Статті в журналах: "Hydrogen deuterium exchange (HDx)"

1

Masson, Glenn R., Sarah L. Maslen, and Roger L. Williams. "Analysis of phosphoinositide 3-kinase inhibitors by bottom-up electron-transfer dissociation hydrogen/deuterium exchange mass spectrometry." Biochemical Journal 474, no. 11 (May 16, 2017): 1867–77. http://dx.doi.org/10.1042/bcj20170127.

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Until recently, one of the major limitations of hydrogen/deuterium exchange mass spectrometry (HDX-MS) was the peptide-level resolution afforded by proteolytic digestion. This limitation can be selectively overcome through the use of electron-transfer dissociation to fragment peptides in a manner that allows the retention of the deuterium signal to produce hydrogen/deuterium exchange tandem mass spectrometry (HDX-MS/MS). Here, we describe the application of HDX-MS/MS to structurally screen inhibitors of the oncogene phosphoinositide 3-kinase catalytic p110α subunit. HDX-MS/MS analysis is able to discern a conserved mechanism of inhibition common to a range of inhibitors. Owing to the relatively minor amounts of protein required, this technique may be utilised in pharmaceutical development for screening potential therapeutics.

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2

Bouyssié, David, Jean Lesne, Marie Locard-Paulet, Renaud Albigot, Odile Burlet-Schiltz, and Julien Marcoux. "HDX-Viewer: interactive 3D visualization of hydrogen–deuterium exchange data." Bioinformatics 35, no. 24 (July 9, 2019): 5331–33. http://dx.doi.org/10.1093/bioinformatics/btz550.

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Abstract Summary With the advent of fully automated sample preparation robots for Hydrogen–Deuterium eXchange coupled to Mass Spectrometry (HDX-MS), this method has become paramount for ligand binding or epitope mapping screening, both in academic research and biopharmaceutical industries. However, bridging the gap between commercial HDX-MS software (for raw data interpretation) and molecular viewers (to map experiment results onto a 3D structure for biological interpretation) remains laborious and requires simple but sometimes limiting coding skills. We solved this bottleneck by developing HDX-Viewer, an open-source web-based application that facilitates and quickens HDX-MS data analysis. This user-friendly application automatically incorporates HDX-MS data from a custom template or commercial HDX-MS software in PDB files, and uploads them to an online 3D molecular viewer, thereby facilitating their visualization and biological interpretation. Availability and implementation The HDX-Viewer web application is released under the CeCILL (http://www.cecill.info) and GNU LGPL licenses and can be found at https://masstools.ipbs.fr/hdx-viewer. The source code is available at https://github.com/david-bouyssie/hdx-viewer.

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3

Gogonea, Valentin, Judith Peters, Gary S. Gerstenecker, Celalettin Topbas, Liming Hou, Jérôme Combet, Joseph A. DiDonato, Jonathan D. Smith, Kerry-Anne Rye, and Stanley L. Hazen. "Protein Backbone and Average Particle Dynamics in Reconstituted Discoidal and Spherical HDL Probed by Hydrogen Deuterium Exchange and Elastic Incoherent Neutron Scattering." Biomolecules 10, no. 1 (January 10, 2020): 121. http://dx.doi.org/10.3390/biom10010121.

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Lipoproteins are supramolecular assemblies of proteins and lipids with dynamic characteristics critically linked to their biological functions as plasma lipid transporters and lipid exchangers. Among them, spherical high-density lipoproteins are the most abundant forms of high-density lipoprotein (HDL) in human plasma, active participants in reverse cholesterol transport, and associated with reduced development of atherosclerosis. Here, we employed elastic incoherent neutron scattering (EINS) and hydrogen-deuterium exchange mass spectrometry (HDX-MS) to determine the average particle dynamics and protein backbone local mobility of physiologically competent discoidal and spherical HDL particles reconstituted with human apolipoprotein A-I (apoA-I). Our EINS measurements indicated that discoidal HDL was more dynamic than spherical HDL at ambient temperatures, in agreement with their lipid-protein composition. Combining small-angle neutron scattering (SANS) with contrast variation and MS cross-linking, we showed earlier that the most likely organization of the three apolipoprotein A-I (apoA-I) chains in spherical HDL is a combination of a hairpin monomer and a helical antiparallel dimer. Here, we corroborated those findings with kinetic studies, employing hydrogen-deuterium exchange mass spectrometry (HDX-MS). Many overlapping apoA-I digested peptides exhibited bimodal HDX kinetics behavior, suggesting that apoA-I regions with the same amino acid composition located on different apoA-I chains had different conformations and/or interaction environments.

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4

Setner, B., M. Wierzbicka, L. Jerzykiewicz, M. Lisowski та Z. Szewczuk. "The unexpected racemization and hydrogen–deuterium exchange of the hydrogen at the α-carbon of proline analogs containing the 5-azoniaspiro[4.4]nonyl-group". Organic & Biomolecular Chemistry 16, № 5 (2018): 825–31. http://dx.doi.org/10.1039/c7ob02926h.

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5

Kazazić, Saša, Zrinka Karačić, Igor Sabljić, Dejan Agić, Marko Tomin, Marija Abramić, Michal Dadlez, Antonija Tomić, and Sanja Tomić. "Conservation of the conformational dynamics and ligand binding within M49 enzyme family." RSC Advances 8, no. 24 (2018): 13310–22. http://dx.doi.org/10.1039/c7ra13059g.

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The hydrogen deuterium exchange (HDX) mass spectrometry combined with molecular dynamics (MD) simulations was employed to investigate conformational dynamics and ligand binding within the M49 family (dipeptidyl peptidase III family).

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6

Song, Xiaowei, Jia Li, Mohammad Mofidfar, and Richard N. Zare. "Distinguishing between Isobaric Ions Using Microdroplet Hydrogen–Deuterium Exchange Mass Spectrometry." Metabolites 11, no. 11 (October 23, 2021): 728. http://dx.doi.org/10.3390/metabo11110728.

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Isobaric ions having the same mass-to-charge ratio cannot be separately identified by mass spectrometry (MS) alone, but this limitation can be overcome by using hydrogen–deuterium exchange (HDX) in microdroplets. Because isobaric ions may contain a varied number of exchangeable sites and different types of functional groups, each one produces a unique MS spectral pattern after droplet spray HDX without the need for MS/MS experiments or introduction of ion mobility measurements. As an example of the power of this approach, isobaric ions in urinary metabolic profiles are identified and used to distinguish between healthy individuals and those having bladder cancer.

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7

Brown, Kerene A., and Derek J. Wilson. "Bottom-up hydrogen deuterium exchange mass spectrometry: data analysis and interpretation." Analyst 142, no. 16 (2017): 2874–86. http://dx.doi.org/10.1039/c7an00662d.

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8

Zhdanova, Polina V., Alexander A. Chernonosov, Daria V. Prokhorova, Grigory A. Stepanov, Lyubov Yu Kanazhevskaya, and Vladimir V. Koval. "Probing the Dynamics of Streptococcus pyogenes Cas9 Endonuclease Bound to the sgRNA Complex Using Hydrogen-Deuterium Exchange Mass Spectrometry." International Journal of Molecular Sciences 23, no. 3 (January 20, 2022): 1129. http://dx.doi.org/10.3390/ijms23031129.

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The Cas9 endonuclease is an essential component of the CRISPR–Cas-based genome editing tools. The attainment of high specificity and efficiency of Cas9 during targetted DNA cleavage is the main problem that limits the clinical application of the CRISPR–Cas9 system. A deep understanding of the Cas9 mechanism and its structural-functional relationships is required to develop strategies for precise gene editing. Here, we present the first attempt to describe the solution structure of Cas9 from S. pyogenes using hydrogen-deuterium exchange mass spectrometry (HDX-MS) coupled to molecular dynamics simulations. HDX data revealed multiple protein regions with deuterium uptake levels varying from low to high. By analysing the difference in relative deuterium uptake by apoCas9 and its complex with sgRNA, we identified peptides involved in the complex formation and possible changes in the protein conformation. The REC3 domain was shown to undergo the most prominent conformational change upon enzyme-RNA interactions. Detection of the HDX in two forms of the enzyme provided detailed information about changes in the Cas9 structure induced by sgRNA binding and quantified the extent of the changes. The study demonstrates the practical utility of HDX-MS for the elucidation of mechanistic aspects of Cas9 functioning.

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9

Dapic, Irena, Ivone Jakasa, Renata Kobetic, and Lidija Brkljacic. "Characterization of Ceramides with Phytosphingosine Backbone by Hydrogen-deuterium Exchange Mass Spectrometry." Croatica chemica acta 92, no. 3 (2019): 411–17. http://dx.doi.org/10.5562/cca3506.

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Ceramides are a lipid subclass of the sphingolipids that show large structural diversity. Structural characterization of the ceramides (CERs) can lead to better understanding of their role and function in the biological system. Here we investigated representatives of NP (CER III, CER IIIB) and AP ceramide classes (CER VI) that contain phytosphingosine (P) backbone. Ceramides were characterized in positive ionization mode by hydrogen-deuterium exchange mass spectrometry (HDX-MS). Fragmentation in positive ionization mode of the CER III and CER VI resulted in abundant ions assigned to phytosphingosine moiety at m/z 282, 300 and 318. HDX-MS of fragments showed increase in m/z of corresponding ions confirming the exchange of deuterium. In negative ionisation spectra multiple fragment ions were assigned to fatty acyl (RCOO–) moiety. Presence of RCOO– allowed unambiguous identification of CER III and CER IIIB which were distinguished by the presence of double bond on fatty acyl chain.

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10

Benhaim, Mark, Kelly K. Lee, and Miklos Guttman. "Tracking Higher Order Protein Structure by Hydrogen-Deuterium Exchange Mass Spectrometry." Protein & Peptide Letters 26, no. 1 (February 13, 2019): 16–26. http://dx.doi.org/10.2174/0929866526666181212165037.

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Background: Structural biology has provided a fundamental understanding of protein structure and mechanistic insight into their function. However, high-resolution structures alone are insufficient for a complete understanding of protein behavior. Higher energy conformations, conformational changes, and subtle structural fluctuations that underlie the proper function of proteins are often difficult to probe using traditional structural approaches. Hydrogen/Deuterium Exchange with Mass Spectrometry (HDX-MS) provides a way to probe the accessibility of backbone amide protons under native conditions, which reports on local structural dynamics of solution protein structure that can be used to track complex structural rearrangements that occur in the course of a protein’s function. Conclusion: In the last 20 years the advances in labeling techniques, sample preparation, instrumentation, and data analysis have enabled HDX to gain insights into very complex biological systems. Analysis of challenging targets such as membrane protein complexes is now feasible and the field is paving the way to the analysis of more and more complex systems.

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11

Eysseric, Emmanuel, Xavier Bellerose, Jean-Michel Lavoie, and Pedro A. Segura. "Post-column hydrogen–deuterium exchange technique to assist in the identification of small organic molecules by mass spectrometry." Canadian Journal of Chemistry 94, no. 9 (September 2016): 781–87. http://dx.doi.org/10.1139/cjc-2016-0281.

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To improve the certainty that a specific small organic molecule has been detected in a given sample by high-resolution mass spectrometry, other techniques that give conclusive evidence about the chemical structure of a compound like nuclear magnetic resonance or complementary information on its composition such as hydrogen–deuterium exchange (HDX) are often necessary. This study presents a systematic investigation that aims to improve the applicability of post-column HDX for those purposes. Key parameters like mobile-phase flow rates, volume percentage of H2O in the mobile phase, and D2O addition flow rates were optimized to provide an isotopic pattern that allows the accurate determination of the number of exchangeable hydrogen atoms in small organic molecules. A loop injection setup was used to emulate chromatographic conditions in the optimization process, and trimethoprim, a widely used anti-infective, was used as test compounds for the experiments. As expected, results showed that deuteration percentage decreased with a higher mobile-phase flow rate and increased with higher D2O flow rate. The post-column HDX technique was then validated with extracts of samples of river water and plants separated by liquid chromatography in hydrophilic interaction or reversed-phase modes. Mass spectra showed a completely visible isotopic pattern that allowed assessing correctly and unambiguously the number of exchangeable hydrogens in the compounds of interest. This study shows that post-column HDX can be used as a complementary technique to identify unknown small organic molecules in complex matrices. The current paper proposes an efficient, cost-effective, versatile technique of HDX that is helpful to assign a unique structure to a given high-resolution mass spectrometry signal.

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12

Lau, Andy M. C., Zainab Ahdash, Chloe Martens, and Argyris Politis. "Deuteros: software for rapid analysis and visualization of data from differential hydrogen deuterium exchange-mass spectrometry." Bioinformatics 35, no. 17 (January 14, 2019): 3171–73. http://dx.doi.org/10.1093/bioinformatics/btz022.

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Abstract Summary Hydrogen deuterium exchange-mass spectrometry (HDX-MS) has emerged as a powerful technique for interrogating the conformational dynamics of proteins and their complexes. Currently, analysis of HDX-MS data remains a laborious procedure, mainly due to the lack of streamlined software to process the large datasets. We present Deuteros which is a standalone software designed to be coupled with Waters DynamX HDX data analysis software, allowing the rapid analysis and visualization of data from differential HDX-MS. Availability and implementation Deuteros is open-source and can be downloaded from https://github.com/andymlau/Deuteros, under the Apache 2.0 license. Written in MATLAB and supported on both Windows and MacOS. Requires the MATLAB runtime library. According to the Wellcome Trust and UK research councils' Common Principles on Data Policy on data, software and materials management and sharing, all data supporting this study will be openly available from the software repository.

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13

Lorenzen, Kristina, and Tony Pawson. "HDX-MS takes centre stage at unravelling kinase dynamics." Biochemical Society Transactions 42, no. 1 (January 23, 2014): 145–50. http://dx.doi.org/10.1042/bst20130250.

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In recent years, HDX-MS (hydrogen–deuterium exchange coupled to MS) on biomolecules has evolved from a niche technique to a powerful method in the investigation of protein dynamics. Protein kinases, in particular, represent an area of active study using this technique owing to their well-characterized protein structures and their relevance to diseases such as cancer, immune disorders and neurodegenerative defects. In the present review, we describe how HDX-MS has revealed important dynamic properties of protein kinases and provided insight into the mechanisms of drug binding.

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14

Zhang, Jianyu, Jeremy L. Balsbaugh, Shuaihua Gao, Natalie G. Ahn, and Judith P. Klinman. "Hydrogen deuterium exchange defines catalytically linked regions of protein flexibility in the catechol O-methyltransferase reaction." Proceedings of the National Academy of Sciences 117, no. 20 (May 5, 2020): 10797–805. http://dx.doi.org/10.1073/pnas.1917219117.

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Human catechol O-methyltransferase (COMT) has emerged as a model for understanding enzyme-catalyzed methyl transfer from S-adenosylmethionine (AdoMet) to small-molecule catecholate acceptors. Mutation of a single residue (tyrosine 68) behind the methyl-bearing sulfonium of AdoMet was previously shown to impair COMT activity by interfering with methyl donor–acceptor compaction within the activated ground state of the wild type enzyme [J. Zhang, H. J. Kulik, T. J. Martinez, J. P. Klinman, Proc. Natl. Acad. Sci. U.S.A. 112, 7954–7959 (2015)]. This predicts the involvement of spatially defined protein dynamical effects that further tune the donor/acceptor distance and geometry as well as the electrostatics of the reactants. Here, we present a hydrogen/deuterium exchange (HDX)-mass spectrometric study of wild type and mutant COMT, comparing temperature dependences of HDX against corresponding kinetic and cofactor binding parameters. The data show that the impaired Tyr68Ala mutant displays similar breaks in Arrhenius plots of both kinetic and HDX properties that are absent in the wild type enzyme. The spatial resolution of HDX below a break point of 15–20 °C indicates changes in flexibility across ∼40% of the protein structure that is confined primarily to the periphery of the AdoMet binding site. Above 20 °C, Tyr68Ala behaves more like WT in HDX, but its rate and enthalpic barrier remain significantly altered. The impairment of catalysis by Tyr68Ala can be understood in the context of a mutationally induced alteration in protein motions that becomes manifest along and perpendicular to the primary group transfer coordinate.

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15

Freato, Nadia, Eduard H. T. M. Ebberink, Josse van Galen, Caroline Fribourg, Mariëtte Boon-Spijker, Floris P. J. van Alphen, Alexander B. Meijer, Maartje van den Biggelaar, and Koen Mertens. "Factor VIII–driven changes in activated factor IX explored by hydrogen-deuterium exchange mass spectrometry." Blood 136, no. 23 (December 3, 2020): 2703–14. http://dx.doi.org/10.1182/blood.2020005593.

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Abstract The assembly of the enzyme-activated factor IX (FIXa) with its cofactor, activated factor VIII (FVIIIa) is a crucial event in the coagulation cascade. The absence or dysfunction of either enzyme or cofactor severely compromises hemostasis and causes hemophilia. FIXa is a notoriously inefficient enzyme that needs FVIIIa to drive its hemostatic potential, by a mechanism that has remained largely elusive to date. In this study, we employed hydrogen–deuterium exchange-mass spectrometry (HDX-MS) to investigate how FIXa responds to assembly with FVIIIa in the presence of phospholipids. This revealed a complex pattern of changes that partially overlaps with those changes that occur upon occupation of the substrate-binding site by an active site-directed inhibitor. Among the changes driven by both cofactor and substrate, HDX-MS highlighted several surface loops that have been implicated in allosteric networks in related coagulation enzymes. Inspection of FVIIIa-specific changes indicated that 3 helices are involved in FIXa–FVIIIa assembly. These are part of a basic interface that is also known as exosite II. Mutagenesis of basic residues herein, followed by functional studies, identified this interface as an extended FVIIIa-interactive patch. HDX-MS was also applied to recombinant FIXa variants that are associated with severe hemophilia B. This revealed that single amino acid substitutions can silence the extended network of FVIIIa-driven allosteric changes. We conclude that HDX-MS has the potential to visualize the functional impact of disease-associated mutations on enzyme–cofactor complexes in the hemostatic system.

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16

Rajabi, Khadijeh. "Microsecond pulsed hydrogen/deuterium exchange of electrosprayed ubiquitin ions stored in a linear ion trap." Physical Chemistry Chemical Physics 17, no. 5 (2015): 3607–16. http://dx.doi.org/10.1039/c4cp04716h.

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17

Meeks, Shannon L., Alexander M. Sevy, John F. Healey, Wei Deng, P. Clint Spiegel, and Renhao Li. "Cooperative Binding Of Anti-Factor VIII Inhibitors and Induced Conformational Change Detected By Hydrogen-Deuterium Exchange Mass Spectrometry." Blood 122, no. 21 (November 15, 2013): 1088. http://dx.doi.org/10.1182/blood.v122.21.1088.1088.

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Abstract The development of anti-factor VIII (fVIII) antibodies (inhibitors) is a significant complication in the management of patients with hemophilia A leading to significant increases in morbidity and treatment cost. Using a panel of anti-fVIII monoclonal antibodies to different epitopes on fVIII, we recently have shown that epitope specificity, inhibitor kinetics, and time to maximum inhibition are more important than inhibitor titer in predicting response to fVIII and the combination of fVIII and recombinant factor VIIa. Thus the ability to quickly map the epitope spectrum of patient plasma using a clinically feasible assay may fundamentally change how clinicians approach the treatment of high-titer inhibitor patients. To this end, we have characterized the binding epitopes of 4 monoclonal antibodies (MAbs) targeted against fVIII C2 domain by hydrogen-deuterium exchange coupled with liquid chromatography-mass spectrometry (HDX-MS). MAbs included both classical (inhibiting binding of fVIII to von Willebrand factor and phospholipid) and non-classical inhibitors (inhibiting activation of fVIII), which target separate regions of fVIII C2 domain and have distinct inhibitory mechanisms. HDX-MS analysis showed clear differences in binding patterns between classical and non-classical inhibitors, centering on the protruding hydrophobic loop at Met2199. The binding epitopes of classical and non-classical inhibitors mapped by HDX-MS agree well with previously reported ones characterized by structural studies and mutagenesis analysis. Classical and non-classical inhibitors could be distinguished by a limited subset of C2-derived peptides, simplifying analysis significantly. In addition, HDX-MS was able to detect subtle differences in binding patterns of various classical inhibitors, based on the HDX protection pattern around the hydrophobic loop at Leu2251. Interestingly, two MAbs, G99 and 3E6, exhibited an observable shift in HDX protection when bound to C2 as a ternary complex, as opposed to when bound individually, thus providing evidence for cooperative binding of these two MAbs (Figure 1). In summary, our results demonstrate the effectiveness and robustness of the HDX-MS method in the rapid epitope mapping of fVIII inhibitors. This method can be expanded to map epitopes of inhibitors against other domains of fVIII, potentially leading to a more personalized treatment of hemophilia A patients.Figure 1Figure 1. Disclosures: No relevant conflicts of interest to declare.

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18

Ozohanics, Oliver, and Attila Ambrus. "Hydrogen-Deuterium Exchange Mass Spectrometry: A Novel Structural Biology Approach to Structure, Dynamics and Interactions of Proteins and Their Complexes." Life 10, no. 11 (November 15, 2020): 286. http://dx.doi.org/10.3390/life10110286.

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Hydrogen/Deuterium eXchange Mass Spectrometry (HDX-MS) is a rapidly evolving technique for analyzing structural features and dynamic properties of proteins. It may stand alone or serve as a complementary method to cryo-electron-microscopy (EM) or other structural biology approaches. HDX-MS is capable of providing information on individual proteins as well as large protein complexes. Owing to recent methodological advancements and improving availability of instrumentation, HDX-MS is becoming a routine technique for some applications. When dealing with samples of low to medium complexity and sizes of less than 150 kDa, conformation and ligand interaction analyses by HDX-MS are already almost routine applications. This is also well supported by the rapid evolution of the computational (software) background that facilitates the analysis of the obtained experimental data. HDX-MS can cope at times with analytes that are difficult to tackle by any other approach. Large complexes like viral capsids as well as disordered proteins can also be analyzed by this method. HDX-MS has recently become an established tool in the drug discovery process and biopharmaceutical development, as it is now also capable of dissecting post-translational modifications and membrane proteins. This mini review provides the reader with an introduction to the technique and a brief overview of the most common applications. Furthermore, the most challenging likely applications, the analyses of glycosylated and membrane proteins, are also highlighted.

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Ständer, Susanne, Laura R. Grauslund, Maria Scarselli, Nathalie Norais, and Kasper Rand. "Epitope Mapping of Polyclonal Antibodies by Hydrogen–Deuterium Exchange Mass Spectrometry (HDX-MS)." Analytical Chemistry 93, no. 34 (July 24, 2021): 11669–78. http://dx.doi.org/10.1021/acs.analchem.1c00696.

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20

Masson, Glenn R., Meredith L. Jenkins, and John E. Burke. "An overview of hydrogen deuterium exchange mass spectrometry (HDX-MS) in drug discovery." Expert Opinion on Drug Discovery 12, no. 10 (August 17, 2017): 981–94. http://dx.doi.org/10.1080/17460441.2017.1363734.

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Vadas, Oscar, and John E. Burke. "Probing the dynamic regulation of peripheral membrane proteins using hydrogen deuterium exchange–MS (HDX–MS)." Biochemical Society Transactions 43, no. 5 (October 1, 2015): 773–86. http://dx.doi.org/10.1042/bst20150065.

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Many cellular signalling events are controlled by the selective recruitment of protein complexes to membranes. Determining the molecular basis for how lipid signalling complexes are recruited, assembled and regulated on specific membrane compartments has remained challenging due to the difficulty of working in conditions mimicking native biological membrane environments. Enzyme recruitment to membranes is controlled by a variety of regulatory mechanisms, including binding to specific lipid species, protein–protein interactions, membrane curvature, as well as post-translational modifications. A powerful tool to study the regulation of membrane signalling enzymes and complexes is hydrogen deuterium exchange–MS (HDX–MS), a technique that allows for the interrogation of protein dynamics upon membrane binding and recruitment. This review will highlight the theory and development of HDX–MS and its application to examine the molecular basis of lipid signalling enzymes, specifically the regulation and activation of phosphoinositide 3-kinases (PI3Ks).

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Marciano, David P., Dana S. Kuruvilla, Bruce D. Pascal та Patrick R. Griffin. "Identification of Bexarotene as a PPARγAntagonist with HDX". PPAR Research 2015 (2015): 1–6. http://dx.doi.org/10.1155/2015/254560.

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The retinoid x receptors (RXRs) are the pharmacological target of Bexarotene, an antineoplastic agent indicated for the treatment of cutaneous T cell lymphoma (CTCL). The RXRs form heterodimers with several nuclear receptors (NRs), including peroxisome proliferator-activated receptor gamma (PPARγ), to regulate target gene expression through cooperative recruitment of transcriptional machinery. Here we have applied hydrogen/deuterium exchange (HDX) mass spectrometry to characterize the effects of Bexarotene on the conformational plasticity of the intact RXRα:PPARγheterodimer. Interestingly, addition of Bexarotene to PPARγin the absence of RXRαinduced protection from solvent exchange, suggesting direct receptor binding. This observation was confirmed using a competitive binding assay. Furthermore, Bexarotene functioned as a PPARγantagonist able to alter rosiglitazone induced transactivation in a cell based promoter:reporter transactivation assay. Together these results highlight the complex polypharmacology of lipophilic NR targeted small molecules and the utility of HDX for identifying and characterizing these interactions.

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Puchała, Weronika, Michał Burdukiewicz, Michał Kistowski, Katarzyna A. Dąbrowska, Aleksandra E. Badaczewska-Dawid, Dominik Cysewski, and Michał Dadlez. "HaDeX: an R package and web-server for analysis of data from hydrogen–deuterium exchange mass spectrometry experiments." Bioinformatics 36, no. 16 (June 24, 2020): 4516–18. http://dx.doi.org/10.1093/bioinformatics/btaa587.

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Abstract Motivation Hydrogen–deuterium mass spectrometry (HDX-MS) is a rapidly developing technique for monitoring dynamics and interactions of proteins. The development of new devices has to be followed with new software suites addressing emerging standards in data analysis. Results We propose HaDeX, a novel tool for processing, analysis and visualization of HDX-MS experiments. HaDeX supports a reproducible analytical process, including data exploration, quality control and generation of publication-quality figures. Availability and implementation HaDeX is available primarily as a web-server (http://mslab-ibb.pl/shiny/HaDeX/), but its all functionalities are also accessible as the R package (https://CRAN.R-project.org/package=HaDeX) and standalone software (https://sourceforge.net/projects/HaDeX/). Supplementary information Supplementary data are available at Bioinformatics online.

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Narang, Dominic, Cristina Lento, and Derek J. Wilson. "HDX-MS: An Analytical Tool to Capture Protein Motion in Action." Biomedicines 8, no. 7 (July 17, 2020): 224. http://dx.doi.org/10.3390/biomedicines8070224.

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Virtually all protein functions in the cell, including pathogenic processes, require coordinated motion of atoms or domains, i.e., conformational dynamics. Understanding protein dynamics is therefore critical both for drug development and to learn about the underlying molecular causes of many diseases. Hydrogen–Deuterium Exchange Mass Spectrometry (HDX-MS) provides valuable information about protein dynamics, which is highly complementary to the static picture provided by conventional high-resolution structural tools (i.e., X-ray crystallography and structural NMR). The amount of protein required to carry out HDX-MS experiments is a fraction of the amount required by alternative biophysical techniques, which are also usually lower resolution. Use of HDX-MS is growing quickly both in industry and academia, and it has been successfully used in numerous drug and vaccine development efforts, with important roles in understanding allosteric effects and mapping binding sites.

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Hossain, Belal M., Douglas A. Simmons, and Lars Konermann. "Do electrospray mass spectra reflect the ligand binding state of proteins in solution?" Canadian Journal of Chemistry 83, no. 11 (November 1, 2005): 1953–60. http://dx.doi.org/10.1139/v05-194.

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Electrospray ionization (ESI) mass spectrometry (MS) has become a popular tool for monitoring ligandprotein and proteinprotein interactions. Due to the "gentle" nature of the ionization process, it is often possible to transfer weakly bound complexes into the gas phase, thus making them amenable to MS detection. One problem with this technique is the potential occurrence of fragmentation events during ESI. Also, some analytes tend to cluster together during ionization, thus forming nonspecific gas-phase assemblies that do not represent solution-phase complexes. In this work, we implemented a hydrogendeuterium exchange (HDX) approach that can reveal whether or not the free and (or) bound constituents of a complex observed in ESI-MS reflect the binding situation in solution. Proteins are subjected to ESI immediately following an isotopic labeling pulse; only ligand-free and ligand-bound protein ions that were formed directly from the corresponding solution-phase species showed different HDX levels. Using myoglobin as a model system, it is demonstrated that this approach can readily distinguish scenarios where the hemeprotein interactions were disrupted in solution from those where dissociation of the complex occurred in the gas phase. Experiments on cytochrome c strongly suggest that dimeric protein ions observed in ESI-MS reflect aggregates that were formed in solution.Key words: electrospray mass spectrometry, ligandprotein interaction, noncovalent complex, hydrogendeuterium exchange, protein folding.

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Nieman, Marvin T., Maria de la Fuente, and Masaru Miyagi. "Analysis of Protease Activated Receptor 4 (PAR4) By Histidine Hydrogen Deuterium Exchange." Blood 124, no. 21 (December 6, 2014): 4166. http://dx.doi.org/10.1182/blood.v124.21.4166.4166.

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Abstract Protease activated receptors (PARs) are G-protein coupled receptors (GPCRs) that have central roles in the cardiovascular system. Numerous studies have focused on the signaling pathways mediated by PARs; however, the structural rearrangements that initiate these pathways are unknown. PARs are activated when proteolytic cleavage exposes a tethered ligand that interacts with a binding pocket on the receptor. Since the ligand is attached to the receptor, there are likely substantial conformational changes that occur during PAR activation. We now describe the first biophysical studies examining this unique mode of activation using PAR4. PAR4 is one of the thrombin receptors on platelets and is required for sustained platelet activation. Recent data from our laboratory and others suggest that PAR4 has the potential to be a therapeutic target. To examine the tethered ligand activation mechanism in general and further examine PAR4 as a target, we have expressed and purified PAR4 that does not contain additional stabilizing sequences from Sf9 cells for biophysical studies. The current study uses histidine-hydrogen deuterium exchange (His-HDX). His-HDX is a structural mass spectrometry technique that relies on the slow exchange of the C2 carbon on the imidazole ring of histidine. PAR4 has nine histidine residues that are well spaced throughout the protein, which gives us a global view of solvent accessible and non-accessible regions. Unique peptides were generated that contained each of the histidine residues (His136, His159, His180, His229, His240, His269, His280, His306, and His380). These peptides were used to determine the t1/2 for each His residue in full length or thrombin cleaved PAR4. The t1/2 values for His159 and His269 were greater than 40 hours in both states indicating that these two histidine residues are not exposed to solvent in either case. In contrast, the thrombin cleaved PAR4 had a 2-fold increase (p > 0.01) in t1/2 values observed for four histidine residues (His180, His229, His240, and His380) demonstrating that these regions have a decrease in solvent accessibility upon thrombin treatment. These residues are spread throughout the protein, which indicates that PAR4 undergoes a global conformational change and the overall structure becomes more rigid. In agreement with these data, thrombin cleaved PAR4 is more resistant to protease digestion by chymotrypsin. The half-life was extended 4-fold over the uncleaved PAR4. We are also able to make conclusions regarding specific regions of PAR4. His229 is located in extracellular loop 2 near the proposed ligand-binding site and has a 2-fold increase in t1/2 following thrombin treatment. These data suggest that the tethered ligand may be binding to this region and protecting it from exchange. The studies described here are the first to examine the tethered ligand activation mechanism for a PAR family member using biophysical approaches. These studies shed light on the overall conformational changes that follow activation of PARs by a protease. Finally, the His-HDX used for purified PAR4 lays important groundwork that will allow us to examine PAR4 and other platelet GPCRs in their native membrane environment using this and related mass spectrometry approaches. Disclosures No relevant conflicts of interest to declare.

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Masson, Glenn R., John E. Burke, Natalie G. Ahn, Ganesh S. Anand, Christoph Borchers, Sébastien Brier, George M. Bou-Assaf, et al. "Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments." Nature Methods 16, no. 7 (June 27, 2019): 595–602. http://dx.doi.org/10.1038/s41592-019-0459-y.

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Rashid, Shaan, Sean Overton, Bihac Mazigh, and Paul M. Mayer. "Dual-spray hydrogen/deuterium exchange (HDX) reactions: A new method of probing protein structure." Rapid Communications in Mass Spectrometry 30, no. 13 (June 8, 2016): 1505–12. http://dx.doi.org/10.1002/rcm.7591.

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Paci, Emanuele, Roman Tuma, Simon Skinner, and Jeanine J. Houwing-Duistermaat. "Can Hydrogen-Deuterium Exchange Rates at Single Residue Level Be Obtained from HDX-MS Data?" Biophysical Journal 116, no. 3 (February 2019): 288a—289a. http://dx.doi.org/10.1016/j.bpj.2018.11.1558.

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30

Alluri, Ravi Kumar, Nicholas Arce, Philip Klenotic, Chih Chia Su, Suman Kundu, Edward Yu, Renhao Li та Keith R. McCrae. "Characterization of Recombinant β2GPI Using Hydrogen-Deuterium Exchange and X-Ray Crystallography". Blood 134, Supplement_1 (13 листопада 2019): 1105. http://dx.doi.org/10.1182/blood-2019-131386.

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Introduction: Antiphospholipid syndrome (APS) is an autoimmune disorder caused by "antiphospholipid" antibodies (aPL) directed against β2-glycoprotein I (β2GPI). However, there remains significant inconsistency in measurements of anti-β2GPI antibodies, possibly due to different sources of β2GPI. To address this issue and others, we have developed a system in which recombinant β2GPI (rβ2GPI) is expressed in mammalian (HEK293) cells using a lentiviral construct with a CMV promoter, in which the signal peptide of β2GPI has been mutated. In previous work, we demonstrated that patient-derived anti-β2GPI antibodies recognize rβ2GPI similarly to purified plasma β2GPI. Plasma Β2GPI has been crystallized and found to be in a "fishhook" or "J" conformation, however the protein was isolated under harsh conditions that included perchloric acid precipitation. Moreover, electron microscopy and other approaches have suggested that β2GPI may adopt several conformations in addition to the fishhook, including circular and other intermediate forms. Methods: cDNA encoding full length APOH (the gene encoding β2GPI), was cloned into pLenti CMV Puro DEST. Lentivirus was produced using the Lentiviral Gateway Expression kit and stable cell lines developed by transducing HEK293 cells with lentivirus encoding APOH. Cell lines were grown in suspension, and supernatants containing secreted full length β2GPI were purified using a hitrap-heparin column. Anti-β2GPI antibody binding to rβ2GPI was determined using a standard β2GPI ELISA. For hydrogen-deuterium exchange (HDX) mass spectrometry, proteins were in 10 mM phosphate buffer, 99.9% D2O in an autosampler at 20 °C. After 10-10,000 seconds of incubation, the reaction was quenched. Sequences of peptic fragments were identified and mass assignment for each peptide without deuterium exchange was checked manually. The average of relative fractional deuterium uptake for each amide proton residue was calculated using HDsite equations. Crystallization of plasma derived and recombinant β2GPI-WT was performed using a hanging-drop vapor diffusion method at 4°C in 150 mM NaCl and 50 mM Tris-HCl pH 7.3. Results: After transduction with lentiviral β2GPI constructs, HEK-293 cells efficiently secreted rβ2GPI-WT, site-directed mutants and several domains constructs. Hydrogen-deuterium exchange (HDX) analysis of plasma derived and rβ2GPI-WT revealed that both have very similar surface deuterium exchange rates and heat map profiles (Figure 1A, B). In both plasma derived and rβ2GPI, limited digestion of domains D-I, II and III by pepsin was observed, consistent with other sushi domain containing proteins. However, solid coverage of domain IV and V was obtained and the heat maps generated for both plasma derived and rβ2GPI-WT were very similar, suggesting similar structure and conformation. Moreover, although crystallization of plasma derived and rβ2GPI-WT revealed differences in crystal size and appearance, X-ray structural analysis to a resolution of 3.1 Å (Figure 1C, D) demonstrated that both plasma derived and rβ2GPI had the same structure and conformation under the conditions employed. Conclusion: These studies demonstrate that recombinant β2GPI produced in mammalian cells following lentiviral transduction has a similar structure and conformation as plasma-derived β2GPI. This methodology may be used for additional mutagenesis studies to better define the underlying structural features regulating the conformation of β2GPI as well as its interactions with other proteins, diagnostic utility and functions. Disclosures McCrae: Sanofi Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Rigel Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Pfizer Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Dova Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

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Zhang, Ying, Xiu Chen, Linzhou Zhang, Quan Shi, Suoqi Zhao, and Chunming Xu. "Specification of the nitrogen functional group in a hydrotreated petroleum molecule using hydrogen/deuterium exchange electrospray ionization high-resolution mass spectrometry." Analyst 145, no. 13 (2020): 4442–51. http://dx.doi.org/10.1039/d0an00772b.

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Harris, Matthew J., Deepika Raghavan, and Antoni J. Borysik. "Quantitative Evaluation of Native Protein Folds and Assemblies by Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS)." Journal of The American Society for Mass Spectrometry 30, no. 1 (October 2, 2018): 58–66. http://dx.doi.org/10.1007/s13361-018-2070-3.

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Petrotchenko, Evgeniy V., and Christoph H. Borchers. "HDX Match Software for the Data Analysis of Top-Down ECD-FTMS Hydrogen/Deuterium Exchange Experiments." Journal of The American Society for Mass Spectrometry 26, no. 11 (July 11, 2015): 1895–98. http://dx.doi.org/10.1007/s13361-015-1213-z.

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Zhu, Shaolong, J. Larry Campbell, Igor Chernushevich, J. C. Yves Le Blanc, and Derek J. Wilson. "Differential Mobility Spectrometry-Hydrogen Deuterium Exchange (DMS-HDX) as a Probe of Protein Conformation in Solution." Journal of The American Society for Mass Spectrometry 27, no. 6 (March 10, 2016): 991–99. http://dx.doi.org/10.1007/s13361-016-1364-6.

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35

Cryar, Adam, Kate Groves, and Milena Quaglia. "Online Hydrogen-Deuterium Exchange Traveling Wave Ion Mobility Mass Spectrometry (HDX-IM-MS): a Systematic Evaluation." Journal of The American Society for Mass Spectrometry 28, no. 6 (April 3, 2017): 1192–202. http://dx.doi.org/10.1007/s13361-017-1633-z.

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Fang, Mulin, Zhe Wang, Kathleen Norris, Judith A. James, Si Wu, and Kenneth Smith. "Hydrogen-Deuterium Exchange Mass Spectrometry Reveals a Novel Binding Region of a Neutralizing Fully Human Monoclonal Antibody to Anthrax Protective Antigen." Toxins 14, no. 2 (January 25, 2022): 92. http://dx.doi.org/10.3390/toxins14020092.

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Anthrax vaccine adsorbed (AVA) containing protective antigen (PA) is the only FDA-approved anthrax vaccine in the United States. Characterization of the binding of AVA-induced anti-PA human antibodies against the PA antigen after vaccination is crucial to understanding mechanisms of the AVA-elicited humoral immune response. Hydrogen deuterium exchange mass spectrometry (HDX-MS) is often coupled with a short liquid chromatography gradient (e.g., 5–10 min) for the characterization of protein interactions. We recently developed a long-gradient (e.g., 90 min), sub-zero temperature, ultra-high performance liquid chromatography HDX-MS (UPLC-HDX-MS) platform that has significantly increased separation power and limited back-exchange for the analysis of protein samples with high complexity. In this study, we demonstrated the utility of this platform for mapping antibody–antigen epitopes by examining four fully human monoclonal antibodies to anthrax PA. Antibody p1C03, with limited neutralizing activity in vivo, bound to a region on domain 1A of PA. p6C04 and p1A06, with no neutralizing activities, bound to the same helix on domain 3 to prevent oligomerization of PA. We found p6C01 strongly bound to domain 3 on a different helix region. We also identified a secondary epitope for p6C01, which likely leads to the blocking of furin cleavage of PA after p6C01 binding. This novel binding of p6C01 results in highly neutralizing activity. This is the first report of this distinct binding mechanism for a highly neutralizing fully human antibody to anthrax protective antigen. Studying such epitopes can facilitate the development of novel therapeutics against anthrax.

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Park, Ji Young, Nguyen Minh Duc, Dong Kyun Kim, Su Youn Lee, Sheng Li, Min-Duk Seo, Virgil L. Woods, and Ka Young Chung. "Different conformational dynamics of PDZ1 and PDZ2 in full-length EBP50 analyzed by hydrogen/deuterium exchange mass spectrometry." Biochemistry and Cell Biology 93, no. 4 (August 2015): 290–97. http://dx.doi.org/10.1139/bcb-2014-0145.

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Ezrin-radixin-moesin-binding protein 50 (EBP50) is a scaffolding protein expressed in polarized epithelial cells in various organs, including the liver, kidney, and small intestine, in which it regulates the trafficking and targeting cellular proteins. EBP50 contains two postsynaptic density-95/disk-large/ZO-1 homology (PDZ) domains (e.g., PDZ1 and PDZ2) and an ezrin/radixin/moesin-binding (EB) domain. PDZ domains are one of the major scaffolding domains regulating protein–protein interactions with critical biological roles in cell polarity, migration, proliferation, recognition, and cell–cell interaction. PDZ1 and PDZ2 in EBP50 have different ligand selectivity, although several high-resolution structural studies of isolated PDZ1 and PDZ2 showed similar structures. We studied the conformations of full-length EBP50 and isolated PDZ1 and PDZ2 using hydrogen/deuterium exchange mass spectrometry (HDX-MS). The deuterium uptake profiles of isolated PDZ1 and PDZ2 were similar to those of full-length EBP50. Interestingly, PDZ1 was more dynamic than PDZ2, and these PDZ domains underwent different conformational changes upon ligand binding. These results might explain the differences in ligand-selectivity between PDZ1 and PDZ2.

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Kulman, John, Susan Tsutakawa, George Bou-Assaf, Steven Berkowitz, David R. Light, Adam Mezo, Deping Wang, et al. "Structural Comparability Between Recombinant FVIII-Fc and Its Isolated FVIII and Fc Constituents." Blood 120, no. 21 (November 16, 2012): 1135. http://dx.doi.org/10.1182/blood.v120.21.1135.1135.

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Abstract Abstract 1135 rFVIIIFc is a recombinant fusion protein comprising human B domain-deleted (BDD) coagulation factor VIII covalently attached to the Fc domain of human immunoglobulin G1 (IgG1). rFVIIIFc is currently in a phase 3 clinical study as a novel long-lasting FVIII molecule designed to potentially reduce dosing frequency and provide prolonged protection from bleeds for patients with hemophilia A receiving prophylaxis or on-demand treatment, or undergoing surgery. In this study, we evaluated the structural comparability of BDD rFVIII and rFVIIIFc using small angle X-ray scattering (SAXS) and hydrogen/deuterium exchange (HDX) mass spectrometry. SAXS analysis of BDD rFVIII and rFVIIIFc, performed at the Advanced Light Source Synchrotron, provided the electron pair radius distribution of each molecule and accurate but inherently low resolution structural information. Both BDD rFVIII and rFVIIIFc were monomeric in solution. BDD rFVIII had a radius of gyration (Rg) of 39.7 Å, compared with a Rg of 38 Å calculated from its published atomic coordinates (2R7E.pdb). rFVIIIFc had a larger Rg of 51 Å, which was consistent with a simple computational model of BDD rFVIII tethered to Fc that predicted an Rg of 50 Å. HDX mass spectrometric analysis yielded 94% coverage of the BDD rFVIII component and 87% coverage of the Fc component. Rates of deuterium exchange within peptides derived from each of these components in the context of the rFVIIIFc fusion protein were very similar to those of the isolated BDD rFVIII and Fc constituents. Taken together, the results of SAXS and HDX analyses support the conclusion that fusion of the Fc moiety to BDD rFVIII does not alter the higher order structure of either component. Disclosures: Kulman: Biogen Idec: Employment. Bou-Assaf:Biogen Idec: Employment. Berkowitz:Biogen Idec: Employment. Light:biogenidec: Employment. Mezo:Biogen Idec: Employment. Wang:Biogen Idec: Employment. Whitlow:Colabrativ Inc.: Employment; Biogen Idec: Consultancy. Culyba:Biogen Idec: Employment. Peters:Biogen Idec: Employment.

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Gramlich, Marius, Henry C. W. Hays, Scott Crichton, Philipp D. Kaiser, Anne Heine, Nicole Schneiderhan-Marra, Ulrich Rothbauer, Dieter Stoll, Sandra Maier, and Anne Zeck. "HDX-MS for Epitope Characterization of a Therapeutic ANTIBODY Candidate on the Calcium-Binding Protein Annexin-A1." Antibodies 10, no. 1 (March 19, 2021): 11. http://dx.doi.org/10.3390/antib10010011.

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Annexin-A1 (ANXA1) belongs to a class of highly homologous Ca2+-dependent phospholipid-binding proteins. Its structure consists of a core region composed of four homologous repeats arranged in a compact, hydrolysis-resistant structure and an N-terminal region with a Ca2+-dependent conformation. ANXA1 is involved in several processes, including cell proliferation, apoptosis, metastasis, and the inflammatory response. Therefore, the development of antibodies blocking selected regions on ANXA1 holds great potential for the development of novel therapeutics treating inflammatory and cancer diseases. Here, we report the interaction site between an ANXA1-specific antibody known to inhibit T cell activation without adverse cytotoxic effects and ANXA1 using amide hydrogen–deuterium exchange mass spectrometry (HDX-MS). For the epitope determination, we applied two bottom-up HDX-MS approaches with pepsin digestion in solution and immobilized on beads. Both strategies revealed the interaction region within domain III of ANXA1 in Ca2+-bound conformation. The antibody-binding region correlates with the hydrophobic binding pocket of the N-terminal domain formed in the absence of calcium. This study demonstrates that even cryptic and flexible binding regions can be studied by HDX-MS, allowing a fast and efficient determination of the binding sites of antibodies which will help to define a mode of action profile for their use in therapy.

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Lento, Cristina, Gerald F. Audette, and Derek J. Wilson. "Time-resolved electrospray mass spectrometry — a brief history." Canadian Journal of Chemistry 93, no. 1 (January 2015): 7–12. http://dx.doi.org/10.1139/cjc-2014-0260.

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This review describes the evolution of time-resolved electrospray ionization mass spectrometry (TRESI-MS), a technology that was developed in large part at Western University. TRESI-MS was initially designed to characterize rapid chemical and biochemical reactions occurring on the millisecond time scale without need for a chromophore. Early TRESI-MS setups usually consisted of continuous-flow rapid mixers with a fixed tee for analysis of a single time point, and later adjustable reaction chamber devices allowing for automatic tracking of the reaction over time. Advances in instrumentation design over the years have resulted in improved time resolution, with microfluidic device implementation allowing for coupling to hydrogen−deuterium exchange (HDX) experiments. Areas of application that will be discussed include the investigation of protein folding intermediates, identification of enzyme−substrate intermediates in the pre-steady state, and the use of time-resolved HDX to study the dynamics of weakly structured protein regions. While some limitations still persist with the method, the continued development of TRESI-MS and related approaches paves the way to a promising future and the study of unexplored application areas.

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Abbattista, Ramona, Ilario Losito, Graziana Basile, Andrea Castellaneta, Giovanni Ventura, Cosima Damiana Calvano, and Tommaso R. I. Cataldi. "Hydrogen/Deuterium Exchange Mass Spectrometry for Probing the Isomeric Forms of Oleocanthal and Oleacin in Extra Virgin Olive Oils." Molecules 28, no. 5 (February 22, 2023): 2066. http://dx.doi.org/10.3390/molecules28052066.

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Reversed-phase liquid chromatography and electrospray ionization with Fourier-transform single and tandem mass spectrometry (RPLC-ESI-FTMS and FTMS/MS) were employed for the structural characterization of oleocanthal (OLEO) and oleacin (OLEA), two of the most important bioactive secoiridoids occurring in extra virgin olive oils (EVOOs). The existence of several isoforms of OLEO and OLEA was inferred from the chromatographic separation, accompanied, in the case of OLEA, by minor peaks due to oxidized OLEO recognized as oleocanthalic acid isoforms. The detailed analysis of the product ion tandem MS spectra of deprotonated molecules ([M-H]−) was unable to clarify the correlation between chromatographic peaks and specific OLEO/OLEA isoforms, including two types of predominant dialdehydic compounds, named Open Forms II, containing a double bond between carbon atoms C8 and C10, and a group of diasteroisomeric closed-structure (i.e., cyclic) isoforms, named Closed Forms I. This issue was addressed by H/D exchange (HDX) experiments on labile H atoms of OLEO and OLEA isoforms, performed using deuterated water as a co-solvent in the mobile phase. HDX unveiled the presence of stable di-enolic tautomers, in turn providing key evidence for the occurrence, as prevailing isoforms, of Open Forms II of OLEO and OLEA, different from those usually considered so far as the main isoforms of both secoiridoids (having a C=C bond between C8 and C9). It is expected that the new structural details inferred for the prevailing isoforms of OLEO and OLEA will help in understanding the remarkable bioactivity exhibited by the two compounds.

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Eisinger, Martin Lorenz, Aline Ricarda Dörrbaum, Hartmut Michel, Etana Padan, and Julian David Langer. "Ligand-induced conformational dynamics of the Escherichia coli Na+/H+ antiporter NhaA revealed by hydrogen/deuterium exchange mass spectrometry." Proceedings of the National Academy of Sciences 114, no. 44 (October 16, 2017): 11691–96. http://dx.doi.org/10.1073/pnas.1703422114.

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Na+/H+ antiporters comprise a family of membrane proteins evolutionarily conserved in all kingdoms of life and play an essential role in cellular ion homeostasis. The NhaA crystal structure of Escherichia coli has become the paradigm for this class of secondary active transporters. However, structural data are only available at low pH, where NhaA is inactive. Here, we adapted hydrogen/deuterium-exchange mass spectrometry (HDX-MS) to analyze conformational changes in NhaA upon Li+ binding at physiological pH. Our analysis revealed a global conformational change in NhaA with two sets of movements around an immobile binding site. Based on these results, we propose a model for the ion translocation mechanism that explains previously controversial data for this antiporter. Furthermore, these findings contribute to our understanding of related human transporters that have been linked to various diseases.

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Trabjerg, Esben, Zeinab E. Nazari, and Kasper D. Rand. "Conformational analysis of complex protein states by hydrogen/deuterium exchange mass spectrometry (HDX-MS): Challenges and emerging solutions." TrAC Trends in Analytical Chemistry 106 (September 2018): 125–38. http://dx.doi.org/10.1016/j.trac.2018.06.008.

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Acter, Thamina, Yunju Cho, Sungji Kim, Arif Ahmed, Byungjoo Kim, and Sunghwan Kim. "Optimization and Application of APCI Hydrogen–Deuterium Exchange Mass Spectrometry (HDX MS) for the Speciation of Nitrogen Compounds." Journal of The American Society for Mass Spectrometry 26, no. 9 (June 27, 2015): 1522–31. http://dx.doi.org/10.1007/s13361-015-1166-2.

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Lin, Yanchun, and Michael L. Gross. "Mass Spectrometry-Based Structural Proteomics for Metal Ion/Protein Binding Studies." Biomolecules 12, no. 1 (January 15, 2022): 135. http://dx.doi.org/10.3390/biom12010135.

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Metal ions are critical for the biological and physiological functions of many proteins. Mass spectrometry (MS)-based structural proteomics is an ever-growing field that has been adopted to study protein and metal ion interactions. Native MS offers information on metal binding and its stoichiometry. Footprinting approaches coupled with MS, including hydrogen/deuterium exchange (HDX), “fast photochemical oxidation of proteins” (FPOP) and targeted amino-acid labeling, identify binding sites and regions undergoing conformational changes. MS-based titration methods, including “protein–ligand interactions by mass spectrometry, titration and HD exchange” (PLIMSTEX) and “ligand titration, fast photochemical oxidation of proteins and mass spectrometry” (LITPOMS), afford binding stoichiometry, binding affinity, and binding order. These MS-based structural proteomics approaches, their applications to answer questions regarding metal ion protein interactions, their limitations, and recent and potential improvements are discussed here. This review serves as a demonstration of the capabilities of these tools and as an introduction to wider applications to solve other questions.

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Lee, Sunghyup, Yunju Cho, and Sunghwan Kim. "Development and Application of a Software Tool for the Interpretation of Organic Mixtures' Spectra - Hydrogen Deuterium Exchange (STORM-HDX) to Interpret APPI HDX MS Spectra." Bulletin of the Korean Chemical Society 35, no. 3 (March 20, 2014): 749–52. http://dx.doi.org/10.5012/bkcs.2014.35.3.749.

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47

Zhu, Shaolong, Peter Liuni, Tricia Chen, Camille Houy, Derek J. Wilson, and D. Andrew James. "Epitope screening using Hydrogen/Deuterium Exchange Mass Spectrometry (HDX‐MS): An accelerated workflow for evaluation of lead monoclonal antibodies." Biotechnology Journal 17, no. 2 (November 21, 2021): 2100358. http://dx.doi.org/10.1002/biot.202100358.

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Wu, Shanshan, Tam T. T. N. Nguyen, Olga V. Moroz, Johan P. Turkenburg, Jens E. Nielsen, Keith S. Wilson, Kasper D. Rand, and Kaare Teilum. "Conformational heterogeneity of Savinase from NMR, HDX-MS and X-ray diffraction analysis." PeerJ 8 (June 26, 2020): e9408. http://dx.doi.org/10.7717/peerj.9408.

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Background Several examples have emerged of enzymes where slow conformational changes are of key importance for function and where low populated conformations in the resting enzyme resemble the conformations of intermediate states in the catalytic process. Previous work on the subtilisin protease, Savinase, from Bacillus lentus by NMR spectroscopy suggested that this enzyme undergoes slow conformational dynamics around the substrate binding site. However, the functional importance of such dynamics is unknown. Methods Here we have probed the conformational heterogeneity in Savinase by following the temperature dependent chemical shift changes. In addition, we have measured changes in the local stability of the enzyme when the inhibitor phenylmethylsulfonyl fluoride is bound using hydrogen-deuterium exchange mass spectrometry (HDX-MS). Finally, we have used X-ray crystallography to compare electron densities collected at cryogenic and ambient temperatures and searched for possible low populated alternative conformations in the crystals. Results The NMR temperature titration shows that Savinase is most flexible around the active site, but no distinct alternative states could be identified. The HDX shows that modification of Savinase with inhibitor has very little impact on the stability of hydrogen bonds and solvent accessibility of the backbone. The most pronounced structural heterogeneities detected in the diffraction data are limited to alternative side-chain rotamers and a short peptide segment that has an alternative main-chain conformation in the crystal at cryo conditions. Collectively, our data show that there is very little structural heterogeneity in the resting state of Savinase and hence that Savinase does not rely on conformational selection to drive the catalytic process.

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Lee, Su Youn, Hee-Seop Yoo, Hye-Seung Choi, Ka Young Chung, and Min-Duk Seo. "Structural and dynamic insights into the subtype-specific IP3-binding mechanism of the IP3 receptor." Biochemical Journal 473, no. 20 (October 11, 2016): 3533–43. http://dx.doi.org/10.1042/bcj20160539.

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There are three subtypes of vertebrate inositol 1,4,5-trisphosphate (IP3) receptor (IP3R), a Ca2+-release channel on the ER membrane — IP3R1, IP3R2, and IP3R3 — each of which has a distinctive role in disease development. To determine the subtype-specific IP3-binding mechanism, we compared the thermodynamics, thermal stability, and conformational dynamics between the N-terminal regions of IP3R1 (IP3R1-NT) and IP3R3 (IP3R3-NT) by performing circular dichroism (CD), isothermal titration calorimetry (ITC), and hydrogen–deuterium exchange mass spectrometry (HDX-MS). Previously determined crystal structures of IP3R1-NT and HDX-MS results from this study revealed that both IP3R1 and IP3R3 adopt a similar IP3-binding mechanism. However, several regions, including the α- and β-interfaces, of IP3R1-NT and IP3R3-NT show significantly different conformational dynamics upon IP3 binding, which may explain the different IP3-binding affinities between the subtypes. The importance of the interfaces for subtype-specific IP3 binding is also supported by the different dynamic conformations of the two subtypes in the apo-states. Furthermore, IP3R1-NT and IP3R3-NT show different IP3-binding affinities and thermal stabilities, but share similar thermodynamic properties for IP3 binding. These results collectively provide new insights into the mechanism underlying IP3 binding to IP3Rs and the subtype-specific regulatory mechanism.

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Park, Kyung-Tae, Maria T. Villar, Antonio Artigues, and Joe Lutkenhaus. "MinE conformational dynamics regulate membrane binding, MinD interaction, and Min oscillation." Proceedings of the National Academy of Sciences 114, no. 29 (June 26, 2017): 7497–504. http://dx.doi.org/10.1073/pnas.1707385114.

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InEscherichia coliMinE induces MinC/MinD to oscillate between the ends of the cell, contributing to the precise placement of the Z ring at midcell. To do this, MinE undergoes a remarkable conformational change from a latent 6β-stranded form that diffuses in the cytoplasm to an active 4β-stranded form bound to the membrane and MinD. How this conformational switch occurs is not known. Here, using hydrogen–deuterium exchange coupled to mass spectrometry (HDX-MS) we rule out a model in which the two forms are in rapid equilibrium. Furthermore, HDX-MS revealed that a MinE mutant (D45A/V49A), previously shown to produce an aberrant oscillation and unable to assemble a MinE ring, is more rigid than WT MinE. This mutant has a defect in interaction with MinD, suggesting it has difficulty in switching to the active form. Analysis of intragenic suppressors of this mutant suggests it has difficulty in releasing the N-terminal membrane targeting sequences (MTS). These results indicate that the dynamic association of the MTS with the β-sheet is fine-tuned to balance MinE’s need to sense MinD on the membrane with its need to diffuse in the cytoplasm, both of which are necessary for the oscillation. The results lead to models for MinE activation and MinE ring formation.

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