Yijie and Cha, Lide and Milikisiyants, Sergey and Smirnova, Tatyana I. and Smirnov, Alex I. and Guo, Yisong and Chang, Wei-chen}, year={2018}, month={Nov}, pages={15190–15193} }
 @article{ou_voinov_smirnov_smirnova_2018, title={Silica-Supported Lipid Bilayers: Electrostatic Effects at Lipid Interfaces as Reported by Spin-Labeling EPR}, volume={114}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/J.BPJ.2017.11.565}, DOI={10.1016/J.BPJ.2017.11.565}, abstractNote={Interfacing biological and artificial systems at the nano-scale level is essential for developing novel living-nonliving biotechnology platforms including biosensors based on biological systems. Despite an impressive progress, the needs remain high to understand the influence of a nanostructured support and nanoconfinement on structure and properties of the membrane-protein interface. We have utilized novel pH-sensitive lipids IMTSL-PE and IKMTSL-PE to assess the phospholipid membrane surface potential. It was shown that bilayers formed from zwitterionic or mixed lipids on silica nanoparticle surfaces possess a higher negative electrostatic potential than the unsupported bilayers with the potential of mixed bilayers containing negatively charged lipids being significantly more sensitive to the silica support. pH-sensitive EPR probes were then employed to label model WALP peptide known to form α-helix when integrated into a lipid bilayer. The effects of silica support on the peptide dynamics and the effective pKa of the ionizable probe have been investigated. It was demonstrated that the silica nanoparticles affect the peptide dynamics and shift the effective pKa of the ionizable nitroxide probe in a membrane depth-dependent manner. Our data also show that upon protonation of the membrane-burred model ionisable sidechain the silica support causes changes in the membrane association of WALP peptide that are not observed when WAPL is integrated into unilamellar phospholipid vesicles. Supported by NSF 1508607 to TIS.}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Ou, Erkang and Voinov, Maxim A. and Smirnov, Alex I. and Smirnova, Tatyana I.}, year={2018}, month={Feb}, pages={96a} }
 @article{chestnut_milikisiyants_koolivand_voynov_smirnova_smirnov_2018, title={Using Hyscore Spectroscopy of Nitroxides to Profile Water Content of Lipid Bilayers with 2 Å Spatial Resolution}, volume={114}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/J.BPJ.2017.11.130}, DOI={10.1016/J.BPJ.2017.11.130}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Chestnut, Melanie and Milikisiyants, Sergey and Koolivand, Amir and Voynov, Maxim A. and Smirnova, Tatyana I. and Smirnov, Alex I.}, year={2018}, month={Feb}, pages={16a} }
 @article{ou_voinov_smirnov_smirnova_2017, title={Effects of Silica Support on Dynamics of Transmembrane Peptides and Effective p K a of Ionisable Sidechains}, volume={112}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/J.BPJ.2016.11.967}, DOI={10.1016/J.BPJ.2016.11.967}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Ou, Erkang and Voinov, Maxim and Smirnov, Alex I. and Smirnova, Tatyana I.}, year={2017}, month={Feb}, pages={175a} }
 @article{mitra_gao_zheng_wu_voinov_smirnov_smirnova_wang_chavala_han_2017, title={Glycol Chitosan Engineered Autoregenerative Antioxidant Significantly Attenuates Pathological Damages in Models of Age-Related Macular Degeneration}, volume={11}, ISSN={1936-0851 1936-086X}, url={http://dx.doi.org/10.1021/ACSNANO.7B00429}, DOI={10.1021/acsnano.7b00429}, abstractNote={Age-related macular degeneration (AMD) is the foremost cause of irreversible blindness in people over the age of 65 especially in developing countries. Therefore, an exploration of effective and alternative therapeutic interventions is an unmet medical need. It has been established that oxidative stress plays a key role in the pathogenesis of AMD, and hence, neutralizing oxidative stress is an effective therapeutic strategy for treatment of this serious disorder. Owing to autoregenerative properties, nanoceria has been widely used as a nonenzymatic antioxidant in the treatment of oxidative stress related disorders. Yet, its potential clinical implementation has been greatly hampered by its poor water solubility and lack of reliable tracking methodologies/processes and hence poor absorption, distribution, and targeted delivery. The water solubility and surface engineering of a drug with biocompatible motifs are fundamental to pharmaceutical products and precision medicine. Here, we report an engineered water-soluble, biocompatible, trackable nanoceria with enriched antioxidant activity to scavenge intracellular reactive oxygen species (ROS). Experimental studies with in vitro and in vivo models demonstrated that this antioxidant is autoregenerative and more active in inhibiting laser-induced choroidal neovascularization by decreasing ROS-induced pro-angiogenic vascular endothelial growth factor (VEGF) expression, cumulative oxidative damage, and recruitment of endothelial precursor cells without exhibiting any toxicity. This advanced formulation may offer a superior therapeutic effect to deal with oxidative stress induced pathogeneses, such as AMD.}, number={5}, journal={ACS Nano}, publisher={American Chemical Society (ACS)}, author={Mitra, Rajendra N. and Gao, Ruijuan and Zheng, Min and Wu, Ming-Jing and Voinov, Maxim A. and Smirnov, Alex I. and Smirnova, Tatyana I. and Wang, Kai and Chavala, Sai and Han, Zongchao}, year={2017}, month={May}, pages={4669–4685} }
 @article{milikisiyants_wang_munro_donohue_ward_bolton_brown_smirnova_ladizhansky_smirnov_2017, title={Oligomeric Structure of Anabaena Sensory Rhodopsin in a Lipid Bilayer Environment by Combining Solid-State NMR and Long-range DEER Constraints}, volume={429}, ISSN={["1089-8638"]}, url={https://doi.org/10.1016/j.jmb.2017.05.005}, DOI={10.1016/j.jmb.2017.05.005}, abstractNote={Oligomerization of membrane proteins is common in nature. Here, we combine spin-labeling double electron-electron resonance (DEER) and solid-state NMR (ssNMR) spectroscopy to refine the structure of an oligomeric integral membrane protein, Anabaena sensory rhodopsin (ASR), reconstituted in a lipid environment. An essential feature of such a combined approach is that it provides structural distance restraints spanning a range of ca 3-60Å while using the same sample preparation (i.e., mutations, paramagnetic labeling, and reconstitution in lipid bilayers) for both ssNMR and DEER. Direct modeling of the multispin effects on DEER signal allowed for the determination of the oligomeric order and for obtaining long-range DEER distance restraints between the ASR trimer subunits that were used to refine the ssNMR structure of ASR. The improved structure of the ASR trimer revealed a more compact packing of helices and side chains at the intermonomer interface, compared to the structure determined using the ssNMR data alone. The extent of the refinement is significant when compared with typical helix movements observed for the active states of homologous proteins. Our combined approach of using complementary DEER and NMR measurements for the determination of oligomeric structures would be widely applicable to membrane proteins where paramagnetic tags can be introduced. Such a method could be used to study the effects of the lipid membrane composition on protein oligomerization and to observe structural changes in protein oligomers upon drug, substrate, and co-factor binding.}, number={12}, journal={JOURNAL OF MOLECULAR BIOLOGY}, publisher={Elsevier BV}, author={Milikisiyants, Sergey and Wang, Shenlin and Munro, Rachel A. and Donohue, Matthew and Ward, Meaghan E. and Bolton, David and Brown, Leonid S. and Smirnova, Tatyana I. and Ladizhansky, Vladimir and Smirnov, Alex I.}, year={2017}, month={Jun}, pages={1903–1920} }
 @article{mccombs_smirnova_ghiladi_2017, title={Oxidation of pyrrole by dehaloperoxidase-hemoglobin: chemoenzymatic synthesis of pyrrolin-2-ones}, volume={7}, ISSN={["2044-4761"]}, url={http://dx.doi.org/10.1039/c7cy00781g}, DOI={10.1039/c7cy00781g}, abstractNote={A biocatalytic approach using the enzyme dehaloperoxidase catalyzes the H<sub>2</sub>O<sub>2</sub>-dependent oxidation of pyrrole to 4-pyrrolin-2-one and notably without polypyrrole formation.}, number={14}, journal={CATALYSIS SCIENCE & TECHNOLOGY}, author={McCombs, Nikolette L. and Smirnova, Tatyana and Ghiladi, Reza A.}, year={2017}, month={Jul}, pages={3104–3118} }
 @article{luo_ou_smirnova_maggard_2016, title={Synthesis of New Mixed-Metal Ammonium Vanadates: Cation Order versus Disorder, and Optical and Photocatalytic Properties}, volume={16}, ISSN={["1528-7505"]}, DOI={10.1021/acs.cgd.6b00851}, abstractNote={Two new ammonium vanadate hydrates, i.e., M3(H2O)2V8O24·2NH4 (M = Mn and Co, I and II, respectively) were synthesized using hydrothermal reaction conditions, and their structures were determined by single crystal X-ray diffraction [I: P2/m (No. 10), Z = 1, a = 8.2011(2) A, b = 3.5207(1) A, c = 9.9129(3) A, β = 110.987(2)°; II: C2/m (No. 12), Z = 2, a = 19.4594(6) A, b = 6.7554(2) A, c = 8.4747(3) A, β = 112.098(2)°]. Interestingly, the two structures are homeotypic, with the structure of I exhibiting an uncommon type of structural disorder between locally-bridging Mn(H2O)22+ (i.e., part of the oxide framework) and nonbridging NH4+ cations over the same site (1:2 ratio), wherein two NH4+ ions occupy the same site as the two H2O molecules when Mn(II) is vacant. The amount of Mn(II) in the formula of I was determined by a combination of techniques, including electron paramagnetic resonance, while the relative amounts of NH4+/H2O in its structure were determined by combined thermogravimetric-mass spectrometry...}, number={10}, journal={CRYSTAL GROWTH & DESIGN}, author={Luo, Lan and Ou, Erkang and Smirnova, Tatyana I. and Maggard, Paul A.}, year={2016}, month={Oct}, pages={5762–5770} }
 @article{roberts_voinov_schmidt_smirnova_sombers_2016, title={The Hydroxyl Radical is a Critical Intermediate in the Voltammetric Detection of Hydrogen Peroxide}, volume={138}, ISSN={0002-7863 1520-5126}, url={http://dx.doi.org/10.1021/JACS.5B13376}, DOI={10.1021/jacs.5b13376}, abstractNote={Cyclic voltammetry is a widely used and powerful tool for sensitively and selectively measuring hydrogen peroxide (H2O2). Herein, voltammetry was combined with electron paramagnetic resonance spectroscopy to identify and define the role of an oxygen-centered radical liberated during the oxidation of H2O2. The spin-trap reagents, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and 2-ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide (EMPO), were employed. Spectra exhibit distinct hyperfine patterns that clearly identify the DMPO(•)-OH and EMPO(•)-OH adducts. Multiple linear regression analysis of voltammograms demonstrated that the hydroxyl radical is a principal contributor to the voltammetry of H2O2, as signal is attenuated when this species is trapped. These data incorporate a missing, fundamental element to our knowledge of the mechanisms that underlie H2O2 electrochemistry.}, number={8}, journal={Journal of the American Chemical Society}, publisher={American Chemical Society (ACS)}, author={Roberts, James G. and Voinov, Maxim A. and Schmidt, Andreas C. and Smirnova, Tatyana I. and Sombers, Leslie A.}, year={2016}, month={Feb}, pages={2516–2519} }
 @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{milikisiyants_wang_munro_donohue_brown_smirnova_ladizhansky_smirnov_2015, title={Determining Oligomeric Order of a Membrane Protein by Double Electron-Electron Resonance Spectroscopy}, volume={108}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/J.BPJ.2014.11.541}, DOI={10.1016/J.BPJ.2014.11.541}, abstractNote={Many different classes of membrane proteins are known to form oligomers in cellular membranes in order to carry out specific cellular functions. Detection and detailed structural characterization of protein oligomers in lipid milieu is by no means a trivial task. Here we demonstrate the use of spin-labeling and Double Electron-Electron Resonance (DEER) spectroscopy to determine the oligomeric order of a membrane protein. Specifically, we investigate oligomerization of a seven-helical membrane photoreceptor Anabaena Sensory Rhodopsin (ASR) from Anabaena sp. PCC7120. Recently, ASR structure has been solved by both x-ray protein crystallography (Science 2004, 306, 1390) and solid-state NMR (Nat Methods 2013, 10, 1007). Here we show that the same spin-labeling sites we employed for paramagnetic relaxation enhancement (PRE) NMR can also be used for DEER experiments. The results demonstrate that DEER restraints can not only differentiate between the dimer (x-ray) and trimer (ssNMR) models that have very different interfaces, but further rule out hypothetical tetramer and other higher order polygon models. The crux of our DEER-based approach relies on taking advantage of the multi-spin effects and analyzing experimental DEER traces by direct fitting to the multispin models. Overall, the observed profound effect of higher order spin correlations on the DEER trace allows for a reliable differentiation between oligomer models. In the specific case of ASR, the DEER trace modeling allowed us to unambiguously discard all but the trimer model. Furthermore, the addition of DEER electron-electron distances to the NMR restraints in the structure calculation protocol improves local RMSD, and allows for refinement of the orientation of helices. Supported by U.S. DOE Contract DE-FG02-02ER15354 to AIS and NSERC Discovery Grants RGPIN-2014-04547 to VL and RGPIN-2013-250202 to LSB.}, number={2}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Milikisiyants, Sergey and Wang, Shenlin and Munro, Rachel and Donohue, Matthew and Brown, Leonid S. and Smirnova, Tatyana I. and Ladizhansky, Vladimir and Smirnov, Alex I.}, year={2015}, month={Jan}, pages={93a} }
 @article{huang_wu_song_xu_smirnova_weare_sommer_2015, title={Ferromagnetic coupling in d(1)-d(3) linear oxido-bridged heterometallic complexes: ground-state models of metal-to-metal charge transfer excited states}, volume={44}, ISSN={["1477-9234"]}, DOI={10.1039/c5dt02719e}, abstractNote={Convenient strategies have been developed to synthesize heterobi/trimetallic oxido complexes containing V(IV)-O-Cr(III), V(IV)-O-Cr(III)-O-Ti(IV) and V(IV)-O-Cr(III)-O-V(IV) cores. These compounds can serve as ground state models for probing the magnetic properties of metal-to-metal charge transfer excited states. Each of these complexes represents the first experimental demonstration of ferromagnetic coupling in a d(1)-d(3) oxido bridged compound, which confirms a long standing theoretical prediction for such a linkage. Structural characterization reveals a similar structure for each of the bi/trimetallic complexes with identical V[double bond, length as m-dash]O bond lengths (∼1.644 Å) and a linear V-O-Cr geometry. The Cr-O distances (1.943-1.964 Å) are significantly influenced by the ligands in the trans axial positions. Ferromagnetic coupling between the V(IV) and Cr(III) of V-O-Cr is measured by temperature-dependent magnetic susceptibility, showing J = +42.5 to +50.7 cm(-1) (H = -2JŜVŜCr). This is further supported by variable temperature X-band EPR. The values of J are found to be consistent with the function J = Ae(βr) (A = 9.221 × 10(8) and β = 8.607 Å(-1)), where r is the Cr-O bond distance. We propose a model that links either ferromagnetic or antiferromagentic exchange coupling with long excited state lifetimes in metal-to-metal charge transfer (MMCT) chromophores.}, number={43}, journal={DALTON TRANSACTIONS}, publisher={Royal Society of Chemistry (RSC)}, author={Huang, Tao and Wu, Xinyuan and Song, Xiao and Xu, Hao and Smirnova, Tatyana I. and Weare, Walter W. and Sommer, Roger D.}, year={2015}, pages={18937–18944} }
 @article{luo_zeng_li_luo_smirnova_maggard_2015, title={Manganese-Vanadate Hybrids: Impact of Organic Ligands on Their Structures, Thermal Stabilities, Optical Properties, and Photocatalytic Activities}, volume={54}, ISSN={["1520-510X"]}, DOI={10.1021/acs.inorgchem.5b00931}, abstractNote={Manganese(II)-vanadate(V)/organic hybrids were prepared in high purity using four different N-donor organic ligands (2,6:2',2″-terpyridine = terpy, 2,2'-bipyrimidine = bpym, o-phenanthroline = o-phen, and 4,4'-bipyridine = 4,4'-bpy), and their crystalline structures, thermal stabilities, optical properties, photocatalytic activities and electronic structures were investigated as a function of the organic ligand. Hydrothermal reactions were employed that targeted a 1:2 molar ratio of Mn(II)/V(V), yielding four hybrid solids with the compositions of Mn(terpy)V2O6·H2O (I), Mn2(bpym)V4O12·0.6H2O (II), Mn(H2O)(o-phen)V2O6 (III), and Mn(4,4'-bpy)V2O6·1.16H2O (IV). The inorganic component within these hybrid compounds, that is, [MnV2O6], forms infinite chains in I and layers in II, III, and IV. In each case, the organic ligand preferentially coordinates to the Mn(II) cations within their respective structures, either as chelating and three-coordinate (mer isomer in I) or two-coordinate (cis isomers in II and III), or as bridging and two coordinate (trans isomer in IV). The terminating ligands in I (terpy) and III (o-phen) yield nonbridged "MnV2O6" chains and layers, respectively, while the bridging ligands in II (bpym) and IV (4,4'-bpy) result in three-dimensional, pillared hybrid networks. The coordination number of the ligand, that is, two- or three-coordinate, has the predominant effect on the dimensionality of the inorganic component, while the connectivity of the combined metal-oxide/organic network is determined by the chelating versus bridging ligand coordination modes. Each hybrid compound decomposes into crystalline MnV2O6 upon heating in air with specific surface areas from ∼7 m(2)/g for III to ∼41 m(2)/g for IV, depending on the extent of structural collapse as the lattice water is removed. All hybrid compounds exhibit visible-light bandgap sizes from ∼1.7 to ∼2.0 eV, decreasing with the increased dimensionality of the [MnV2O6] network in the order of I > II ≈ III > IV. These bandgap sizes are smaller by ∼0.1-0.4 eV in comparison to related vanadate hybrids, owing to the addition of the higher-energy 3d orbital contributions from the Mn(II) cations. Each compound also exhibits temperature-dependent photocatalytic activities for hydrogen production under visible-light irradiation in 20% methanol solutions, with threshold temperatures of ∼30 °C for III, ∼36 °C for I, and ∼40 °C for II, IV, and V4O10(o-phen)2. Hydrogen production rates are ∼142 μmol H2 g(-1)·h(-1), ∼673 μmol H2 g(-1)·h(-1), ∼91 μmol H2 g(-1)·h(-1), and ∼218 μmol H2 g(-1)·h(-1) at 40 °C, for I, II, III, and IV, respectively, increasing with the oxide/organic network connectivity. In contrast, the related V4O10(o-phen)2 exhibits a much lower photocatalytic rate of ∼36 H2 g(-1)·h(-1). Electronic structure calculations based on density-functional theory methods show that the valence band edges are primarily derived from the half-filled Mn 3d(5) orbitals in each, while the conduction band edges are primarily comprised of contributions from the empty V 3d(0) orbitals in I and II and from ligand π* orbitals in III. Thus, the coordinating organic ligands are shown to significantly affect the local and extended structural features, which has elucidated the underlying relationships to their photocatalytic activities, visible-light bandgap sizes, electronic structures, and thermal stabilities.}, number={15}, journal={INORGANIC CHEMISTRY}, author={Luo, Lan and Zeng, Yuhan and Li, Le and Luo, Zhixiang and Smirnova, Tatyana I. and Maggard, Paul A.}, year={2015}, month={Aug}, pages={7388–7401} }
 @article{donohue_voynov_milikisiyants_smirnov_smirnova_2015, title={“Snorkeling” of the Charged Sidechain of a Transmembrane Peptide as Directly Observed by Double Electron-Electron Resonance Experiment}, volume={108}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/J.BPJ.2014.11.1120}, DOI={10.1016/J.BPJ.2014.11.1120}, abstractNote={While hydrophobic amino acids constitute the bulk of transmembrane protein domains, polar and even charged amino acids are not uncommon and often play significant roles in membrane protein function. Positioning a polar residue within the bilayer core is highly unfavorable thermodynamically; however, the free energy penalty could be minimized by “stretching” the side chain of the amino acid to bring the charged moiety closer to the bilayer surface while keeping the rest of the side chain inside the hydrophobic core. This biophysical phenomenon is known as “snorkeling”. Here we report experimental observations of “snorkeling” for nitroxide-modified side-chains upon protonation, its dependence upon the location along the transmembrane peptide helix, and how this snorkeling is affected by the membrane electrostatic surface potential. pH sensitive spin labels, either IMTSL or IKMTSL (JPCB 2009, 113, 3453) were attached to two cysteine residues positioned equidistant from the center of the WALP peptide so that the primary sequence of each peptide is palindromic, thus, ensuring symmetric location of the labels with respect to the bilayer. The change in protonation states of the nitroxide was directly observed from EPR spectra. The distance between two nitroxide moieties was measured by Q-band double electron-electron resonance (DEER) experiment. Upon protonation, the distance between the two IMTSL probes increased compared to that of the neutral forms, by approximately 3 A indicating displacements of the charged nitroxide sidechain towards the polar head region. The “snorkeling” of the label was observed to be depth dependent - no changes in the positioning of the sidechain upon protonation was observed for labels located within 10-8 A from the center of the bilayer. Supported by NSF-0843632 to TIS.}, number={2}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Donohue, Matthew and Voynov, Maxim and Milikisiyants, Sergey and Smirnov, Alex I. and Smirnova, Tatyana I.}, year={2015}, month={Jan}, pages={203a} }
 @article{luo_lin_li_smirnova_maggard_2014, title={Copper-Organic/Octamolybdates: Structures, Bandgap Sizes, and Photocatalytic Activities}, volume={53}, ISSN={0020-1669 1520-510X}, url={http://dx.doi.org/10.1021/IC402910A}, DOI={10.1021/IC402910A}, abstractNote={The structures, optical bandgap sizes, and photocatalytic activities are described for three copper-octamolybdate hybrid solids prepared using hydrothermal methods, [Cu(pda)]4[β-Mo8O26] (I; pda = pyridazine), [Cu(en)2]2[γ-Mo8O26] (II; en = ethylenediamine), and [Cu(o-phen)2]2[α-Mo8O26] (III; o-phen = o-phenanthroline). The structure of I consists of a [Cu(pda)]4(4+) tetramer that bridges to neighboring [β-Mo8O26](4-) octamolybdate clusters to form two-dimensional layers that stack along the a axis. The previously reported structures of II and III are constructed from [Cu2(en)4Mo8O26] and [Cu2(o-phen)4Mo8O26] clusters. The optical bandgap sizes were measured by UV-vis diffuse reflectance techniques to be ∼1.8 eV for I, ∼3.1 eV for II, and ∼3.0 eV for III. Electronic structure calculations show that the smaller bandgap size of I originates primarily from an electronic transition between the valence and conduction band edges comprised of filled 3d(10) orbitals on Cu(I) and empty 4d(0) orbitals on Mo(VI). Both II and III contain Cu(II) and exhibit larger bandgap sizes. Accordingly, aqueous suspensions of I exhibit visible-light photocatalytic activity for the production of oxygen at a rate of ∼90 μmol O2 g(-1) h(-1) (10 mg samples; radiant power density of ∼1 W/cm(2)) and a turnover frequency per calculated surface [Mo8O26](4-) cluster of ∼36 h(-1). Under combined ultraviolet and visible-light irradiation, I also exhibits photocatalytic activity for hydrogen production in 20% aqueous methanol of ∼316 μmol H2 g(-1) h(-1). By contrast, II decomposed during the photocatalysis measurements. The molecular [Cu2(o-phen)4(α-Mo8O26)] clusters of III dissolve into the aqueous methanol solution under ultraviolet irradiation and exhibit homogeneous photocatalytic rates for hydrogen production of up to ∼8670 μmol H2·g(-1) h(-1) and a turnover frequency of 17 h(-1). The clusters of III can be precipitated out by evaporation and redispersed into solution with no apparent decrease in photocatalytic activity. During the photocatalysis measurements, the dissolution of the clusters in III is found to occur with the reduction of Cu(II) to Cu(I), followed by subsequent detachment from the octamolybdate cluster. The lower turnover frequency, but higher photocatalytic rate, of III arises from the net contribution of all dissolved [Cu2(o-phen)4(α-Mo8O26)] clusters, compared to only the surface clusters for the heterogeneous photocatalysis of I.}, number={7}, journal={Inorganic Chemistry}, publisher={American Chemical Society (ACS)}, author={Luo, Lan and Lin, Haisheng and Li, Le and Smirnova, Tatyana I. and Maggard, Paul A.}, year={2014}, month={Mar}, pages={3464–3470} }
 @article{meng_smirnova_song_moore_ren_kelley_park_tilotta_2014, title={Identification of free radicals in pyrolysis oil and their impact on bio-oil stability}, volume={4}, ISSN={["2046-2069"]}, DOI={10.1039/c4ra02007c}, abstractNote={The existence of radicals in pyrolysis oil generated from loblolly pine in three different reactor systems was verified with electron paramagnetic resonance (EPR) spectroscopy.}, number={56}, journal={RSC ADVANCES}, author={Meng, Jiajia and Smirnova, Tatyana I. and Song, Xiao and Moore, Andrew and Ren, Xueyong and Kelley, Stephen and Park, Sunkyu and Tilotta, David}, year={2014}, pages={29840–29846} }
 @article{donohue_voynov_smirnov_smirnova_2014, title={Profiling the Dielectric Constant at the Membrane-Peptide Interface using Ionizable EPR Probes}, volume={106}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/J.BPJ.2013.11.2841}, DOI={10.1016/J.BPJ.2013.11.2841}, abstractNote={Polarity, electric potentials, and hydration are the major physico-chemical characteristics of lipid membranes that govern membrane-protein and protein-protein interactions as well as small molecules transport. Insertion of transmembrane proteins perturbs membrane structure altering local dielectric environment and hydration at the membrane-protein interface. The significance of distorting local membrane structure at the lipid-protein interface for modulating protein-protein interactions should not be overlooked. In this work we report on employing pH-sensitive ionizable EPR labels to profile a heterogeneous dielectric environment along the α-helix of a WALP peptide integrated in a lipid bilayer. Labels were attached to two cysteine residues positioned equidistant from the center of the peptide so that the primary sequence of each peptide is palindromic, thus insuring symmetric location of the labels with respect to the bilayer center. The change in protonation state of the nitroxide was directly observed by EPR. Q-band double electron-electron resonance (DEER) experiments were carried out to determine the distance between spin labels when imbedded in lipid bilayers to provide information about the label location. Thus, for the first time measurements of local electrostatics at peptide-bilayer interface were based on direct distance measurements rather than on assumptions on the probe location. Two pH sensitive spin labels, methanethiosulfonic acid S-(1-oxyl-2,2,3,5,5-pentamethyl-imidazolidin-4-ylmethyl) ester (IMTSL) and S-4-(4-(dimethylamino)-2-ethyl-5,5-dimethyl-1-oxyl-2,5-dihydro-1H-imidazol-2-yl) benzylmethanethiosulfonate (IKMTSL), with intrinsic pKa's differing by approximately 2 pH units were used to expand the pH range of the titration experiments. This provided the opportunity to vary the lipid composition in order to investigate effect of the surface charge on dielectric profile at peptide-membrane interface. Water penetration at the peptide-membrane interface was assessed by hyperfine sublevel correlation spectroscopy (HYSCORE) experiment in which the hyperfine coupling between the nitroxide and hydrogen/deuterium atom of water is measured. Supported by NSF-0843632 to TIS.}, number={2}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Donohue, Matthew and Voynov, Maxim A. and Smirnov, Alex I. and Smirnova, Tatyana I.}, year={2014}, month={Jan}, pages={508a} }
 @article{quinones_bankaitis_smirnova_2014, title={Structure, Dynamics, and Electrostatic Effects on Membrane Binding of Nod Peptides}, volume={106}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/J.BPJ.2013.11.1719}, DOI={10.1016/J.BPJ.2013.11.1719}, abstractNote={A Sec14p-nodulin domain phosphatidylinositol transfer protein has been shown to polarize membrane growth of root hairs. To understand the mechanism of regulation, we have synthesized three peptides mimicking the sequence thought to be responsible for the targeted membrane binding containing single Cys substitutions. Binding of the peptides to model membranes was studied by spin-labeling EPR, ITC and DLS as a function of the bilayer lipid composition, with emphasis on the presence of phosphatidylinositol (PI) and brain PI(4,5)P2. For EPR studies, peptides were labeled with thiol-specific spin label (2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)methyl methanesulfonothioate (MTSL) and membrane binding was characterized from changes in EPR spectra. Binding data were analyzed using a comprehensive model that includes description of electrostatic interaction by the Gouy-Chapman theory. Analysis of local polarity and accessibility of the EPR label for the bound form of the peptides was used to assess the location of bound peptides within model phospholipid bilayers. Substantial differences in binding behavior of these charged peptides were observed despite the similarity in sequences. Binding of the peptides did not induce liposome leakage or fusion. Unusual temperature dependent reversible effect of the peptides on liposome aggregation was observed and showed strong dependence upon membrane composition, especially the presence of PI(4,5)P2 lipids. Supported by NSF 0843632 to TIS.}, number={2}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Quinones, Mary Chely and Bankaitis, Vytas A. and Smirnova, Tatyana I.}, year={2014}, month={Jan}, pages={295a} }
 @article{li_wang_smirnova_khade_zhang_oldfield_2013, title={Isoprenoid Biosynthesis: Ferraoxetane or Allyl Anion Mechanism for IspH Catalysis?}, volume={52}, ISSN={["1433-7851"]}, DOI={10.1002/anie.201302343}, abstractNote={The catalytic mechanism of the enzyme IspH, involved in formation of isopentenyl diphosphate and dimethylallyl diphosphate, was investigated by using HYSCORE spectroscopy combined with DFT. The results indicate the formation of an allyl anion bound to a HiPIP-like oxidized 4Fe–4S cluster, rather than formation of a cyclic, ferraoxetane intermediate, as has been proposed elsewhere.}, number={25}, journal={ANGEWANDTE CHEMIE-INTERNATIONAL EDITION}, author={Li, Jikun and Wang, Ke and Smirnova, Tatyana I. and Khade, Rahul L. and Zhang, Yong and Oldfield, Eric}, year={2013}, pages={6522–6525} }
 @article{donohue_voynov_smirnov_smirnova_2013, title={Molecular pH Probes at a Protein-Lipid Interface: Assessment of Local Dielectric Environment for Transmembrane Peptide}, volume={104}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/j.bpj.2012.11.2073}, DOI={10.1016/j.bpj.2012.11.2073}, abstractNote={Spin-labeling EPR spectroscopy has found many applications in studying structure and dynamics of proteins and biological membranes. Recently, there has been substantial interest in utilizing EPR to characterize local effects of polarity and hydrogen bonding in these systems. Here we report on employing an pH-sensitive EPR probe IMSTL (S-(1-oxyl-2,2,3,5,5-pentamethylimidazolidin-4-ylmethyl) ester) to profile heterogeneous dielectric environments along the α-helix of a WALP peptide integrated in a lipid bilayer. A series of symmetrically positioned double cysteine mutants were labeled with a pH-sensitive nitroxide and the protonation state of IMTSL was directly observed by EPR. Q-band DEER experiments with double-labeled WALPs were employed to derive nitroxide-nitroxide distances of nitroxides before and after the protonation and, therefore, the positions of pH probes with respect to lipid bialyer. Thus, for the first time measurements of local electrostatics at peptide-bilayer interface were based on direct distance measurements rather than on assumptions on the probe location. For double-labeled WALP consecutive protonation of symmetrically positioned nitroxide tags was observed. The difference in observable pKa values was interpreted in terms of electrostatic interaction energy between titratable probes allowing us to estimate effective dielectric constant. Supported by NSF-0843632 to TIS.}, number={2}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Donohue, Matthew and Voynov, Maxim A. and Smirnov, Alex I. and Smirnova, Tatyana I.}, year={2013}, month={Jan}, pages={373a} }
 @article{young_donohue_smirnova_smirnov_zhou_2013, title={The UDP-diacylglucosamine Pyrophosphohydrolase LpxH in Lipid A Biosynthesis Utilizes Mn2+ Cluster for Catalysis}, volume={288}, ISSN={["1083-351X"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84884550173&partnerID=MN8TOARS}, DOI={10.1074/jbc.m113.497636}, abstractNote={In Escherichia coli and the majority of β- and γ-proteobacteria, the fourth step of lipid A biosynthesis, i.e. cleavage of the pyrophosphate group of UDP-2,3-diacyl-GlcN, is carried out by LpxH. LpxH has been previously suggested to contain signature motifs found in the calcineurin-like phosphoesterase (CLP) family of metalloenzymes; however, it cleaves a pyrophosphate bond instead of a phosphoester bond, and its substrate contains nucleoside diphosphate moieties more common to the Nudix family rather than to the CLP family. Furthermore, the extent of biochemical data fails to demonstrate a significant level of metal activation in enzymatic assays, which is inconsistent with the behavior of a metalloenzyme. Here, we report cloning, purification, and detailed enzymatic characterization of Haemophilus influenzae LpxH (HiLpxH). HiLpxH shows over 600-fold stimulation of hydrolase activity in the presence of Mn(2+). EPR studies reveal the presence of a Mn(2+) cluster in LpxH. Finally, point mutants of residues in the conserved metal-binding motifs of the CLP family greatly inhibit HiLpxH activity, highlighting their importance in enzyme function. Contrary to previous analyses of LpxH, we find HiLpxH does not obey surface dilution kinetics. Overall, our work unambiguously establishes LpxH as a calcineurin-like phosphoesterase containing a Mn(2+) cluster coordinated by conserved residues. These results set the scene for further structural investigation of the enzyme and for design of novel antibiotics targeting lipid A biosynthesis.}, number={38}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, publisher={American Society for Biochemistry & Molecular Biology (ASBMB)}, author={Young, Hayley E. and Donohue, Matthew P. and Smirnova, Tatyana I. and Smirnov, Alex I. and Zhou, Pei}, year={2013}, month={Sep}, pages={26987–27001} }
 @article{dumarieh_d'antonio_deliz-liang_smirnova_svistunenko_ghiladi_2013, title={Tyrosyl Radicals in Dehaloperoxidase}, volume={288}, ISSN={0021-9258 1083-351X}, url={http://dx.doi.org/10.1074/JBC.M113.496497}, DOI={10.1074/JBC.M113.496497}, abstractNote={Dehaloperoxidase (DHP) from Amphitrite ornata, having been shown to catalyze the hydrogen peroxide-dependent oxidation of trihalophenols to dihaloquinones, is the first oxygen binding globin that possesses a biologically relevant peroxidase activity. The catalytically competent species in DHP appears to be Compound ES, a reactive intermediate that contains both a ferryl heme and a tyrosyl radical. By simulating the EPR spectra of DHP activated by H2O2, Thompson et al. (Thompson, M. K., Franzen, S., Ghiladi, R. A., Reeder, B. J., and Svistunenko, D. A. (2010) J. Am. Chem. Soc. 132, 17501-17510) proposed that two different radicals, depending on the pH, are formed, one located on either Tyr-34 or Tyr-28 and the other on Tyr-38. To provide additional support for these simulation-based assignments and to deduce the role(s) that tyrosyl radicals play in DHP, stopped-flow UV-visible and rapid-freeze-quench EPR spectroscopic methods were employed to study radical formation in DHP when three tyrosine residues, Tyr-28, Tyr-34, and Tyr-38, were replaced either individually or in combination with phenylalanines. The results indicate that radicals form on all three tyrosines in DHP. Evidence for the formation of DHP Compound I in several tyrosine mutants was obtained. Variants that formed Compound I showed an increase in the catalytic rate for substrate oxidation but also an increase in heme bleaching, suggesting that the tyrosines are necessary for protecting the enzyme from oxidizing itself. This protective role of tyrosines is likely an evolutionary adaptation allowing DHP to avoid self-inflicted damage in the oxidative environment.}, number={46}, journal={Journal of Biological Chemistry}, publisher={American Society for Biochemistry & Molecular Biology (ASBMB)}, author={Dumarieh, Rania and D'Antonio, Jennifer and Deliz-Liang, Alexandria and Smirnova, Tatyana and Svistunenko, Dimitri A. and Ghiladi, Reza A.}, year={2013}, month={Oct}, pages={33470–33482} }
 @article{dumarieh_d'antonio_deliz-liang_smirnova_svistunenko_ghiladi_2013, title={Tyrosyl radicals in dehaloperoxidase how nature deals with evolving an oxygen-binding globin to a biologically relevant peroxidase}, volume={288}, number={46}, journal={Journal of Biological Chemistry}, author={Dumarieh, R. and D'Antonio, J. and Deliz-Liang, A. and Smirnova, T. and Svistunenko, D. A. and Ghiladi, R. A.}, year={2013}, pages={33470–33482} }
 @article{smirnova_voynov_poluektov_smirnov_2012, title={Probing Dielectric and Hydrogen Bonding Gradients in Biological Membranes}, volume={102}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/j.bpj.2011.11.2262}, DOI={10.1016/j.bpj.2011.11.2262}, abstractNote={Nitroxide spin-labeling in combination with EPR spectroscopy has found many applications in studying structure and dynamics of proteins and biological membranes. Recently, there has been a substantial interest in utilizing EPR to characterize local effects of polarity and hydrogen bonding in proteins and biological membrane systems. Here we report on employing an arsenal of advanced spin-labeling EPR methods to profile heterogeneous dielectric and hydrogen bonding environment along the α-helical chain of an alanine-rich WALP peptide that is anchored in a lipid bilayer in a transmembrane orientation. A series of WALP cysteine mutants was labeled with a pH-sensitive nitroxide IMSTL (S-(1-oxyl-2,2,3,5,5-pentamethylimidazolidin-4-ylmethyl) ester) that is similar in molecular volume to phenylalanine. The protonation state of this nitroxide could be directly observed by EPR allowing us to follow proton gradient across the membrane in the vicinity of the WALP α-helix, and, thus, to reconstruct the gradient in the effective dielectric constant. These experiments were complemented by assessing local polarity from characteristic changes in EPR spectra that were enhanced by the use of perdeuterated and 15N-substituted nitroxides and high field EPR at 130 GHz (D-band). Formation of hydrogen bonds between the nitroxides and membrane-penetrating water molecules was observed directly in HYSCORE X-band experiments. Such measurements allowed us to derive experimental profiles of heterogeneous dielectric and hydrogen bonding environment along a typical transmembrane α-helix. Supported by: NSF-0843632 to TIS and NIH 1R01GM072897 to AIS.}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Smirnova, Tatyana I. and Voynov, Maxim A. and Poluektov, Oleg G. and Smirnov, Alex I.}, year={2012}, month={Jan}, pages={414a} }
 @article{wang_wang_li_nellutla_smirnova_oldfield_2011, title={An ENDOR and HYSCORE Investigation of a Reaction Intermediate in IspG (GcpE) Catalysis}, volume={133}, ISSN={["0002-7863"]}, DOI={10.1021/ja200763a}, abstractNote={IspG is a 4Fe–4S protein that carries out an essential reduction step in isoprenoid biosynthesis. Using electron–nuclear double resonance (ENDOR) and hyperfine sublevel correlation (HYSCORE) spectroscopies on labeled samples, we have specifically assigned the hyperfine interactions in a reaction intermediate. These results help clarify the nature of the reaction intermediate, supporting a direct interaction between the unique fourth Fe in the cluster and C2 and O3 of the ligand.}, number={22}, journal={JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, author={Wang, Weixue and Wang, Ke and Li, Jikun and Nellutla, Saritha and Smirnova, Tatyana I. and Oldfield, Eric}, year={2011}, month={Jun}, pages={8400–8403} }
 @article{wang_li_wang_smirnova_oldfield_2011, title={Pyridine Inhibitor Binding to the 4Fe-4S Protein A. aeolicus IspH (LytB): A HYSCORE Investigation}, volume={133}, ISSN={["0002-7863"]}, DOI={10.1021/ja2008455}, abstractNote={IspH is a 4Fe-4S protein that carries out an essential reduction step in isoprenoid biosynthesis. Using hyperfine sublevel correlation (HYSCORE) spectroscopy, we show that pyridine inhibitors of IspH directly bind to the unique fourth Fe in the 4Fe-4S cluster, opening up new routes to inhibitor design, of interest in the context of both anti-bacterial as well as anti-malarial drug discovery.}, number={17}, journal={JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, author={Wang, Weixue and Li, Jikun and Wang, Ke and Smirnova, Tatyana I. and Oldfield, Eric}, year={2011}, month={May}, pages={6525–6528} }
 @article{schaaf_dynowski_mousley_shah_yuan_winklbauer_campos_trettin_quinones_smirnova_et al._2011, title={Resurrection of a functional phosphatidylinositol transfer protein from a pseudo-Sec14 scaffold by directed evolution}, volume={22}, ISSN={["1939-4586"]}, DOI={10.1091/mbc.e10-11-0903}, abstractNote={Sec14-superfamily proteins integrate the lipid metabolome with phosphoinositide synthesis and signaling via primed presentation of phosphatidylinositol (PtdIns) to PtdIns kinases. Sec14 action as a PtdIns-presentation scaffold requires heterotypic exchange of phosphatidylcholine (PtdCho) for PtdIns, or vice versa, in a poorly understood progression of regulated conformational transitions. We identify mutations that confer Sec14-like activities to a functionally inert pseudo-Sec14 (Sfh1), which seemingly conserves all of the structural requirements for Sec14 function. Unexpectedly, the “activation” phenotype results from alteration of residues conserved between Sfh1 and Sec14. Using biochemical and biophysical, structural, and computational approaches, we find the activation mechanism reconfigures atomic interactions between amino acid side chains and internal water in an unusual hydrophilic microenvironment within the hydrophobic Sfh1 ligand-binding cavity. These altered dynamics reconstitute a functional “gating module” that propagates conformational energy from within the hydrophobic pocket to the helical unit that gates pocket access. The net effect is enhanced rates of phospholipid-cycling into and out of the Sfh1* hydrophobic pocket. Taken together, the directed evolution approach reveals an unexpectedly flexible functional engineering of a Sec14-like PtdIns transfer protein—an engineering invisible to standard bioinformatic, crystallographic, and rational mutagenesis approaches.}, number={6}, journal={MOLECULAR BIOLOGY OF THE CELL}, author={Schaaf, Gabriel and Dynowski, Marek and Mousley, Carl J. and Shah, Sweety D. and Yuan, Peihua and Winklbauer, Eva M. and Campos, Marilia K. F. and Trettin, Kyle and Quinones, Mary-Chely and Smirnova, Tatyana I. and et al.}, year={2011}, month={Mar}, pages={892–905} }
 @article{smirnova_chadwick_bankaitis_schaaf_poluektov_smirnov_2011, title={Role of Electrostatic and Hydrogen Bonding Environment in Sequestering Lipids from Membranes Into the Sec14 Protein Cavity}, volume={100}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/j.bpj.2010.12.3215}, DOI={10.1016/j.bpj.2010.12.3215}, abstractNote={Sec14p is a major yeast phosphatidylinositol/phosophatidylcholine (PtdIns/PtdCho) transfer protein that promotes transfer of PtdIns or PtdCho between lipid bilayers in vitro in energy-independent manner. The exact biophysical mechanism of such a process is unknown at this moment. Here we report on employing an arsenal of advanced spin-labeling EPR methods to probe local electrostatic and hydrogen bonding environment that govern binding of lipids by Sec14 protein. n-doxyl PtdCho (where n=5,7,10,12, and 16 reflects position of nitroxide along the acyl chain) were used as EPR active probes. The local polarity and hydrogen bonding profile inside the lipid binding cavity of Sec14p were assessed from characteristic changes in high field EPR at 130 GHz (D-band). The data indicate that the phospholipid-binding cavity of Sec14p with the likely sequestered water molecules provides a close match for the polarity profile along the bound PtdCho molecule and the one determined for membrane bilayers. This polarity match rationalizes the efficient energy-independent partitioning of a lipid molecule from a bilayer into the Sec14p phospholipid-binding pocket. Further, we have developed a direct method for observing formation of a hydrogen bond between sequestered water molecules and a spin-labeled site by applying pulsed Hyperfine Sub-level Correlation (HYSCORE) spectroscopy. Funded by: NSF-0843632 to TIS and NIH 1R01GM072897 to AIS.}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Smirnova, Tatyana I. and Chadwick, Thomas G. and Bankaitis, Vytas A. and Schaaf, Gabriel and Poluektov, Oleg G. and Smirnov, Alex I.}, year={2011}, month={Feb}, pages={552a–553a} }
 @article{voinov_pagán jason o. sosa_morrison_smirnova_smirnov_2011, title={Surface-Mediated Production of Hydroxyl Radicals as a Mechanism of Iron Oxide Nanoparticle Biotoxicity}, volume={133}, ISSN={0002-7863 1520-5126}, url={http://dx.doi.org/10.1021/ja104683w}, DOI={10.1021/ja104683w}, abstractNote={Emerging applications of nanosized iron oxides in nanotechnology introduce vast quantities of nanomaterials into the human environment, thus raising some concerns. Here we report that the surface of γ-Fe(2)O(3) nanoparticles 20-40 nm in diameter mediates production of highly reactive hydroxyl radicals (OH(•)) under conditions of the biologically relevant superoxide-driven Fenton reaction. By conducting comparative spin-trapping EPR experiments, we show that the free radical production is attributed primarily to the catalytic reactions at the nanoparticles' surface rather than being caused by the dissolved metal ions released by the nanoparticles as previously thought. Moreover, the catalytic centers on the nanoparticle surface were found to be at least 50-fold more effective in OH(•) radical production than the dissolved Fe(3+) ions. Conventional surface modification methods such as passivating the nanoparticles' surface with up to 935 molecules of oleate or up to 18 molecules of bovine serum albumin per iron oxide core were found to be rather ineffective in suppressing production of the hydroxyl radicals. The experimental protocols developed in this study could be used as one of the approaches for developing analytical assays for assessing the free radical generating activity of a variety of nanomaterials that is potentially related to their biotoxicity.}, number={1}, journal={Journal of the American Chemical Society}, publisher={American Chemical Society (ACS)}, author={Voinov, Maxim A. and Pagán Jason O. Sosa and Morrison, Erin and Smirnova, Tatyana I. and Smirnov, Alex I.}, year={2011}, month={Jan}, pages={35–41} }
 @article{smirnova_voynov_poluektov_smirnov_2010, title={Heterogeneous Dielectric and Hydrogen Bonding Environment of Transmembrane Peptides}, volume={98}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/j.bpj.2009.12.494}, DOI={10.1016/j.bpj.2009.12.494}, abstractNote={A complex set of hydrogen bonding and hydrophobic interactions between the protein side chains and cellular membrane components is primary responsible for such important biophysical processes as initial protein binding/docking to cellular membranes, membrane insertion, folding, and the final adaptation of the correct transmembrane position. Although general concepts of membrane protein folding and thermodynamic stability are beginning to emerge, the experimental data on the transmembrane profile of the effective dielectric constant and the local hydrogen bond network formed by membrane protein side chains remain severely limited. Here we describe the use of an arsenal of modern spin-labeling EPR methods to profile heterogeneous dielectric and hydrogen bonding environment along a series of the alpha-helical chain of the alanine-rich WALP peptide that adopts a transmembrane orientation. Firstly, we have employed a recently described pH-sensitive cysteine-specific spin-label IMSTL (methanethiosulfonic acid S-(1-oxyl-2,2,3,5,5-pentamethylimidazolidin-4-ylmethyl) ester) to label a series of WALP cysteine mutants. EPR titrations of such peptides reconstituted into anionic lipid bilayers yield the magnitude of relative changes in the effective dielectric constant across the bilayer in the vicinity of the peptide alpha-helix. Secondly, perdeuterated and 15N-substituted nitroxides in combination with High Field EPR at 130 GHz (D-band) were used to assess local polarity and formation of hydrogen bonds for the same series of spin-labeled WALP mutants. Finally, the nature of the hydrogen bonds observed by EPR was ascertained by a series of HYSCORE X-band measurements. It was concluded that such combination of EPR techniques significantly expands the capabilities of spin-labeling methods in studies of membrane proteins as demonstrated by deriving profiles of heterogeneous dielectric and hydrogen bonding environment along a typical transmembrane alpha-helix. Supported by NSF-0843632 to TIS and NIH 1R01GM072897 to AIS.}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Smirnova, Tatyana I. and Voynov, Maxim A. and Poluektov, Oleg G. and Smirnov, Alex I.}, year={2010}, month={Jan}, pages={87a} }
 @article{d’antonio_d’antonio_thompson_bowden_franzen_smirnova_ghiladi_2010, title={Spectroscopic and Mechanistic Investigations of Dehaloperoxidase B fromAmphitrite ornata}, volume={49}, ISSN={0006-2960 1520-4995}, url={http://dx.doi.org/10.1021/bi100407v}, DOI={10.1021/bi100407v}, abstractNote={Dehaloperoxidase (DHP) from the terebellid polychaete Amphitrite ornata is a bifunctional enzyme that possesses both hemoglobin and peroxidase activities. Of the two DHP isoenzymes identified to date, much of the recent focus has been on DHP A, whereas very little is known pertaining to the activity, substrate specificity, mechanism of function, or spectroscopic properties of DHP B. Herein, we report the recombinant expression and purification of DHP B, as well as the details of our investigations into its catalytic cycle using biochemical assays, stopped-flow UV-visible, resonance Raman, and rapid freeze-quench electron paramagnetic resonance spectroscopies, and spectroelectrochemistry. Our experimental design reveals mechanistic insights and kinetic descriptions of the dehaloperoxidase mechanism which have not been previously reported for isoenzyme A. Namely, we demonstrate a novel reaction pathway in which the products of the oxidative dehalogenation of trihalophenols (dihaloquinones) are themselves capable of inducing formation of oxyferrous DHP B, and an updated catalytic cycle for DHP is proposed. We further demonstrate that, unlike the traditional monofunctional peroxidases, the oxyferrous state in DHP is a peroxidase-competent starting species, which suggests that the ferric oxidation state may not be an obligatory starting point for the enzyme. The data presented herein provide a link between the peroxidase and oxygen transport activities which furthers our understanding of how this bifunctional enzyme is able to unite its two inherent functions in one system.}, number={31}, journal={Biochemistry}, publisher={American Chemical Society (ACS)}, author={D’Antonio, Jennifer and D’Antonio, Edward L. and Thompson, Matthew K. and Bowden, Edmond F. and Franzen, Stefan and Smirnova, Tatyana and Ghiladi, Reza A.}, year={2010}, month={Aug}, pages={6600–6616} }
 @article{feducia_dumarieh_gilvey_smirnova_franzen_ghiladi_2009, title={Characterization of Dehaloperoxidase Compound ES and Its Reactivity with Trihalophenols†}, volume={48}, ISSN={0006-2960 1520-4995}, url={http://dx.doi.org/10.1021/bi801916j}, DOI={10.1021/bi801916j}, abstractNote={Dehaloperoxidase (DHP), the oxygen transport hemoglobin from the terebellid polychaete Amphitrite ornata, is the first globin identified to possess a biologically relevant peroxidase activity. DHP has been shown to oxidize trihalophenols to dihaloquinones in a dehalogenation reaction that uses hydrogen peroxide as a substrate. Herein, we demonstrate that the first detectable intermediate following the addition of hydrogen peroxide to ferric DHP contains both a ferryl heme and a tyrosyl radical, analogous to Compound ES of cytochrome c peroxidase. Furthermore, we provide a detailed kinetic description for the reaction of preformed DHP Compound ES with the substrate 2,4,6-trichlorophenol and demonstrate the catalytic competency of this intermediate in generating the product 2,4-dichloroquinone. Using rapid-freeze-quench electron paramagnetic resonance spectroscopy, we detected a g ≈ 2.0058 signal confirming the presence of a protein radical in DHP Compound ES. In the absence of substrate, DHP Compound ES evolves to a new species, Compound RH, which is functionally unique to dehaloperoxidase. We propose that this intermediate plays a protective role against heme bleaching. While unreactive toward further oxidation, Compound RH can be reduced and subsequently bind dioxygen, generating oxyferrous DHP, which may represent the catalytic link between peroxidase and oxygen transport activities in this bifunctional protein.}, number={5}, journal={Biochemistry}, publisher={American Chemical Society (ACS)}, author={Feducia, Jeremiah and Dumarieh, Rania and Gilvey, Lauren B. G. and Smirnova, Tatyana and Franzen, Stefan and Ghiladi, Reza A.}, year={2009}, month={Feb}, pages={995–1005} }
 @article{chadwick_rabah_davies_smirnova_2009, title={Membrane insertion of peptides mimicking E2 domain of Sindbis virus is modulated by cholesterol}, volume={96}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/j.bpj.2008.12.2910}, DOI={10.1016/j.bpj.2008.12.2910}, abstractNote={In the process of assembly Sindbis enveloped virus uses a host-derived membrane bilayer that is “sandwiched” between the concentric protein shells. The transmembrane domains of three glycoproteins penetrate the bilayer and are capable of assembling in two strikingly different membranes: mammalian membranes that contain up to 40% of cholesterol and insects membranes that contain larger fraction of shorter unsaturated lipids and no cholesterol. Recently, it was shown that mutations in the transmembrane domain of the Sindbis virus E2 protein produce deferential alterations in the protein association with the lipid bilayer: some mutants were able to grow in insect cells, but not in mammalian cells [1,2]. The Sindbis virus with STM-16 deletion mutation of the E2 transmembrane domain shows the most pronounced differential growth in mammal and insect cells while STM-18 shows almost wild-type behaviour. We have investigated the interaction of synthetic peptides mimicking E2 domain mutants with lipid bilayers with the goal to understand constraints placed upon membrane spanning domains for correct integration into the bilayer. The phospholipid composition was chosen to represent mammalian and insects' membranes. Results of EPR spin-labeling experiments show that both STM-16 and STM-18 peptides adopt a transmembrane configuration in bilayers with lipid composition mimicking that of insects. In mammalian cell mimicking membranes and containing cholesterol the STM-16 peptide aggregates at the surface of the bilayer. Both peptides exhibit transmembrane orientation in bilayers consisting of “mammalian” lipid mixture but without cholesterol. Thus, we show that cholesterol content of the lipid mixture modulates insertion of the peptides into bilayer mimicking mammalian cell membrane. Supported by NSF grant MCB-0451510 to TIS.[1] Hernandez, R., et. al. J Virol 2003 77(23), 12710-9.[2] West, J., et. al. J. Virol., 2006 80:4458-4468.}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Chadwick, Thomas G. and Rabah, Ghada A. and Davies, Brian R. and Smirnova, Tatyana I.}, year={2009}, month={Feb}, pages={389a–390a} }
 @article{ghiladi_dumarieh_thompson_wang_smirnova_franzen_2009, title={Spectroscopic Probes of the Reactive Intermediates of Dehaloperoxidase from Amphitrite ornata}, volume={96}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/j.bpj.2008.12.2240}, DOI={10.1016/j.bpj.2008.12.2240}, abstractNote={The enzyme intermediates of dehaloperoxidase (DHP) from the marine worm Amphitrite ornata are unique within both the globin and cytochrome c peroxidase superfamilies. DHP has been shown to oxidize trihalophenols to dihaloquinones in a dehalogenation reaction that uses hydrogen peroxide as a substrate. We show that the initially formed heme intermediate in this reaction is not Compound I as is often the case in peroxidases, but rather is a combination of Compound II and a tyrosyl radical that has similarity to the Compound ES intermediate of cytochrome c peroxidase. Using stopped-flow UV-visible spectroscopy, we provide a detailed kinetic description for the reaction of pre-formed DHP Compound II and tyrosyl radical with the substrate 2,4,6-trichlorophenol, and demonstrate the catalytic competency of this intermediate in generating the product 2,4-dichloroquinone. Furthermore, using rapid-freeze-quench electron paramagnetic resonance spectroscopy, we detected a signal at g ≈ 2.0058, confirming the presence of a protein radical in DHP Compound II, and assign it as a tyrosyl radical based upon mutagenesis studies and structural arguments. In the absence of a halophenol substrate, the DHP Compound II + tyrosyl radical intermediate decomposes to a new and significantly less active species, termed Compound RH, which is unique to dehaloperoxidase. We propose that this intermediate plays a protective role against heme bleaching. While unreactive toward further oxidation, Compound RH can be reduced and subsequently bind dioxygen, generating oxyferrous DHP, which may represent the catalytic link between the peroxidase and oxygen-transport activities in this bifunctional protein.}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Ghiladi, Reza A. and Dumarieh, Rania and Thompson, Matthew and Wang, Zao and Smirnova, Tatyana and Franzen, Stefan}, year={2009}, month={Feb}, pages={437a} }
 @article{smirnova_davis_weber_franzen_2009, title={Substrate binding triggers a switch in the iron coordination in dehaloperoxidase from Amphitrite Ornate}, volume={96}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/j.bpj.2008.12.2241}, DOI={10.1016/j.bpj.2008.12.2241}, abstractNote={We have explored the effect of substrate binding on the heme iron conformation in the enzyme dehaloperoxidase (DHP). DHP is a dimeric hemoglobin that also has significant peroxidase activity under physiological conditions and has been shown to oxidize trihalophenols to dihaloquinones in a dehalogenation reaction that uses hydrogen peroxide as a cosubstrate. Hyperfine sublevel correlation spectroscopic (HYSCORE) analysis of the ferric form of DHP was carried out to characterize effects of the substrate 2,4,6-trifluorophenol (TFP) binding on the iron coordination in order to elucidate molecular mechanisms responsible for switching the protein function from a globin to a peroxidase. The CW EPR spectrum shows that at pH 6.0 DHP heme iron exists in a highly axial high spin (HS) state that could be interpreted as arising from two different populations of the HS iron centers. Substrate binding does not change the spin state at pH 6.0, however, affects the magnetic parameters of the signal. HYSCORE spectra recorded at magnetic field corresponding to g = 2 revealed the presence of exchangeable protons with hyperfine coupling of ca. 6 MHz, consistent with a water molecule being the sixth ligand in the iron coordination. These protons' spectral features disappeared upon substrate binding. At pH 9.6 the EPR spectrum from heme iron of DHP shows the presence of both high- and low-spin states with the low spin signal characteristic of hydroxyl form. Upon TFP binding the low spin signal disappears. HYSCORE spectra at pH 9.6 also show the presence of exchangeable protons that disappear upon substrate binding. This observation highlights the proposed role of molecules in the distal pocket to control the peroxidase function of DHP.Supported by the NSF Grant MCB-0451510 to T.I.S. and ARO grant 52278-LS to S.F.}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Smirnova, Tatyana I. and Davis, Mike F. and Weber, Ralph T. and Franzen, Stefan}, year={2009}, month={Feb}, pages={437a} }
 @article{smirnova_smirnov_chadwick_walker_2008, title={Characterization of magnetic and electronic properties of trimetallic nitride endohedral fullerenes by SQUID magnetometry and electron paramagnetic resonance}, volume={453}, ISSN={["1873-4448"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-44549085132&partnerID=MN8TOARS}, DOI={10.1016/j.cplett.2008.01.036}, abstractNote={Magnetic and electronic properties of high-purity trimetallic nitride endohedral fullerenes Er3[email protected]80, Lu3[email protected]80, Sc3[email protected]80 diluted in a diamagnetic host were characterized by SQUID DC magnetometry and electron paramagnetic resonance (EPR) at multiple frequencies. The Er3[email protected]80 sample followed the Curie–Weiss law with negligible Weiss temperature of 0.16 K. Based on temperature dependence and isothermal saturation magnetization the effective magnetic moment of Er3[email protected]80 was estimated as 10.2 μB. The magnetic behavior of these fullerenes is ascribed to quenching of the orbital moment of the metal ions due to interactions between the metal centers with the nitrogen and the fullerene cage.}, number={4-6}, journal={CHEMICAL PHYSICS LETTERS}, publisher={Elsevier BV}, author={Smirnova, Tatyana I. and Smirnov, Alex I. and Chadwick, Thomas G. and Walker, Kenneth L.}, year={2008}, month={Mar}, pages={233–237} }
 @article{smirnova_weber_davis_franzen_2008, title={Substrate binding triggers a switch in the iron coordination in dehaloperoxidase from Amphitrite ornata: HYSCORE experiments}, volume={130}, ISSN={["0002-7863"]}, DOI={10.1021/ja0772952}, abstractNote={Dehaloperoxidase (DHP) from the terebellid polychaete Amphitrite ornata is the first known hemoglobin to exhibit efficient peroxidase activity in the oxidation of phenolic substrates. Hyperfine sublevel correlation spectroscopic (HYSCORE) analysis of the ferric form of DHP was carried out to characterize effects of the substrate 2,4,6-trifluorophenol (TFP) binding on the iron coordination in order to elucidate the molecular mechanism of the change in protein function from a globin to a peroxidase. Continuous wave EPR spectra show that heme iron of DHP at pH 6.0 exists in the high spin state. HYSCORE spectra recorded at magnetic field corresponding to g = 2 revealed the presence of exchangeable protons with hyperfine coupling of ca. 6 MHz, consistent with a water molecule being the sixth ligand in the iron coordination. These protons' spectral features disappeared upon substrate binding. This observation highlights the proposed role of the substrate as a trigger for the switch from hemoglobin to peroxidase function.}, number={7}, journal={JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, author={Smirnova, Tatyana I. and Weber, Ralph T. and Davis, Mike F. and Franzen, Stefan}, year={2008}, month={Feb}, pages={2128-+} }
 @article{smirnova_2007, title={Binding of MRI contrast agents to albumin: a high-field EPR study}, volume={31}, ISSN={["1613-7507"]}, DOI={10.1007/BF03166594}, number={3-4}, journal={APPLIED MAGNETIC RESONANCE}, author={Smirnova, T. I.}, year={2007}, pages={431–446} }
 @article{smirnova_smirnov_paschenko_poluektov_2007, title={Geometry of hydrogen bonds formed by lipid bilayer nitroxide probes: A high-frequency pulsed ENDOR/EPR study}, volume={129}, ISSN={["1520-5126"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-33947673323&partnerID=MN8TOARS}, DOI={10.1021/ja068395v}, abstractNote={Solvent effects on magnetic parameters of nitroxide spin labels in combination with side-directed spin-labeling EPR methods provide very useful means for elucidating polarity profiles in lipid bilayers and mapping local electrostatic effects in complex biomolecular systems. One major contributor to these solvent effects is the hydrogen bonds that could be formed between the nitroxide moiety and water and/or the available hydroxyl groups. Here, formation of hydrogen bonds between a lipid bilayer spin probe5-doxyl stearic acid, 5DSAand hydrogen-bond donors has been studied using high-frequency (HF) pulsed ENDOR and EPR. A hydrogen-bonded deuteron was directly detected in HF ENDOR (130 GHz) spectra of 5DSA dissolved in several deuterated alcohols, while the characteristic signal was absent in nonpolar toluene-d8. The length of the hydrogen bond, 1.74 ± 0.06 Å, and its geometry were found to be essentially the same for all four alcohols studied, indicating that nearly identical hydrogen bonds have been formed regardless of the solvent dielectric constant. This strengthens a hypothesis that HF EPR spectra are exclusively sensitive to formation of hydrogen bonds and could be used for probing the hydrogen-bond network in complex biomolecular assemblies and lipid bilayers with site-directed spin-labeling methods.}, number={12}, journal={JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, publisher={American Chemical Society (ACS)}, author={Smirnova, Tatyana I. and Smirnov, Alex I. and Paschenko, Serguei V. and Poluektov, Oleg G.}, year={2007}, month={Mar}, pages={3476-+} }
 @article{smirnova_chadwick_voinov_poluektov_tol_ozarowski_schaaf_ryan_bankaitis_2007, title={Local polarity and hydrogen bonding inside the Sec14p phospholipid-binding cavity: High-field multi-frequency electron paramagnetic resonance studies}, volume={92}, ISSN={["1542-0086"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-34247863841&partnerID=MN8TOARS}, DOI={10.1529/biophysj.106.097899}, abstractNote={Sec14p promotes the energy-independent transfer of either phosphatidylinositol (PtdIns) or phosphatidylcholine (PtdCho) between lipid bilayers in vitro and represents the major PtdIns/PtdCho transfer protein in the budding yeast Saccharomyces cerevisiae. Herein, we employ multi-frequency high-field electron paramagnetic resonance (EPR) to analyze the electrostatic and hydrogen-bonding microenvironments for series of doxyl-labeled PtdCho molecules bound by Sec14p in a soluble protein-PtdCho complex. A structurally similar compound, 5-doxyl stearic acid dissolved in a series of solvents, was used for experimental calibration. The experiments yielded two-component rigid limit 130- and 220-GHz EPR spectra with excellent resolution in the gx region. Those components were assigned to hydrogen-bonded and nonhydrogen-bonded nitroxide species. Partially resolved 130-GHz EPR spectra from n-doxyl-PtdCho bound to Sec14p were analyzed using this two-component model and allowed quantification of two parameters. First, the fraction of hydrogen-bonded nitroxide species for each n-doxyl-PtdCho was calculated. Second, the proticity profile along the phospholipid-binding cavity of Sec14p was characterized. The data suggest the polarity gradient inside the Sec14p cavity is a significant contributor to the driving molecular forces for extracting a phospholipid from the bilayer. Finally, the enhanced g-factor resolution of EPR at 130 and 220 GHz provides researchers with a spectroscopic tool to deconvolute two major contributions to the x-component of the nitroxide g-matrix: hydrogen-bond formation and local electrostatic effects.}, number={10}, journal={BIOPHYSICAL JOURNAL}, author={Smirnova, Tatyana I. and Chadwick, Thomas G. and Voinov, Maxim A. and Poluektov, Oleg and Tol, Johan and Ozarowski, Andrzej and Schaaf, Gabriel and Ryan, Margaret M. and Bankaitis, Vytas A.}, year={2007}, month={May}, pages={3686–3695} }
 @article{smirnov_smirnova_macarthur_good_hall_2006, title={Cryogen-free superconducting magnet system for multifrequency electron paramagnetic resonance up to 12.1T}, volume={77}, ISSN={0034-6748 1089-7623}, url={http://dx.doi.org/10.1063/1.2182571}, DOI={10.1063/1.2182571}, abstractNote={Multifrequency and high field/high frequency (HF) electron paramagnetic resonance (EPR) is a powerful spectroscopy for studying paramagnetic spin systems ranging from organic-free radicals to catalytic paramagnetic metal ion centers in metalloproteins. Typically, HF EPR experiments are carried out at resonant frequencies ν=95–300GHz and this requires magnetic fields of 3.4–10.7T for electronic spins with g≈2.0. Such fields could be easily achieved with superconducting magnets, but, unlike NMR, these magnets cannot operate in a persistent mode in order to satisfy a wide range of resonant fields required by the experiment. Operating and maintaining conventional passively cooled superconducting magnets in EPR laboratories require frequent transfer of cryogens by trained personnel. Here we describe and characterize a versatile cryogen-free magnet system for HF EPR at magnetic fields up to 12.1T that is suitable for ramping the magnetic field over the entire range, precision scans around the target field, and/or holding the field at the target value. We also demonstrate that in a nonpersistent mode of operation the magnetic field can be stabilized to better than 0.3ppm∕h over 15h period by employing a transducer-controlled power supply. Such stability is sufficient for many HF EPR experiments. An important feature of the system is that it is virtually maintenance-free because it is based on a cryogen-free technology and therefore does not require any liquid cryogens (liquid helium or nitrogen) for operation. We believe that actively cooled superconducting magnets are ideally suited for a wide range of HF EPR experiments including studies of spin-labeled nucleic acids and proteins, single-molecule magnets, and metalloproteins.}, number={3}, journal={Review of Scientific Instruments}, publisher={AIP Publishing}, author={Smirnov, Alex I. and Smirnova, Tatyana I. and MacArthur, Ryan L. and Good, Jeremy A. and Hall, Renny}, year={2006}, month={Mar}, pages={035108} }
 @article{smirnova_chadwick_macarthur_poluektov_song_ryan_schaaf_bankaitis_2006, title={The chemistry of phospholipid binding by the Saccharomyces cerevisiae phosphatidylinositol transfer protein Sec14p as determined by EPR spectroscopy}, volume={281}, ISSN={["1083-351X"]}, DOI={10.1074/jbc.M603054200}, abstractNote={The major yeast phosphatidylinositol/phosphatidylcholine transfer protein Sec14p is the founding member of a large eukaryotic protein superfamily. Functional analyses indicate Sec14p integrates phospholipid metabolism with the membrane trafficking activity of yeast Golgi membranes. In this regard, the ability of Sec14p to rapidly exchange bound phospholipid with phospholipid monomers that reside in stable membrane bilayers is considered to be important for Sec14p function in cells. How Sec14p-like proteins bind phospholipids remains unclear. Herein, we describe the application of EPR spectroscopy to probe the local dynamics and the electrostatic microenvironment of phosphatidylcholine (PtdCho) bound by Sec14p in a soluble protein-PtdCho complex. We demonstrate that PtdCho movement within the Sec14p binding pocket is both anisotropic and highly restricted and that the C5 region of the sn-2 acyl chain of bound PtdCho is highly shielded from solvent, whereas the distal region of that same acyl chain is more accessible. Finally, high field EPR reports on a heterogeneous polarity profile experienced by a phospholipid bound to Sec14p. Taken together, the data suggest a headgroup-out orientation of Sec14p-bound PtdCho. The data further suggest that the Sec14p phospholipid binding pocket provides a polarity gradient that we propose is a primary thermodynamic factor that powers the ability of Sec14p to abstract a phospholipid from a membrane bilayer. The major yeast phosphatidylinositol/phosphatidylcholine transfer protein Sec14p is the founding member of a large eukaryotic protein superfamily. Functional analyses indicate Sec14p integrates phospholipid metabolism with the membrane trafficking activity of yeast Golgi membranes. In this regard, the ability of Sec14p to rapidly exchange bound phospholipid with phospholipid monomers that reside in stable membrane bilayers is considered to be important for Sec14p function in cells. How Sec14p-like proteins bind phospholipids remains unclear. Herein, we describe the application of EPR spectroscopy to probe the local dynamics and the electrostatic microenvironment of phosphatidylcholine (PtdCho) bound by Sec14p in a soluble protein-PtdCho complex. We demonstrate that PtdCho movement within the Sec14p binding pocket is both anisotropic and highly restricted and that the C5 region of the sn-2 acyl chain of bound PtdCho is highly shielded from solvent, whereas the distal region of that same acyl chain is more accessible. Finally, high field EPR reports on a heterogeneous polarity profile experienced by a phospholipid bound to Sec14p. Taken together, the data suggest a headgroup-out orientation of Sec14p-bound PtdCho. The data further suggest that the Sec14p phospholipid binding pocket provides a polarity gradient that we propose is a primary thermodynamic factor that powers the ability of Sec14p to abstract a phospholipid from a membrane bilayer. Phosphatidylinositol transfer proteins (PITPs) 5The abbreviations used are: PITP, phosphatidylinositol transfer protein; PtdIns, 1,2-dioleoyl-sn-glycero-3-phosphoinositol (ammonium salt); 10-doxyl-PtdCho, 1-acyl-2-(10-(doxyl)stearoyl)-sn-glycero-3-phosphocholine; 16-doxyl-PtdCho, 1-acyl-2-(16-(doxyl)stearoyl)-sn-glycero-3-phosphocholine. 5The abbreviations used are: PITP, phosphatidylinositol transfer protein; PtdIns, 1,2-dioleoyl-sn-glycero-3-phosphoinositol (ammonium salt); 10-doxyl-PtdCho, 1-acyl-2-(10-(doxyl)stearoyl)-sn-glycero-3-phosphocholine; 16-doxyl-PtdCho, 1-acyl-2-(16-(doxyl)stearoyl)-sn-glycero-3-phosphocholine. represent a subset of a larger group of phospholipid transfer proteins that are operationally defined by their ability to mobilize phospholipids between membrane bilayers in vitro. PITPs catalyze such energy-independent transfer of either phosphatidylinositol (PtdIns) or phosphatidylcholine (PtdCho) between membrane bilayers in vitro, with PtdIns representing the preferred ligand in the transfer reactions (1Cleves A.E. McGee T.P. Bankaitis V.A. Trends Cell Biol. 1991; 1: 30-34Abstract Full Text PDF PubMed Scopus (134) Google Scholar, 2Phillips S.E. Vincent P. Rizzieri K. Schaaf G. Gaucher E.A. Bankaitis V.A. Crit. Rev. Biochem. Mol. Biol. 2006; 41: 1-28Crossref Google Scholar). PITPs themselves are highly conserved across the eukaryotic kingdom and fall into two distinct classes based upon primary sequence and structural fold (reviewed in Ref. 2Phillips S.E. Vincent P. Rizzieri K. Schaaf G. Gaucher E.A. Bankaitis V.A. Crit. Rev. Biochem. Mol. Biol. 2006; 41: 1-28Crossref Google Scholar). These two classes are defined as the Sec14p-like PITPs and the unrelated metazoan PITPs. Deficiencies in metazoan PITP functions are either cell-lethal in mammals or, in the case of PITP-α isoform, result in neurodegenerative, glucose homeostatic, and intestinal malabsorbtion diseases in mice (3Alb J.G. Cortese Jr., J.D. Phillips S.E. Albin R.L. Nagy T.R. Hamilton B.A. Bankaitis V.A. J. Biol. Chem. 2003; 278: 33501-33518Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Sec14p domain proteins are also of medical interest, since inherited disorders, such as vitamin E-responsive ataxia, ataxia/dystonia in the jittery mouse, etc., are associated with individual defects in them (4Yokota T. Igarashi K. Uchihara T. Jishage K. Tomita H. Inaba A. Li Y. Arita M. Suzuki H. Mizusawa H. Arai H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 15185-15190Crossref PubMed Scopus (221) Google Scholar, 5Bomar J.M. Benke P.J. Slattery E.L. Puttagunta R. Taylor L.P. Seong E. Nystuen A. Chen W. Albin R.L. Patel P.D. Kittles R.A. Sheffield V.C. Burmeister M. Nat. Genet. 2003; 35: 264-269Crossref PubMed Scopus (119) Google Scholar). Moreover, the developmental switch from yeast to mycelial growth modes, a morphogenetic program whose execution is essential for the pathogenesis of dimorphic yeast, is also regulated by Sec14p isoforms (6Carmen-Lopez M. Nicaud J.-M. Skinner H.B. Vergnolle C. Kader J.C. Bankaitis V.A. Gaillardin C. J. Cell Biol. 1994; 124: 113-127Crossref Scopus (90) Google Scholar, 7Monteoliva L. Sanchez M. Pla J. Gil C. Nombela C. Yeast. 1996; 12: 1097-1105Crossref PubMed Scopus (37) Google Scholar). The preponderance of evidence indicates that PITPs integrate the action of specific phospholipid metabolic reactions with regulation of specific membrane trafficking processes.The largest and most widely distributed PITP class is represented by the Sec14p-like proteins, so named on the basis of the founding member of the family, the yeast Sec14p PITP. The Sec14p-like PITP class consists of nearly 300 members, and it is appropriate to consider this large group of proteins as the Sec14p superfamily (2Phillips S.E. Vincent P. Rizzieri K. Schaaf G. Gaucher E.A. Bankaitis V.A. Crit. Rev. Biochem. Mol. Biol. 2006; 41: 1-28Crossref Google Scholar). The Sec14p lipid binding domain is employed in a binding strategy for a diverse set of hydrophobic ligands (e.g. phospholipids, phosphoinositides, sterol precursors, retinal, and tocopherols) and can be expressed either as a free standing domain or as one that is integrated into multidomain modules. The simple budding yeast S. cerevisiae expresses six Sec14ps, and this protein class is more highly expanded in higher eukaryotes (e.g. at least 31 members in Arabidopsis) (8Vincent P. Chua M. Nogue F. Fairbrother A. Mekheel H. Xu Y. Allen N. Bibikova T.N. Gilroy S. Bankaitis V.A. J. Cell Biol. 2005; 168: 801-812Crossref PubMed Scopus (167) Google Scholar).The available Sec14p crystal structure for Sec14p identifies a novel fold that forms a hydrophobic pocket of sufficient volume to accommodate a single molecule of PtdIns or PtdCho (Fig. 1) (9Sha B. Phillips S.E. Bankaitis V.A. Luo M. Nature. 1998; 391: 506-510Crossref PubMed Scopus (228) Google Scholar). The crystallizing unit is an apo-Sec14p loaded with two molecules of the detergent β-octyl glucoside. This particular structure is interpreted to describe a transitional Sec14p conformer that exists only transiently on the membrane surface as Sec14p undergoes phospholipid exchange. Although an understanding of how Sec14p functions at the molecular level is incomplete, the structural model spawned several hypotheses regarding intramolecular dynamic aspects of Sec14p function (9Sha B. Phillips S.E. Bankaitis V.A. Luo M. Nature. 1998; 391: 506-510Crossref PubMed Scopus (228) Google Scholar). First, an unusual surface helix (A10/T4) is identified as an attractive candidate for the structural element that “gates” the hydrophobic pocket (Fig. 1A). Second, a string motif that contains a series of four tightly wound 310 helices wraps up the Sec14p fold and is suggested to play an important role in regulating conformational changes that accompany the phospholipid exchange reaction (Fig. 1B).How Sec14p actually binds its phospholipid substrates remains an important and unanswered question. The available model provides considerable insight into how Sec14p might bind target membranes and individual phospholipids. With regard to substrate binding in the soluble Sec14p-phospholipid complex, bound phospholipid is predicted to orient with the acyl-chains packed into the hydrophobic interior of the pocket, whereas the phospholipid headgroup is disposed toward solvent. Although this hypothetical “headgroup-out” disposition of the bound phospholipid is both an attractive and an intuitive one, solution of a phospholipid-bound Sec14p structure is required to resolve this question, and no such information is yet available. Resolution of this issue is important, since it speaks to one of several potential mechanisms for how Sec14p (and Sec14p-like proteins) may regulate phospholipid metabolism. The uncertainty surrounding whether the intuitive orientation of phospholipid within the Sec14p pocket is indeed the correct one is emphasized by structural analyses of phospholipid-bound metazoan PITPs. These proteins bind phospholipid substrates in the reverse “headgroup-in” orientation (10Yoder M.D. Thomas L.M. Tremblay J.M. Oliver R.L. Yarbrough L.R. Helmkamp Jr., G.M. J. Biol. Chem. 2001; 276: 9246-9252Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 11Tilley S.J. Skippen A. Murray-Rust J. Swigart P.M. Stewart A. Morgan C.P. Cockcroft S. McDonald N.Q. Structure. 2004; 12: 317-326Abstract Full Text PDF PubMed Scopus (77) Google Scholar).In this report, we describe our application of EPR spectroscopy to probe the local dynamics and the electrostatic microenvironment of a series of spin-labeled n-doxyl-PtdCho molecules incorporated into the Sec14p phospholipid binding pocket. We demonstrate that motion of PtdCho within the Sec14p binding pocket, while anisotropic and highly restricted, nonetheless exhibits a progressive increase in the mobility of the sn-2 acyl chain as distance from the headgroup/backbone region increases. Moreover, we show that the C5 region of the sn-2 acyl chain is highly shielded from solvent, whereas the distal region of the acyl chain is less so. Finally, we describe evidence for a polar protic microenvironment at position C5 of the sn-2 acyl chain that becomes increasingly aprotic as one moves away from the headgroup/backbone region of the bound phospholipid and returns to a more polar/protic environment at the distal end of the chain. These collective data are most consistent with a headgroup-out orientation of Sec14p-bound PtdCho and further indicate that the Sec14p phospholipid binding pocket provides a hydrophobic matching to accommodate the PtdCho molecule. We propose this polarity gradient is a primary thermodynamic factor that drives the ability of Sec14p to abstract a phospholipid from a membrane bilayer.EXPERIMENTAL PROCEDURESExpression and Purification of Sec14p—Recombinant His6-Sec14p was purified from Escherichia coli essentially as previously described (9Sha B. Phillips S.E. Bankaitis V.A. Luo M. Nature. 1998; 391: 506-510Crossref PubMed Scopus (228) Google Scholar). Briefly, Sec14p expression in E. coli strain KK2186 (Δ(lac-pro) supE thi strA sbcB-15 endA/F′(traD36 lacIQ lacZΔ15)) was driven by a derivative of the pQE31 plasmid (Qiagen, Hilden, Germany) and induced with isopropyl β-thiogalactoside (1 mm final concentration). Bacterial cultures were incubated for an additional 5 h with shaking at 37 °C. Cells were harvested by centrifugation, and the cell pellet was resuspended in ice-cold lysis buffer (50 mm sodium phosphate, pH 7.1, 300 mm NaCl, 1 mm NaN3, 0.2 mm phenylmethylsulfonyl fluoride). Lysozyme was added, and the suspension was incubated at 25 °C for an additional 10 min. Cells were disrupted by collision with 0.1-mm diameter glass beads in a cooled bead beater (Biospec Products, Bartlesville, OK). Cell lysates were subsequently clarified by differential centrifugation series at 1000 × g, 14,000 × g, and 100,000 × g, respectively. The clarified supernatant was applied to Talon™ Sepharose resin (Clontech), the resin was washed extensively with lysis buffer, and bound protein was eluted with a linear imidazole gradient (0–200 mm) reconstituted in lysis buffer. Peak fractions were pooled and dialyzed extensively against lysis buffer at 4 °C, and a second round of affinity purification and dialysis was subsequently performed. Protein purity was monitored by SDS-PAGE and Coomassie Blue staining of gels. Sec14p purity exceeded 95%, and purified protein exhibited robust PtdIns and PtdCho transfer activity.Preparation of EPR Samples—Nitroxide-labeled PtdCho species, 1-acyl-2-(n-(4,4-dimethyloxazolidine-N-oxyl)stearoyl)-sn-glycero-3-phosphocholine (n-doxyl-PtdCho), with the spin label positioned at n = 5, 7, 10, 12, and 16, were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL) (Fig. 2) as chloroform solutions. Organic solvents for calibrating high field EPR spectra for polarity and hydrogen-bonding effects included methanol, isopropyl alcohol, and hexane (Sigma). All solvents were analytical grade or higher and were used as received. Nickel(II) ethylenediamine-N,N′-diacetic acid (NiEDDA) was synthesized as described (12Altenbach C. Greenhalgh D.A. Khorana H.G. Hubbell W.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1667-1671Crossref PubMed Scopus (403) Google Scholar). NiEDDA 200 mm stock solution was prepared in lysis buffer. 5-Doxyl-stearic acid (5-doxyl-SA) was purchased from TCI America (Portland, OR). Solutions of 5-doxyl-SA were typically prepared at concentrations ≤1 mm (i.e. below the critical micellar concentration). A solution of 5-doxyl-PtdCho in isopropyl alcohol was prepared at 0.1 mm concentration.FIGURE 2Structure of lipids and selected spin probes used in this work: PtdCho (a), PtdIns (b), 5-doxyl-PtdCho (c), 10-doxyl-PtdCho (d), 16-doxyl-PtdCho (e).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Preparation of n-Doxyl-PtdCho Dispersions and n-Doxyl-PtdCho Binding to Sec14p—Multilamellar aqueous dispersions of n-doxyl-PtdCho (20% by weight) were prepared as described (13Smirnova T.I. Smirnov A.I. Clarkson R.B. Belford R.L. J. Am. Chem. Soc. 1998; 120: 5060-5072Crossref Scopus (28) Google Scholar). Previous studies have shown that spin-labeled lipids form bilayers when dispersed in water and exhibit similar phase behavior as biological lipids (14Chen S.C. Sturtevant J.M. Conklin K. Gaffney B.J. Biochemistry. 1982; 21: 5096-5101Crossref PubMed Scopus (8) Google Scholar).To load Sec14p with n-doxyl-PtdCho, a 95 μm protein solution was mixed with a small molar excess of 100% spin-labeled multilamellar liposomes and incubated at room temperature for at least 2 h. As an assay for completeness of binding, we monitored binding of n-doxyl-PtdCho to Sec14p by X-band EPR until no further changes in the signal amplitude were observed. This assay relies on our observation that the EPR signal from an isolated spin label typically exhibits much narrower features and is easily distinguished from EPR spectra originating from n-doxylPtdCho liposomes (see “Results”).EPR Spectroscopy and Spectral Analysis—Continuous wave (CW) X-band (9.0–9.5 GHz) EPR spectra were acquired with a Century Series Varian E-109 (Varian Associates, Palo Alto, CA) EPR spectrometer and digitized to 2000 data points/spectrum. Typical spectrometer settings were as follows: microwave power 2 milliwatts, field modulation frequency 100 kHz, and amplitude less than 1 G to avoid overmodulation. Specific ranges of magnetic field scans are indicated in the figures. Variable temperature EPR spectra were obtained using a nitrogen flow system connected to a Varian variable temperature controller. Sample temperature was measured with a VWR International (West Chester, PA) digital thermometer equipped with a stainless steel microprobe positioned in the cavity just above the sample. The VWR thermometer has a resolution of 0.001 °C and accuracy of ±0.05 °C. In all experiments, temperature was controlled better than ±0.5 °C.High field (HF) EPR spectra were acquired with a 130 GHz EPR spectrometer constructed and installed in the Argonne National Laboratory (Argonne, IL). Field-swept echo-detected EPR spectra from spin-labeled samples were recorded at a temperature of 25 K using a two-pulse sequence. Typically, π /2 pulses of 50 ns in length were separated by a 350-ns delay, and the repetition rate was set to 500 Hz. For detecting low temperature rigid limit HF EPR spectra, we employed an echo-detected field-swept mode over the conventional continuous wave scheme for several reasons. First, the pulse detection eliminates signals originating from spin-labeled n-doxyl-PtdCho bilayers (i.e. phospholipids aggregated in a solution and not bound to Sec14p), because spins of the former molecules have much shorter phase memory T2 relaxation time due to strong magnetic interactions with the neighboring spins. Second, low temperature echo-detected HF EPR spectra do not have microwave phase distortions, allowing for more accurate line shape and g-factor analysis.For HF EPR, spin-labeled samples were drawn by capillary action into clear fused quartz tubes (inner diameter = 0.5 mm, outer diameter = 0.6 mm; VitroCom, Mountain Lakes, NJ), sealed with a Critoseal clay (purchased from Fisher), and loaded into precooled cryostat at 25 K. Temperature of the EPR resonator and the sample was controlled by an ITC-4 temperature controller coupled to a flow cryostat (all supplied by Oxford Instruments, Concord, MA).Saturation recovery (SR) pulsed EPR experiments were conducted at 4 °C with a Bruker Biospin (Billerica, MA) X-band ElexSys 680 EPR spectrometer installed at the National High Magnetic Field Laboratory (Tallahassee, FL). The SR measurements were performed using a 0.16-μs pump pulse and an amplifier output of 10 watts. Shot repetition time was 204 μs. Each SR curve was digitized to 1024 points. Data were acquired at the field position corresponding to the maximum EPR intensity (mI = 0 hydrogen hyperfine transition). Reference signal was acquired at the 50-G shift in the lower field. Data on NiEDDA accessibility to 5-doxyl-SA in a water/ethanol mixture were provided by Dr. J. Widomska (National Biomedical EPR Center, Milwaukee, WI).Lorentzian line broadening induced by NiEDDA was measured from CW X-band spectra using a one-parameter model (13Smirnova T.I. Smirnov A.I. Clarkson R.B. Belford R.L. J. Am. Chem. Soc. 1998; 120: 5060-5072Crossref Scopus (28) Google Scholar, 15Smirnov A.I. Belford R.L. J. Magn. Reson. A. 1995; 98: 65-73Crossref Scopus (76) Google Scholar, 16Smirnova T.I. Smirnov A.I. Clarkson R.B. Belford R.L. Magn. Reson. Med. 1995; 33: 801-810Crossref PubMed Scopus (31) Google Scholar). In brief, first derivative EPR spectra measured in the presence of NiEDDA were least squares fitted using the Levenberg-Marquardt algorithm to a convolution of the corresponding experimental spectra recorded in the absence of paramagnetic relaxant and a Lorentzian broadening function. A modification of this approach for elucidating very small broadening effects has been described (17Smirnova T.I. Smirnov A.I. J. Phys. Chem. B. 2003; 107: 7212-7215Crossref Scopus (7) Google Scholar).RESULTSBinding of n-Doxyl-PtdCho to Sec14p—Binding of n-doxyl-PtdCho to Sec14p was monitored from changes in EPR spectra collected at room temperature. Fig. 3A (dots) shows a typical room temperature EPR signal obtained for 5-doxyl-PtdCho liposomes. For all n-doxyl-PtdCho species studied, the signal appeared as a single line of ∼25–27 G peak-to-peak width. Such a spectrum is characteristic of strong dipole-dipole and exchange interactions.FIGURE 3Room temperature X-band CW EPR spectra from 5-doxyl-PtdCho. A, in a form of multilamellar lipid dispersion (field modulation amplitude 8G) (B, solid line) after mixing with 95 μm Sec14p in ∼1:1 molar ratio. Least squares fit of the spectrum (A) to a Lorentzian function is shown as a dashed line (B). C, signal from 5-doxyl-PtdCho incorporated into the Sec14p binding pocket obtained by the spectral subtraction.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The single line EPR spectrum of spin-labeled lipids is well modeled by a Lorentzian function as illustrated for 5-doxyl-PtdCho in Fig. 3A; least squares simulations are shown as a solid line that very closely follows the experimental spectrum. The spectrum in Fig. 3A also demonstrates that the EPR signals from nonaggregated n-doxyl-PtdCho (i.e. n-doxyl-PtdCho resident in the aqueous buffer) are too weak to be detected under these conditions. This is expected, because the critical micelle concentration for n-doxyl-PtdCho is in the nanomolar range, resulting in a concentration of n-doxyl-PtdCho monomers in aqueous buffer that is well below the typical sensitivity of X-band EPR spectrometers.When Sec14p loads with a phospholipid from a membrane bilayer, the bound phospholipid becomes encapsulated in the protein cavity. Such a loading event effectively eliminates spin-spin interactions for the bound n-doxyl-PtdCho and produces significant line shape changes. Fig. 3B (solid line) depicts a typical EPR spectrum observed in experiments where Sec14p was mixed with multilamellar 5-doxyl-PtdCho bilayers and allowed to equilibrate for ∼2 h. A sharp decrease in spin-spin interactions was observed for Sec14p-bound 5-doxyl-PtdCho as reported by the line narrowing. This line narrowing results in a significant (∼6-fold) increase in the peak-to-peak amplitude of the corresponding EPR spectrum as compared with the initial signal from liposomal 5-doxyl-PtdCho. The presence of the signal from aggregated lipids is particularly noticeable from broad wings that extend far away from characteristic features (i.e. outside the dashed lines in Fig. 3B). Thus, the signal from Sec14p-bound 5-doxyl-PtdCho dominates the first derivative EPR spectrum observed in CW experiments, thereby simplifying initial analyses of local spin label dynamics. In time domain experiments, such as saturation recovery and echo-detected field-swept HF EPR, little if any contribution from the liposomal 5-doxyl-PtdCho is recorded, because the latter spin species have much shorter electronic relaxation times. However, for accurate line shape least squares simulation analyses, the contribution from the remaining liposomal 5-doxyl-PtdCho (dashed line) is subtracted from the experimental spectrum (Fig. 3B, solid line), yielding the Sec14p-bound 5-doxyl-PtdCho spectrum (Fig. 3C). In this particular experiment, the fraction of liposomal 5-doxyl-PtdCho molecules was determined to be ∼30%.Local Dynamics of n-Doxyl-PtdCho Incorporated into the Sec14p Phospholipid Binding Pocket—The data described above demonstrate that Sec14p loads with n-doxyl-PtdCho and that the bound n-doxyl-PtdCho becomes sequestered within the protein cavity. Since CW EPR spectra of spin-labeled phospholipids are sensitive to rotational motion of the nitroxide moiety, this method was used to investigate the flexibility of the n-doxyl-PtdCho acyl chain within the Sec14p lipid binding pocket. A series of room temperature (22 °C) CW X-band spectra from n-doxyl-PtdCho (where n = 5, 7, 10, 12, and 16) bound to Sec14p are shown in Fig. 4. For all label positions analyzed, the contribution of liposomal n-doxyl-PtdCho registered only as a broad line most readily observed at the wings. This component was subtracted from experimental spectra and will not be discussed further. Instead, we focus on the dominant EPR spectrum that originates from n-doxyl-PtdCho sequestered within the Sec14p cavity.FIGURE 4Room temperature X-band CW EPR spectra from Sec14p-bound n-doxyl-PtdCho. Spectra are corrected for the background signal as illustrated in Fig. 2 and are intensity-normalized using double integration.View Large Image Figure ViewerDownload Hi-res image Download (PPT)All EPR spectra depicted in Fig. 4 report an intermediate to slow motional regime and anisotropic rotation of the nitroxide spin label. The absence of any abnormal broadening and/or substantial drop in intensity of these spectra indicative of spin-spin interactions is consistent with a Sec14p molecule loading with a single n-doxyl-PtdCho molecule. A particularly informative parameter of such spectra is the peak-to-peak width ΔHp-p (mI = 0) of the central nitrogen hyperfine component (where mI = 0 is the nitrogen spin quantum number). When tumbling of a spin label falls into an intermediate to slow motional regime, this width is approximately proportional to the rotational correlation time τc. In studies of local dynamics of protein side chains, an inverse of this width ΔHp−p−1 (mI=0) is typically reported as a “mobility” parameter (18Mchaourab H.S. Lietzow M.A. Hideg K. Hubbell W.L. Biochemistry. 1996; 35: 7692-7704Crossref PubMed Scopus (528) Google Scholar). This parameter reliably estimates mobility of spin-labeled amino acid side chains in proteins, and a smaller mobility parameter indicates slower rotation and a longer rotational correlation time τc. Other useful parameters for characterizing rotational tumbling of a nitroxide are the effective hyperfine splitting constants Aout and Ain as defined in Fig. 4. A plot of the mobility parameter,ΔHp−p−1 (mI=0), as well as Aout and Ain, all indicate a progressive increase in spin label local mobility as the nitroxide label is moved toward the headgroup-distal end of the sn-2 acyl chain from position C5 to C12 (Fig. 5). At position C16, however, motion of the label becomes more restricted, very much like at position C5. We used the definition of McConnell and Hubbell to calculate the effective order parameter (Seff) to describe the anisotropy of spin label motion (19McConnell H.M. Hubbell W.L. J. Am. Chem. Soc. 1971; 93: 314-326Crossref PubMed Scopus (1427) Google Scholar, 20Griffith O.H. Jost P.C. Berliner L.J. Spin Labeling: Theory and Applications. Academic Press, Inc., New York1976: 454-523Google Scholar),Seff=(A∥−A⊥)A013(A∥+2A⊥)ΔA where A∥ = Aout (i.e. a half of the outer hyperfine splitting), and A⊥ is calculated from Ain, a half of the inner hyperfine splitting expressed in gauss,A⊥=(Ain+0.85) for Sapp<0.45A⊥=Ain+1.32+1.86log(1−Sapp) for Sapp>0.45Sapp=(Aout−Ain)/ΔA where A0 is the isotropic nitrogen hyperfine coupling constant, and ΔA is the maximum extent of the axial nitrogen hyperfine anisotropy (21Schorn K. Marsh D. Spectrochim. Acta A. Mol. Biomol. Spectrosc. 1997; 53: 2235-2240Crossref Scopus (22) Google Scholar, 22Bartucci R. Belsito S. Sportelli L. Chem. Phys. Lipids. 2003; 124: 111-122Crossref PubMed Scopus (11) Google Scholar). Although the values of ΔA and A0 vary by a few percent with the position of the label along the sn-2 acyl chain of Sec14p-bound n-doxyl-PtdCho, we found it convenient to fix A0 and ΔA for all n-doxyl-PtdCho isomers to those values observed for 5-doxyl-PtdCho in isopropyl alcohol. In doing so, we introduced less than 3% error into the Seff calculations. Effective order parameters Seff calculated from room temperature X-band EPR spectra of Sec14p-bound n-doxyl-PtdCho are reported in Fig. 6 and are compared with Seff values measured for n-doxyl-PtdCho in either a fluid dimyristoyl-PtdCho membrane bilayer environment (T = 27 °C) or in a crystalline bilayer environment (T = 5 °C).FIGURE 5Empirical motional characteristics of nitroxide spin label as determined from room temperature T = 22 °C X-band CW EPR spectra of n-doxyl-PtdCho bound to Sec14p as function of the label position along the sn-2 acyl chain. A, Aout; B, Ain; C, mobility parameter,ΔHp−p−1 (mI=0).View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 6Effective order parameters Seff calculated from X-band CW EPR spectra as a function of the label position. Filled circles, n-doxyl-PtdCho bound to Sec14p (T = 27 °C); open circles, Seff for n-doxyl-PtdCho in dimyristoyl-PtdCho bilayers in a crystalline phase (T = 5 °C); open squares, Seff for n-doxyl-PtdCho in dimyristoyl-PtdCho bilayers in fluid phase (T = 27 °C)View Large Image Figure ViewerDownload Hi-res image Download (PPT)Substantial alterations in the label dynamic and order parameters are recorded as a function of the spin label position along the sn-2 acyl chain. Qualitativ}, number={46}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Smirnova, Tatyana I. and Chadwick, Thomas G. and MacArthur, Ryan and Poluektov, Oleg and Song, Likai and Ryan, Margaret M. and Schaaf, Gabriel and Bankaitis, Vytas A.}, year={2006}, month={Nov}, pages={34897–34908} }
 @article{smirnova_smirnov_2003, title={Dynamic molecular oxygen accessibility to a buried Mn2+ protein site: A high-field EPR experiment}, volume={107}, ISSN={["1520-6106"]}, DOI={10.1021/jp0349637}, abstractNote={A high-field (W-band, 3.35 T, 95 GHz) electron paramagnetic resonance (EPR) experiment to measure dynamic molecular oxygen accessibility to manganese(II) ions in liquids is described. The method is based on the direct observation of magnetic interactions between molecular oxygen and the manganese(II) ion in solution. The effect is observed as a Lorentzian broadening of the EPR line. The observation of this effect is facilitated by narrowing the manganese(II) EPR signal at high magnetic field and utilizing elevated oxygen pressures of up to 3 atm. The magnitude of the broadening effect is dependent on both the oxygen permeability of the solvent and the coordination of the manganese(II) ion and is independent of the frequency of the EPR experiment. The latter indicates that Heisenberg spin exchange between the electronic spins of oxygen and manganese(II) during bimolecular collisions is the likely broadening mechanism. The method can be also used to study dynamic molecular oxygen accessibility to manganese(II) sites in biological macromolecules, as demonstrated by an example of a buried site in Concanavalin A lectin. It was found that the oxygen accessibility of this site is significantly smaller (by a factor of 4) than that of an aqua ion and is recovered upon denaturing of the protein.}, number={29}, journal={JOURNAL OF PHYSICAL CHEMISTRY B}, publisher={American Chemical Society (ACS)}, author={Smirnova, TI and Smirnov, AI}, year={2003}, month={Jul}, pages={7212–7215} }
 @article{smirnov_smirnova_2001, title={Resolving domains of interdigitated phospholipid membranes with 95 GHz spin labeling EPR}, volume={21}, ISSN={["1613-7507"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0035528064&partnerID=MN8TOARS}, DOI={10.1007/BF03162420}, number={3-4}, journal={APPLIED MAGNETIC RESONANCE}, author={Smirnov, AI and Smirnova, TI}, year={2001}, pages={453–467} }
 @article{clarkson_smirnov_smirnova_kang_belford_earle_freed_1998, title={Multi-frequency EPR determination of zero field splitting of high spin species in liquids: Gd(III) chelates in water}, volume={95}, ISSN={0026-8976 1362-3028}, url={http://dx.doi.org/10.1080/00268979809483262}, DOI={10.1080/00268979809483262}, abstractNote={Multi-frequency EPR spectroscopy at 9.5, 35, 94, and 249 GHz has been employed to investigate the zero field splitting (ZFS) of high spin ions in liquids. In particular, experiments are reported on aqueous solutions of DTPA and DOTA chelates of Gd(III), and on the uncomplexed ion, which are relevant to the effectiveness of paramagnetic contrast agents for magnetic resonance imaging (MRI). The field dependence of the centroid of the resonance line, characterized by an effective g factor, geff, has been analysed in order to determine δ1, the trace of the square of the ZFS matrix. Analysis of the variation in transverse electron spin relaxation (T 2e) with experimental frequency provides yet another route to measure δ2 from EPR data. This analysis also gives δv, a correlation time describing the time-dependent ZFS effect. The ZFS parameters so obtained agree well with results obtained by the analysis of proton nuclear magnetic relaxation dispersion. At 94 GHz, partially resolved spectra from chelated and unchelated Gd(III) were observed. The shifts in resonance field for Gd(III) in these two compounds are due primarily to differences in the magnitude of ZFS. The spectral resolution as a function of frequency exhibits a maximum in the range of our experiments; the resolution disappeared at either higher or lower resonance frequency. Study of ZFS by EPR at multiple high fields offers a new and sensitive route to probe water interactions and chelate dynamics in biologically relevant systems having high spin ions.}, number={6}, journal={Molecular Physics}, publisher={Informa UK Limited}, author={Clarkson, R. B. and Smirnov, Alex I. and Smirnova, T. I. and Kang, H. and Belford, R. L. and Earle, K. and Freed, Jack H.}, year={1998}, month={Dec}, pages={1325–1332} }