@article{milton_draughn_bobay_stowe_olson_feldmann_thompson_myers_santoro_kearns_et al._2020, title={The Solution Structures and Interaction of SinR and SinI: Elucidating the Mechanism of Action of the Master Regulator Switch for Biofilm Formation in Bacillus subtilis}, volume={432}, ISSN={["1089-8638"]}, DOI={10.1016/j.jmb.2019.08.019}, abstractNote={Bacteria have developed numerous protection strategies to ensure survival in harsh environments, with perhaps the most robust method being the formation of a protective biofilm. In biofilms, bacterial cells are embedded within a matrix that is composed of a complex mixture of polysaccharides, proteins, and DNA. The gram-positive bacterium Bacillus subtilis has become a model organism for studying regulatory networks directing biofilm formation. The phenotypic transition from a planktonic to biofilm state is regulated by the activity of the transcriptional repressor, SinR, and its inactivation by its primary antagonist, SinI. In this work, we present the first full-length structural model of tetrameric SinR using a hybrid approach combining high-resolution solution nuclear magnetic resonance (NMR), chemical cross-linking, mass spectrometry, and molecular docking. We also present the solution NMR structure of the antagonist SinI dimer and probe the mechanism behind the SinR-SinI interaction using a combination of biochemical and biophysical techniques. As a result of these findings, we propose that SinI utilizes a residue replacement mechanism to block SinR multimerization, resulting in diminished DNA binding and concomitant decreased repressor activity. Finally, we provide an evidence-based mechanism that confirms how disruption of the SinR tetramer by SinI regulates gene expression.}, number={2}, journal={JOURNAL OF MOLECULAR BIOLOGY}, author={Milton, Morgan E. and Draughn, G. Logan and Bobay, Benjamin G. and Stowe, Sean D. and Olson, Andrew L. and Feldmann, Erik A. and Thompson, Richele J. and Myers, Katherine H. and Santoro, Michael T. and Kearns, Daniel B. and et al.}, year={2020}, month={Jan}, pages={343–357} }
 @article{draughn_milton_feldmann_bobay_roth_olson_thompson_actis_davies_cavanagh_2018, title={The Structure of the Biofilm-controlling Response Regulator BfmR from Acinetobacter baumannii Reveals Details of Its DNA-binding Mechanism}, volume={430}, ISSN={0022-2836}, url={http://dx.doi.org/10.1016/J.JMB.2018.02.002}, DOI={10.1016/j.jmb.2018.02.002}, abstractNote={The rise of drug-resistant bacterial infections coupled with decreasing antibiotic efficacy poses a significant challenge to global health care. Acinetobacter baumannii is an insidious, emerging bacterial pathogen responsible for severe nosocomial infections aided by its ability to form biofilms. The response regulator BfmR, from the BfmR/S two-component system, is the master regulator of biofilm initiation in A. baumannii and is a tractable therapeutic target. Here we present the structure of A. baumannii BfmR using a hybrid approach combining X-ray crystallography, nuclear magnetic resonance spectroscopy, chemical crosslinking mass spectrometry, and molecular modeling. We also show that BfmR binds the previously proposed bfmRS promoter sequence with moderate affinity. While BfmR shares many traits with other OmpR/PhoB family response regulators, some unusual properties were observed. Most importantly, we observe that when phosphorylated, BfmR binds this promoter sequence with a lower affinity than when not phosphorylated. All other OmpR/PhoB family members studied to date show an increase in DNA-binding affinity upon phosphorylation. Understanding the structural and biochemical mechanisms of BfmR will aid in the development of new antimicrobial therapies.}, number={6}, journal={Journal of Molecular Biology}, publisher={Elsevier BV}, author={Draughn, G. Logan and Milton, Morgan E. and Feldmann, Erik A. and Bobay, Benjamin G. and Roth, Braden M. and Olson, Andrew L. and Thompson, Richele J. and Actis, Luis A. and Davies, Christopher and Cavanagh, John}, year={2018}, month={Mar}, pages={806–821} }
 @article{stowe_thompson_peng_su_blackledge_draughn_coe_johannes_lapham_mackenzie_et al._2015, title={Membrane-Permeabilizing Activity of Reverse-Amide 2-Aminoimidazole Antibiofilm Agents Against Acinetobacter baumannii}, volume={12}, ISSN={["1875-5704"]}, DOI={10.2174/1567201811666140924125740}, abstractNote={Acinetobacter baumannii has quickly become one of the most insidious and prevalent nosocomial infections. Recently, the reverse-amide class of 2-aminoimidazole compounds (RA-2AI) was found both to prevent A. baumannii biofilm formation and also to disperse preexisting formations, putatively through interactions with cytosolic response regulators. Here we focus on how this class of antibiofilm agent traverses cellular membranes. Following the discovery of dosage-dependent growth rate changes, the cellular effects of RA-2AI were investigated using a combination of molecular assays and microscopic techniques. It was found that RA-2AI exposure has measureable effects on the bacterial membranes, resulting in a period of increased permeability and visible structural aberrations. Based on these results, we propose a model that describes how the structure of RA-2AI allows it to insert itself into and disrupt the fluidity of the membrane, creating an opportunity for increased molecular permeability.}, number={2}, journal={CURRENT DRUG DELIVERY}, author={Stowe, Sean D. and Thompson, Richele J. and Peng, Lingling and Su, Zhaoming and Blackledge, Meghan S. and Draughn, G. Logan and Coe, William H. and Johannes, Eva and Lapham, Valerie K. and Mackenzie, John and et al.}, year={2015}, pages={223–230} }
 @article{tucker_bobay_banse_olson_soderblom_moseley_thompson_varney_losick_cavanagh_2014, title={A DNA Mimic: The Structure and Mechanism of Action for the Anti-Repressor Protein AbbA}, volume={426}, ISSN={["1089-8638"]}, DOI={10.1016/j.jmb.2014.02.010}, abstractNote={Bacteria respond to adverse environmental conditions by switching on the expression of large numbers of genes that enable them to adapt to unfavorable circumstances. In Bacillus subtilis, many adaptive genes are under the negative control of the global transition state regulator, the repressor protein AbrB. Stressful conditions lead to the de-repression of genes under AbrB control. Contributing to this de-repression is AbbA, an anti-repressor that binds to and blocks AbrB from binding to DNA. Here, we have determined the NMR structure of the functional AbbA dimer, confirmed that it binds to the N-terminal DNA-binding domain of AbrB, and have provided an initial description for the interaction using computational docking procedures. Interestingly, we show that AbbA has structural and surface characteristics that closely mimic the DNA phosphate backbone, enabling it to readily carry out its physiological function.}, number={9}, journal={JOURNAL OF MOLECULAR BIOLOGY}, author={Tucker, Ashley T. and Bobay, Benjamin G. and Banse, Allison V. and Olson, Andrew L. and Soderblom, Erik J. and Moseley, M. Arthur and Thompson, Richele J. and Varney, Kristen M. and Losick, Richard and Cavanagh, John}, year={2014}, month={May}, pages={1911–1924} }
 @article{olson_thompson_melander_cavanagh_2014, title={Chemical shift assignments and secondary structure prediction of the C-terminal domain of the response regulator BfmR from Acinetobacter baumannii}, volume={8}, ISSN={["1874-270X"]}, DOI={10.1007/s12104-012-9454-2}, abstractNote={Acinetobacter baumannii is a Gram-negative pathogen responsible for severe nocosomial infections by forming biofilms in healthcare environments. The two-domain response regulator BfmR has been shown to be the master controller for biofilm formation. Inactivation of BfmR resulted in an abolition of pili production and consequently biofilm creation. Here we report backbone and sidechain resonance assignments and secondary structure prediction for the C-terminal domain of BfmR (residues 130–238) from A. baumannii.}, number={1}, journal={BIOMOLECULAR NMR ASSIGNMENTS}, author={Olson, Andrew L. and Thompson, Richele J. and Melander, Christian and Cavanagh, John}, year={2014}, month={Apr}, pages={67–70} }
 @article{bobay_thompson_milton_cavanagh_2014, title={Chemical shift assignments and secondary structure prediction of the phosphorelay protein VanU from Vibrio anguillarum}, volume={8}, ISSN={["1874-270X"]}, DOI={10.1007/s12104-013-9478-2}, abstractNote={Vibrio anguillarum is a biofilm forming Gram-negative bacterium that survives prolonged periods in seawater and causes vibriosis in marine life. A quorum-sensing signal transduction pathway initiates biofilm formation in response to environmental stresses. The phosphotransferase protein VanU is the focal point of the quorum-sensing pathway and facilitates the regulation between independent phosphorelay systems that activate or repress biofilm formation. Here we report the 1H, 13C, and 15N backbone and side chain resonance assignments and secondary structure prediction for VanU from V. anguillarum.}, number={1}, journal={BIOMOLECULAR NMR ASSIGNMENTS}, author={Bobay, Benjamin G. and Thompson, Richele J. and Milton, Debra L. and Cavanagh, John}, year={2014}, month={Apr}, pages={177–179} }
 @article{brackett_melander_an_krishnamurthy_thompson_cavanagh_melander_2014, title={Small-Molecule Suppression of beta-Lactam Resistance in Multidrug-Resistant Gram-Negative Pathogens}, volume={57}, ISSN={["1520-4804"]}, DOI={10.1021/jm501050e}, abstractNote={Recent efforts toward combating antibiotic resistance in bacteria have focused on Gram-positive bacteria; however, multidrug-resistant Gram-negative bacteria pose a significant risk to public health. An orthogonal approach to the development of new antibiotics is to develop adjuvant compounds that enhance the susceptibility of drug-resistant strains of bacteria to currently approved antibiotics. This paper describes the synthesis and biological activity of a library of aryl amide 2-aminoimidazoles based on a lead structure from an initial screen. A small molecule was identified from this library that is capable of lowering the minimum inhibitory concentration of β-lactam antibiotics by up to 64-fold.}, number={17}, journal={JOURNAL OF MEDICINAL CHEMISTRY}, author={Brackett, Christopher M. and Melander, Roberta J. and An, Il Hwan and Krishnamurthy, Aparna and Thompson, Richele J. and Cavanagh, John and Melander, Christian}, year={2014}, month={Sep}, pages={7450–7458} }
 @article{olson_tucker_bobay_soderblom_moseley_thompson_cavanagh_2014, title={Structure and DNA-Binding Traits of the Transition State Regulator AbrB}, volume={22}, ISSN={["1878-4186"]}, DOI={10.1016/j.str.2014.08.018}, abstractNote={The AbrB protein from Bacillus subtilis is a DNA-binding global regulator controlling the onset of a vast array of protective functions under stressful conditions. Such functions include biofilm formation, antibiotic production, competence development, extracellular enzyme production, motility, and sporulation. AbrB orthologs are known in a variety of prokaryotic organisms, most notably in all infectious strains of Clostridia, Listeria, and Bacilli. Despite its central role in bacterial response and defense, its structure has been elusive because of its highly dynamic character. Orienting its N- and C-terminal domains with respect to one another has been especially problematic. Here, we have generated a structure of full-length, tetrameric AbrB using nuclear magnetic resonance, chemical crosslinking, and mass spectrometry. We note that AbrB possesses a strip of positive electrostatic potential encompassing its DNA-binding region and that its C-terminal domain aids in DNA binding.}, number={11}, journal={STRUCTURE}, author={Olson, Andrew L. and Tucker, Ashley T. and Bobay, Benjamin G. and Soderblom, Erik J. and Moseley, M. Arthur and Thompson, Richele J. and Cavanagh, John}, year={2014}, month={Nov}, pages={1650–1656} }
 @article{stowe_tucker_thompson_piper_richards_rogers_mathies_melander_cavanagh_2012, title={Evaluation of the toxicity of 2-aminoimidazole antibiofilm agents using both cellular and model organism systems}, volume={35}, ISSN={["1525-6014"]}, DOI={10.3109/01480545.2011.614620}, abstractNote={Biofilm formation is a ubiquitous bacterial defense mechanism and has been shown to be a primary element in the antibiotic resistance of many human diseases, especially in the case of nosocomial infections. Recently, we have developed several compound libraries that are extremely effective at both dispersing preexisting biofilms and also inhibiting their initial formation. In addition to their antibiofilm properties, some of these molecules are able to resensitize resistant bacterial strains to previously ineffective antibiotics and are being assessed as adjuvants. In this study, we evaluated the toxic effects of three of our most effective 2-aminoimidazole compounds (dihydrosventrin, RA, and SPAR) using a rapid pipeline that combines a series of assays. A methylthiazolyldiphenyl-tetrazolium assay, using the HaCaT keratinocyte cell line was used to determine epidermal irritants and was combined with Caenorhabditis elegans fecundity assays that demonstrated the effects of environmental exposure to various concentrations of these molecules. In each case, the assays showed that the compounds did not exhibit toxicity until they reached well above their current biofilm dispersion/inhibition concentrations. The most effective antibiofilm compound also had significant effects when used in conjunction with several standard antibiotics against resistant bacteria. Consequently, it was further investigated using the C. elegans assay in combination with different antibiotics and was found to maintain the same low level of toxicity as when acting alone, bolstering its candidacy for further testing as an adjuvant.}, number={3}, journal={DRUG AND CHEMICAL TOXICOLOGY}, author={Stowe, Sean D. and Tucker, Ashley T. and Thompson, Richele and Piper, Amanda and Richards, Justin J. and Rogers, Steven A. and Mathies, Laura D. and Melander, Christian and Cavanagh, John}, year={2012}, month={Jul}, pages={310–315} }
 @article{thompson_bobay_stowe_olson_peng_su_actis_melander_cavanagh_2012, title={Identification of BfmR, a Response Regulator Involved in Biofilm Development, as a Target for a 2-Aminoimidazole-Based Antibiofilm Agent}, volume={51}, ISSN={["0006-2960"]}, DOI={10.1021/bi3015289}, abstractNote={2-Aminoimidazoles (2AIs) have been documented to disrupt bacterial protection mechanisms, including biofilm formation and genetically encoded antibiotic resistance traits. Using Acinetobacter baumannii, we provide initial insight into the mechanism of action of a 2AI-based antibiofilm agent. Confocal microscopy confirmed that the 2AI is cell permeable, while pull-down assays identified BfmR, a response regulator that is the master controller of biofilm formation, as a target for this compound. Binding assays demonstrated specificity of the 2AI for response regulators, while computational docking provided models for 2AI-BfmR interactions. The 2AI compound studied here represents a unique small molecule scaffold that targets bacterial response regulators.}, number={49}, journal={BIOCHEMISTRY}, author={Thompson, Richele J. and Bobay, Benjamin G. and Stowe, Sean D. and Olson, Andrew L. and Peng, Lingling and Su, Zhaoming and Actis, Luis A. and Melander, Christian and Cavanagh, John}, year={2012}, month={Dec}, pages={9776–9778} }
 @article{bobay_stewart_tucker_thompson_varney_cavanagh_2012, title={Structural insights into the calcium-dependent interaction between calbindin-D28K and caspase-3}, volume={586}, ISSN={["0014-5793"]}, DOI={10.1016/j.febslet.2012.08.032}, abstractNote={Calbindin‐D28K and Caspase‐3 bind by isothermal titration calorimetry (View interaction)}, number={20}, journal={FEBS LETTERS}, author={Bobay, Benjamin G. and Stewart, Amanda L. and Tucker, Ashley T. and Thompson, Richele J. and Varney, Kristen M. and Cavanagh, John}, year={2012}, month={Oct}, pages={3582–3589} }
 @misc{stowe_richards_tucker_thompson_melander_cavanagh_2011, title={Anti-biofilm compounds derived from marine sponges}, volume={9}, number={10}, journal={Marine Drugs}, author={Stowe, S. D. and Richards, J. J. and Tucker, A. T. and Thompson, R. and Melander, C. and Cavanagh, J.}, year={2011}, pages={2010–2035} }
 @article{bobay_thompson_hoch_cavanagh_2010, title={Long range dynamic effects of point-mutations trap a response regulator in an active conformation}, volume={584}, ISSN={["0014-5793"]}, DOI={10.1016/j.febslet.2010.08.051}, abstractNote={When a point‐mutation in a protein elicits a functional change, it is most common to assign this change to local structural perturbations. Here we show that point‐mutations, distant from an essential highly dynamic kinase recognition loop in the response regulator Spo0F, lock this loop in an active conformation. This ‘conformational trapping’ results in functionally hyperactive Spo0F. Consequently, point‐mutations are seen to affect functionally critical motions both close to and far from the mutational site.}, number={19}, journal={FEBS LETTERS}, author={Bobay, Benjamin G. and Thompson, Richele J. and Hoch, James A. and Cavanagh, John}, year={2010}, month={Oct}, pages={4203–4207} }
 @article{hobbs_bobay_thompson_perego_cavanagh_2010, title={NMR Solution Structure and DNA-binding Model of the DNA-binding Domain of Competence Protein A}, volume={398}, ISSN={["0022-2836"]}, DOI={10.1016/j.jmb.2010.03.003}, abstractNote={Competence protein A (ComA) is a response regulator protein involved in the development of genetic competence in the Gram-positive spore-forming bacterium Bacillus subtilis, as well as the regulation of the production of degradative enzymes and antibiotic synthesis. ComA belongs to the NarL family of proteins, which are characterized by a C-terminal transcriptional activator domain that consists of a bundle of four helices, where the second and third helices (alpha 8 and alpha 9) form a helix-turn-helix DNA-binding domain. Using NMR spectroscopy, the high-resolution 3D solution structure of the C-terminal DNA-binding domain of ComA (ComAC) has been determined. In addition, surface plasmon resonance and NMR protein-DNA titration experiments allowed for the analysis of the interaction of ComAC with its target DNA sequences. Combining the solution structure and biochemical data, a model of ComAC bound to the ComA recognition sequences on the srfA promoter has been developed. The model shows that for DNA binding, ComA uses the conserved helix-turn-helix motif present in other NarL family members. However, the model reveals also that ComA might use a slightly different part of the helix-turn-helix motif and there appears to be some associated domain re-orientation. These observations suggest a basis for DNA binding specificity within the NarL family.}, number={2}, journal={JOURNAL OF MOLECULAR BIOLOGY}, author={Hobbs, Carey A. and Bobay, Benjamin G. and Thompson, Richele J. and Perego, Marta and Cavanagh, John}, year={2010}, month={Apr}, pages={248–263} }
 @article{hobbs_deterding_perera_bobay_thompson_darden_cavanagh_tomer_2009, title={Structural Characterization of the Conformational Change in Calbindin-D-28k upon Calcium Binding Using Differential Surface Modification Analyzed by Mass Spectrometry}, volume={48}, ISSN={["0006-2960"]}, DOI={10.1021/bi900350q}, abstractNote={Calbindin-D28k is a calcium binding protein with six EF hand domains. Calbindin-D28k is unique in that it functions as both a calcium buffer and a sensor protein. It is found in many tissues, including brain, pancreas, kidney, and intestine, playing important roles in each. Calbindin-D28k is known to bind four calcium ions and upon calcium binding undergoes a conformational change. The structure of apo calbindin-D28k is in an ordered state, transitioning into a disordered state as calcium is bound. Once fully loaded with four calcium ions, it again takes on an ordered state. The solution structure of disulfide-reduced holo-calbindin-D28k has been determined by NMR, while the structure of apo calbindin-D28k has yet to be determined. Differential surface modification of lysine and histidine residues analyzed by mass spectrometry has been used in this study to identify, for the first time, the specific regions of calbindin-D28k undergoing conformational changes between the holo and apo states. Using differential surface modification in combination with mass spectrometry, EF hands 1 and 4 as well as the linkers before EF hand 1 and the linkers between EF hands 4 and 5 and EF hands 5 and 6 were identified as regions of conformational change between apo and holo calbindin-D28k. Under the experimental conditions employed, EF hands 2 and 6, which are known not to bind calcium, were unaffected in either form. EF hand 2 is highly accessible; however, EF hand 6 was determined not to be surface accessible in either form. Previous research has identified a disulfide bond between cysteines 94 and 100 in the holo state. Until now, it was unknown whether this bond also exists in the apo form. Our data confirm the presence of the disulfide bond between cysteines 94 and 100 in the holo form and indicate that there is predominantly no disulfide bond between these residues in the apoprotein.}, number={36}, journal={BIOCHEMISTRY}, author={Hobbs, Carey A. and Deterding, Leesa J. and Perera, Lalith and Bobay, Benjamin G. and Thompson, Richele J. and Darden, Thomas A. and Cavanagh, John and Tomer, Kenneth B.}, year={2009}, month={Sep}, pages={8603–8614} }
 @article{kojetin_mclaughlin_thompson_dubnau_prepiak_rance_cavanagh_2009, title={Structural and Motional Contributions of the Bacillus subtilis ClpC N-Domain to Adaptor Protein Interactions}, volume={387}, ISSN={["1089-8638"]}, DOI={10.1016/j.jmb.2009.01.046}, abstractNote={The AAA + (ATPases associated with a variety of cellular activities) superfamily protein ClpC is a key regulator of cell development in Bacillus subtilis. As part of a large oligomeric complex, ClpC controls an array of cellular processes by recognizing, unfolding, and providing misfolded and aggregated proteins as substrates for the ClpP peptidase. ClpC is unique compared to other HSP100/Clp proteins, as it requires an adaptor protein for all fundamental activities. The NMR solution structure of the N-terminal repeat domain of ClpC (N-ClpCR) comprises two structural repeats of a four-helix motif. NMR experiments used to map the MecA adaptor protein interaction surface of N-ClpCR reveal that regions involved in the interaction possess conformational flexibility and conformational exchange on the microsecond-to-millisecond timescale. The electrostatic surface of N-ClpCR differs substantially from the N-domain of Escherichia coli ClpA and ClpB, suggesting that the electrostatic surface characteristics of HSP100/Clp N-domains may play a role in adaptor protein and substrate interaction specificity, and perhaps contribute to the unique adaptor protein requirement of ClpC.}, number={3}, journal={JOURNAL OF MOLECULAR BIOLOGY}, author={Kojetin, Douglas J. and McLaughlin, Patrick D. and Thompson, Richele J. and Dubnau, David and Prepiak, Peter and Rance, Mark and Cavanagh, John}, year={2009}, month={Apr}, pages={639–652} }
 @article{sullivan_bobay_kojetin_thompson_rance_strauch_cavanagh_2008, title={Insights into the Nature of DNA Binding of AbrB-like Transcription Factors}, volume={16}, ISSN={["1878-4186"]}, DOI={10.1016/j.str.2008.08.014}, abstractNote={Understanding the DNA recognition and binding by the AbrB-like family of transcriptional regulators is of significant interest since these proteins enable bacteria to elicit the appropriate response to diverse environmental stimuli. Although these “transition-state regulator” proteins have been well characterized at the genetic level, the general and specific mechanisms of DNA binding remain elusive. We present RDC-refined NMR solution structures and dynamic properties of the DNA-binding domains of three Bacillus subtilis transition-state regulators: AbrB, Abh, and SpoVT. We combined previously investigated DNase I footprinting, DNA methylation, gel-shift assays, and mutagenic and NMR studies to generate a structural model of the complex between AbrBN55 and its cognate promoter, abrB8. These investigations have enabled us to generate a model for the specific nature of the transition-state regulator-DNA interaction, a structure that has remained elusive thus far.}, number={11}, journal={STRUCTURE}, author={Sullivan, Daniel M. and Bobay, Benjamin G. and Kojetin, Douglas J. and Thompson, Richele J. and Rance, Mark and Strauch, Mark A. and Cavanagh, John}, year={2008}, month={Nov}, pages={1702–1713} }
 @article{kojetin_mclaughlin_thompson_venters_rance_cavanagh_2007, title={NMR assignment of the N-terminal repeat domain of Bacillus subtilis ClpC}, volume={1}, ISSN={["1874-2718"]}, DOI={10.1007/s12104-007-9046-8}, abstractNote={The HSP100/AAA+ superfamily protein ClpC is a key regulator of cell development in Bacillus subtilis. We present here the backbone and side-chain assignments of the N-terminal repeat domain (residues 1-145) of ClpC from Bacillus subtilis.}, number={2}, journal={BIOMOLECULAR NMR ASSIGNMENTS}, author={Kojetin, Douglas J. and McLaughlin, Patrick D. and Thompson, Richele J. and Venters, Ronald A. and Rance, Mark and Cavanagh, John}, year={2007}, month={Dec}, pages={163–165} }
 @article{kordys_bobay_thompson_venters_cavanagh_2007, title={Peptide binding proclivities of calcium loaded calbindin-D28k}, volume={581}, ISSN={["0014-5793"]}, DOI={10.1016/j.febslet.2007.09.004}, abstractNote={Calbindin‐D28k is known to function as a calcium‐buffering protein in the cell. Moreover, recent evidence shows that it also plays a role as a sensor. Using circular dichroism and NMR, we show that calbindin‐D28k undergoes significant conformational changes upon binding calcium, whereas only minor changes occur when binding target peptides in its Ca2+‐loaded state. NMR experiments also identify residues that undergo chemical shift changes as a result of peptide binding. The subsequent use of computational protein–protein docking protocols produce a model describing the interaction interface between calbindin‐D28k and its target peptides.}, number={24}, journal={FEBS LETTERS}, author={Kordys, David R. and Bobay, Benjamin G. and Thompson, Richele J. and Venters, Ronald A. and Cavanagh, John}, year={2007}, month={Oct}, pages={4778–4782} }
 @article{mclaughlin_bobaya_regel_thompson_hoch_cavanagh_2007, title={Predominantly buried residues in the response regulator Spo0F influence specific sensor kinase recognition}, volume={581}, ISSN={["0014-5793"]}, DOI={10.1016/j.febslet.2007.02.061}, abstractNote={Several alanine mutations in the response regulator Spo0F induce hypersporulation in Bacillus subtilis. L66A, I90A and H101A mutants are purported to be involved in contacts stabilizing the orientation of the α4‐helix and hence the β4–α4 kinase recognition loop. Y13A is thought to affect the orientation of the α1‐helix and consequently phosphatase action. Using comparative NMR chemical shift analyses for these mutants, we have confirmed these suppositions and isolated residues in Spo0F critical in sensor kinases discrimination. In addition, we discuss how buried residues and intra‐protein communication networks contribute to precise molecular recognition by ensuring that the correct surface is presented.}, number={7}, journal={FEBS LETTERS}, author={McLaughlin, Patrick D. and Bobaya, Benjamm G. and Regel, Erin J. and Thompson, Richele J. and Hoch, James A. and Cavanagh, John}, year={2007}, month={Apr}, pages={1425–1429} }
 @article{bobay_mueller_thompson_murzin_venters_strauch_cavanagh_2006, title={NMR structure of AbhN and comparison with AbrBN - First insights into the DNA binding promiscuity and specificity of AbrB-like transition state regulator proteins}, volume={281}, ISSN={["1083-351X"]}, DOI={10.1074/jbc.M601963200}, abstractNote={Understanding the molecular mechanisms of transition state regulator proteins is critical, since they play a pivotal role in the ability of bacteria to cope with changing environments. Although much effort has focused on their genetic characterization, little is known about their structural and functional conservation. Here we present the high resolution NMR solution structure of the N-terminal domain of the Bacillus subtilis transition state regulator Abh (AbhN), only the second such structure to date. We then compare AbhN to the N-terminal DNA-binding domain of B. subtilis AbrB (AbrBN). This is the first such comparison between two AbrB-like transition state regulators. AbhN and AbrBN are very similar, suggesting a common structural basis for their DNA binding. However, we also note subtle variances between the AbhN and AbrBN structures, which may play important roles in DNA target specificity. The results of accompanying in vitro DNA-binding studies serve to highlight binding differences between the two proteins.}, number={30}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Bobay, Benjamin G. and Mueller, Geoffrey A. and Thompson, Richele J. and Murzin, Alexey G. and Venters, Ronald A. and Strauch, Mark A. and Cavanagh, John}, year={2006}, month={Jul}, pages={21399–21409} }
 @article{kojetin_venters_kordys_thompson_kumar_cavanagh_2006, title={Structure, binding interface and hydrophobic transitions of Ca2+-loaded calbindin-D-28K}, volume={13}, DOI={10.1038/nsmb1112d}, number={7}, journal={Nature Structural & Molecular Biology}, author={Kojetin, D. J. and Venters, R. A. and Kordys, D. R. and Thompson, R. J. and Kumar, R. and Cavanagh, J.}, year={2006}, pages={641–647} }
 @article{kojetin_thompson_benson_naylor_waterman_davies_opperman_stephenson_hoch_cavanagh_2005, title={Structural analysis of divalent metals binding to the Bacillus subtilis response regulator Spo0F: the possibility for In vitro metalloregulation in the initiation of sporulation}, volume={18}, ISSN={["1572-8773"]}, DOI={10.1007/s10534-005-4303-8}, abstractNote={The presence of a divalent metal ion in a negatively charged aspartic acid pocket is essential for phosphorylation of response regulator proteins. Here, we present metal binding studies of the Bacillus subtilis response regulator Spo0F using NMR and microESI-MS. NMR studies show that the divalent metals Ca(2+), Mg(2+) and Mn(2+) primarily bind, as expected, in the Asp pocket phosphorylation site. However, identical studies with Cu(2+) show distinct binding effects in three specific locations: (i) the Asp pocket, (ii) a grouping of charged residues at a site opposite of the Asp pocket, and (iii) on the beta 4-alpha 4 loop and the beta 5/alpha 5 interface, particularly around and including H101. microESI-MS studies stoichiometrically confirm the NMR studies and demonstrate that most divalent metal ions bind to Spo0F primarily in a 1:1 ratio. Again, in the case of Cu(2+), multiple metal-bound species are observed. Subsequent experiments reveal that Mg(2+) supports phosphotransfer between KinA and Spo0F, while Cu(2+) fails to support KinA phosphotransfer. Additionally, the presence of Cu(2+) at non-lethal concentrations in sporulation media for B. subtilis and the related organism Pasteuria penetrans was found to inhibit spore formation while continuing to permit vegetative growth. Depending on the type of divalent metal ion present, in vitro phosphorylation of Spo0F by its cognate kinase KinA can be inhibited.}, number={5}, journal={BIOMETALS}, author={Kojetin, DJ and Thompson, RJ and Benson, LM and Naylor, S and Waterman, J and Davies, KG and Opperman, CH and Stephenson, K and Hoch, JA and Cavanagh, J}, year={2005}, month={Oct}, pages={449–466} }
 @article{bobay_benson_naylor_feeney_clark_goshe_strauch_thompson_cavanagh_2004, title={Evaluation of the DNA Binding Tendencies of the Transition State Regulator AbrB†}, volume={43}, ISSN={0006-2960 1520-4995}, url={http://dx.doi.org/10.1021/bi048399h}, DOI={10.1021/bi048399h}, abstractNote={Global transition state regulator proteins represent one of the most diverse classes of prokaryotic transcription factors. One such transition state regulator, AbrB from Bacillus subtilis, is known to bind more than 60 gene targets yet displays specificity within this target set by binding each promoter with a different affinity. Microelectrospray ionization mass spectrometry (microESI-MS), circular dichroism, fluorescence, UV spectroscopy, and molecular modeling were used to elucidate differences among AbrB, DNA, and AbrB-DNA complexes. MicroESI-MS analysis of AbrB confirmed its stable macromolecular state as being tetrameric and verified the same stoichiometric state in complex with DNA targets. MicroESI-MS, circular dichroism, and fluorescence provided relative binding affinities for AbrB-DNA interactions in a qualitative manner. UV spectroscopy was used in a quantitative manner to determine solution phase dissociation constants for AbrB-DNA complexes. General DNA structural parameters for all known natural AbrB binding sequences were also studied and significant similarities in topological constraints (stretch, opening, and propeller twist) were observed. It is likely that these parameters contribute to the differential binding proclivities of AbrB. In addition to providing an improved understanding of transition state regulator-DNA binding properties and structural tendencies of target promoters, this comprehensive and corroborative spectroscopic study endorses the use of microESI-MS for rapidly ascertaining qualitative binding trends in noncovalent systems in a high-throughput manner.}, number={51}, journal={Biochemistry}, publisher={American Chemical Society (ACS)}, author={Bobay, Benjamin G. and Benson, Linda and Naylor, Stephen and Feeney, Brett and Clark, A. Clay and Goshe, Michael B. and Strauch, Mark A. and Thompson, Richele and Cavanagh, John}, year={2004}, month={Dec}, pages={16106–16118} }
 @article{ulrich_thompson_bassler_cavanagh_loria_2004, title={H-1, N-15, and C-13 chemical shift assignments of the Vibrio harveyi histidine phosphotransferase protein LuxU}, volume={29}, ISSN={["1573-5001"]}, DOI={10.1023/B:JNMR.0000034349.22942.e1}, number={4}, journal={JOURNAL OF BIOMOLECULAR NMR}, author={Ulrich, DL and Thompson, R and Bassler, B and Cavanagh, J and Loria, JP}, year={2004}, month={Aug}, pages={551–552} }
 @article{kojetin_thompson_cavanagh_2004, title={Sub-classification of response regulators using the surface characteristics of their receiver domains (FEBS 27785) (vol 554, pg 231, 2003)}, volume={560}, ISSN={["1873-3468"]}, DOI={10.1016/s0014-5793(04)00063-8}, number={1-3}, journal={FEBS LETTERS}, author={Kojetin, DJ and Thompson, RJ and Cavanagh, J}, year={2004}, month={Feb}, pages={227–228} }
 @article{cavanagh_benson_thompson_naylor_2003, title={In-line desalting mass spectrometry for the study of noncovalent biological complexes}, volume={75}, ISSN={["1520-6882"]}, DOI={10.1021/ac030182q}, abstractNote={Electrospray ionization-mass spectrometry is becoming widely used as a high-throughput method for the study of biomolecular interactions. It allows for the analysis of complexes from heterogeneous mixtures with high sensitivity and selectivity. In many cases, biomolecules and their complexes must be stored in nonvolatile salt buffers and other solubilizing agents, such as organics or detergents, to maintain stability and integrity. To ensure an efficient electrospray process, desalting and exchanging the biomolecular solutions into a volatile buffer is imperative. Current off-line or on-line methods to accomplish this are time-consuming, frequently disrupt noncovalent interactions, and can result in considerable sample loss. Here we describe a simple, general, and highly efficient, rapid in-line desalting approach using a small gel cartridge to assist in the mass spectrometric analysis of biomolecules and their complexes. Though the method has broad applicability, we focus our analysis on proteins and demonstrate its usefulness by examining protein-metal, protein-protein, protein-DNA, and protein-RNA interactions. The method is shown to provide rapid direct analysis of analyte solutions containing salts, glycerol, organics, and involatile buffers without deleterious effects.}, number={14}, journal={ANALYTICAL CHEMISTRY}, author={Cavanagh, J and Benson, LM and Thompson, R and Naylor, S}, year={2003}, month={Jul}, pages={3281–3286} }
 @article{kojetin_thompson_cavanagh_2003, title={Sub-classification of response regulators using the surface characteristics of their receiver domains}, volume={554}, ISSN={["0014-5793"]}, DOI={10.1016/S0014-5793(03)01167-0}, abstractNote={The omnipresent bacterial switch known as a two‐component system is comprised of a response regulator and a sensor kinase with which it interacts. Sensor kinases have been classified and further sub‐classified into groups based on their sequence similarity, loop lengths and domain organization. Response regulators have been classified predominantly by the identity and function of their output domains. Here, comparative based homology modeling of the receiver domains of the OmpR sub‐family of response regulators in Bacillus subtilis and Escherichia coli suggests further sub‐classification is possible. A color‐coded scale is used to show trends in surface hydrophobicity. For the OmpR receiver domains modeled these trends allow further sub‐classification. The specific surface regions used for this sub‐classification procedure correlate with clusters of residues that are important for interaction with cognate four helix bundle HisKA/Hpt domains.}, number={3}, journal={FEBS LETTERS}, author={Kojetin, DJ and Thompson, RJ and Cavanagh, J}, year={2003}, month={Nov}, pages={231–236} }
 @article{venters_benson_craig_paul_kordys_thompson_naylor_kumar_cavanagh_2003, title={The effects of Ca2+ binding on the conformation of calbindin D-28K: A nuclear magnetic resonance and microelectrospray mass spectrometry study}, volume={317}, ISSN={["0003-2697"]}, DOI={10.1016/S0003-2697(03)00084-8}, abstractNote={Calbindin D(28K) is a six-EF-hand calcium-binding protein found in the brain, peripheral nervous system, kidney, and intestine. There is a paucity of information on the effects of calcium binding on calbindin D(28K) structure. To further examine the mechanism and structural consequences of calcium binding to calbindin D(28K) we performed detailed complementary heteronuclear NMR and microelectrospray mass spectrometry investigations of the calcium-induced conformational changes of calbindin D(28K). The combined use of these two powerful analytical techniques clearly and very rapidly demonstrates the following: (i). apo-calbindin D(28K) has an ordered structure which changes to a notably different ordered conformation upon Ca(2+) loading, (ii). calcium binding is a sequential process and not a simultaneous event, and (iii). EF-hands 1, 3, 4, and 5 take up Ca(2+), whereas EF-hands 2 and 6 do not. Our results support the opinion that calbindin D(28K) has characteristics of both a calcium sensor and a buffer.}, number={1}, journal={ANALYTICAL BIOCHEMISTRY}, author={Venters, RA and Benson, LM and Craig, TA and Paul, KH and Kordys, DR and Thompson, R and Naylor, S and Kumar, R and Cavanagh, J}, year={2003}, month={Jun}, pages={59–66} }
 @article{venters_thompson_cavanagh_2002, title={Current approaches for the study of large proteins by NMR}, volume={602}, ISSN={["1872-8014"]}, DOI={10.1016/s0022-2860(01)00690-1}, abstractNote={An overview of current methods employed for characterizing larger (>25 kDa) proteins by NMR is presented. These techniques include: the attenuation of T2 relaxation effects by offsetting dipole–dipole and chemical shift anisotropy relaxation mechanisms (TROSY); the extraction of residual dipolar couplings from partially oriented molecules; the elimination of relaxation pathways by incorporating deuterium nuclei into protein samples; the easing of resonance overlap by isotopically labeling only specific protein segments; and the decrease of rotational correlation times by dissolving proteins in low viscosity solvents.}, journal={JOURNAL OF MOLECULAR STRUCTURE}, author={Venters, RA and Thompson, R and Cavanagh, J}, year={2002}, month={Jan}, pages={275–292} }
 @article{benson_vaughn_strauch_bobay_thompson_naylor_cavanagh_2002, title={Macromolecular assembly of the transition state regulator AbrB in its unbound and complexed states probed by microelectrospray ionization mass spectrometry}, volume={306}, ISSN={["1096-0309"]}, DOI={10.1006/abio.2002.5704}, abstractNote={The Bacillus subtilis global transition-state regulator AbrB specifically recognizes over 60 different DNA regulatory regions of genes expressed during cellular response to suboptimal environments. Most interestingly the DNA regions recognized by AbrB share no obvious consensus base sequence. To more clearly understand the functional aspects of AbrB activity, microelectrospray ionization mass spectrometry has been employed to resolve the macromolecular assembly of unbound and DNA-bound AbrB. Analysis of the N-terminal DNA binding domain of AbrB (AbrBN53, residues 1-53) demonstrates that AbrBN53 is a stable dimer, showing no apparent exchange with a monomeric form as a function of pH, ionic strength, solvent, or protein concentration. AbrBN53 demonstrates a capacity for DNA binding, underscoring the role of the N-terminal domain in both DNA recognition and dimerization. Full-length AbrB is shown to exist as a homotetramer. An investigation of the binding of AbrBN53 and AbrB to the natural DNA target element sinIR shows that AbrBN53 binds as a dimer and AbrB binds as a tetramer. This study represents the first detailed characterization of the stoichiometry of a transition-state regulator binding to one of its target promoters.}, number={2}, journal={ANALYTICAL BIOCHEMISTRY}, author={Benson, LM and Vaughn, JL and Strauch, MA and Bobay, BG and Thompson, R and Naylor, S and Cavanagh, J}, year={2002}, month={Jul}, pages={222–227} }
 @article{cavanagh_thompson_bobay_benson_naylor_2002, title={Stoichiometries of protein - Protein/DNA binding and conformational changes for the transition-state regulator AbrB measured by pseudo cell-size exclusion chromatography-mass spectrometry}, volume={41}, ISSN={["0006-2960"]}, DOI={10.1021/bi0202225}, abstractNote={We have developed on-line pseudo cell-size exclusion chromatography-mass spectrometry (PsC-SEC-MS) for the rapid, real time analyses of noncovalently bound protein complexes. The methodology can be used to determine constituent components of such complexes, as well as exact stoichiometries. Furthermore, it enables the efficient determination of gross conformational changes upon complexation. The power of the new approach is demonstrated in the analysis of the global transition-state regulator AbrB and its complex with a target DNA sequence from the promoter sinIR. Using PsC-SEC-MS, we confirm that AbrB is assembled as a homotetramer and not as a homohexamer as previously suggested. Additionally, we show that AbrB binds to the sinIR DNA target element as a homotetramer, affording a 4:1 protein:DNA stoichiometry. Finally, we demonstrate that when the complex binds to sinIR, the hydrodynamic volume (size) of the complex is notably reduced compared to that of the apoprotein, indicating a protein conformational change.}, number={25}, journal={BIOCHEMISTRY}, author={Cavanagh, J and Thompson, R and Bobay, B and Benson, LM and Naylor, S}, year={2002}, month={Jun}, pages={7859–7865} }
 @article{sehgal_tompson_cavanagh_kelly_2002, title={Structural and catalytic response to temperature and cosolvents of carboxylesterase EST1 from the extremely thermoacidophilic archaeon Sulfolobus solfataricus P1}, volume={80}, ISSN={["0006-3592"]}, DOI={10.1002/bit.10433}, abstractNote={Abstract}, number={7}, journal={BIOTECHNOLOGY AND BIOENGINEERING}, author={Sehgal, AC and Tompson, R and Cavanagh, J and Kelly, RM}, year={2002}, month={Dec}, pages={784–793} }