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Hydrogen exchange mass spectrometry

Erik Schneider, Katrina Brendle, Patrick Jäger, Patrick Weis, Manfred M Kappes
We present gas-phase structures of dimers of MnIII and FeIII meso-tetra(4-sulfonatophenyl)porphyrin multianions with various amounts of sodium and hydrogen counterions. The structural assignments are achieved by combining mass spectrometry, ion mobility measurements, quantum chemical calculations, and trajectory method collision cross section calculations. For a common charge state, we observe significant topological variations in the dimer structures of [(MTPPS)2 +nX](6-n)- (M=MnIII , FeIII ; X=H, Na; n = 1-3) induced by replacing hydrogen counterions by sodium...
April 17, 2018: Journal of the American Society for Mass Spectrometry
Siva K Angalakurthi, Connie A Tenorio, Michael Blaber, C Russell Middaugh
In this study, we examined the local dynamics of acidic fibroblast growth factor (FGF-1) as well as the binding sites of various polyanions including poly-sulfates (heparin and low MW heparin) and poly-phosphates (phytic acid and ATP) using hydrogen-deuterium exchange mass spectrometry (HX-MS). For local dynamics, results are analyzed at the peptide level as well as in terms of buried amides employing crystallographic B-factors and compared with a residue level heat map generated from HX-MS results. Results show that strand 4 and 5 and the turn between them to be the most flexible regions as was previously seen by NMR...
April 12, 2018: Protein Science: a Publication of the Protein Society
Cesar A Ramirez-Sarmiento, Elizabeth A Komives
Hydrogen-deuterium exchange mass spectrometry (HDXMS) has emerged as a powerful approach for revealing folding and allostery in protein-protein interactions. The advent of higher resolution mass spectrometers combined with ion mobility separation and ultra-high liquid chromatographic separations have allowed the complete coverage of large protein sequences and multi-protein complexes. Liquid-handling robots have improved the reproducibility and accurate temperature control of the sample preparation. Many researchers are also appreciating the power of combining biophysical approaches such as stopped-flow fluorescence, single molecule FRET, and molecular dynamics simulations with HDXMS...
April 5, 2018: Methods: a Companion to Methods in Enzymology
Thomas E Bohl, Pek Ieong, John K Lee, Thomas Lee, Jayakanth Kankanala, Ke Shi, Özlem Demir, Kayo Kurahashi, Rommie E Amaro, Zhengqiang Wang, Hideki Aihara
Gram-negative bacteria are surrounded by a secondary membrane of which the outer leaflet is composed of the glycolipid lipopolysaccharide (LPS), which guards against hydrophobic toxins, including many antibiotics. Therefore, LPS synthesis in bacteria is an attractive target for antibiotic development. LpxH is a pyrophosphatase involved in LPS synthesis, and previous structures revealed that LpxH has a helical cap that binds its lipid substrates. Here, crystallography and hydrogen-deuterium exchange mass spectrometry provided evidence for a highly flexible substrate-binding cap in LpxH...
April 6, 2018: Journal of Biological Chemistry
Dominic Narang, Wei Chen, Clarisse G Ricci, Elizabeth A Komives
The main NFκB transcription factor family members RelA-p50 heterodimer and RelA homodimer have different biological functions and show different transcriptional activation profiles. To investigate whether the two family members adopt a similar conformation in their free states, we performed hydrogen-deuterium exchange mass spectrometry (HDXMS), all-atom molecular dynamics (MD) simulations and stopped-flow binding kinetics experiments. Surprisingly, the N-terminal DNA binding domains adopt an open conformation in RelA-p50 but a closed conformation in RelA homodimer...
April 3, 2018: Journal of Molecular Biology
Christina Dal Magro, Patrick Keller, Annika Kotter, Stephan Werner, Victor Duarte, Virginie Marchand, Michael Ignarski, Anja Freiwald, Roman-Ulrich Müller, Christoph Dieterich, Yuri Motorin, Falk Butter, Mohammed Atta, Mark Helm
Recently discovered new chemical entities in RNA modifications have provided surprises with respect to functional groups that enlarge the chemical space of RNA. Using LC-MS, we found over one hundred signals of RNA constituents that contained a ribose moiety in tRNAs from E. coli. Feeding experiments with variegated stable isotope labeled compounds identified 37 compounds, representing new structures of RNA modifications. One structure was elucidated by deuterium exchange and high resolution mass spectrometry...
April 6, 2018: Angewandte Chemie
Aaron A Cook, Wei Deng, Jinqi Ren, Renhao Li, John Sondek, Wolfgang Bergmeier
Platelets are recruited to sites of vascular injury where they are activated and aggregate to form a hemostatic plug. This process requires the activation of the small GTPase Rap1B by its cognate guanine nucleotide exchange factor CalDAG-GEFI. Studies on platelet function suggest that CalDAG-GEFI activity is regulated by changes in cytosolic calcium, but the exact molecular mechanism is poorly understood. Here, we show that purified CalDAG-GEFI is autoinhibited and directly regulated by calcium. Substitutions of putative calcium-binding residues within the canonical EF hands of CalDAG-GEFI diminish its capacity to activate Rap1B...
April 5, 2018: Journal of Biological Chemistry
Danilo Donnarumma, Claudio Maestri, Pietro Ivan Giammarinaro, Luigi Capriotti, Erika Bartolini, Daniele Veggi, Roberto Petracca, Maria Scarselli, Nathalie Norais
Hydrogen Deuterium exchange (HDx) associated with Mass Spectrometry (MS) is emerging as a powerful tool to provide conformational information on membrane proteins. Unfortunately, as for X-ray diffraction and NMR, HDx performed on reconstituted in vitro systems might not always reflect the in vivo environment. Outer Membrane Vesicles naturally released by E. coli were used to carry out analysis of native OmpF through HDx-MS. A new protocol compatible with HDx analysis and avoiding hindrance from the lipid contents was set-up...
April 5, 2018: Journal of Proteome Research
Brent A Kochert, Roxana E Iacob, Thomas E Wales, Alexandros Makriyannis, John R Engen
Hydrogen-deuterium exchange (HDX) mass spectrometry (MS) can provide valuable information about binding, allostery, and other conformational effects of interaction in protein complexes. For protein-ligand complexes, where the ligand may be a small molecule, peptide, nucleotide, or another protein(s), a typical experiment measures HDX in the protein alone and then compares that with HDX for the protein when part of the complex. Multiple factors are critical in the design and implementation of such experiments, including thoughtful consideration of the percent protein bound, the effects of the labeling protocol on the protein complex, and the dynamic range of the analysis method...
2018: Methods in Molecular Biology
Yury Kostyukevich, Thamina Acter, Alexander Zherebker, Arif Ahmed, Sunghwan Kim, Eugene Nikolaev
The isotopic exchange approach is in use since the first observation of such reactions in 1933 by Lewis. This approach allows the investigation of the pathways of chemical and biochemical reactions, determination of structure, composition, and conformation of molecules. Mass spectrometry has now become one of the most important analytical tools for the monitoring of the isotopic exchange reactions. Investigation of conformational dynamics of proteins, quantitative measurements, obtaining chemical, and structural information about individual compounds of the complex natural mixtures are mainly based on the use of isotope exchange in combination with high resolution mass spectrometry...
March 30, 2018: Mass Spectrometry Reviews
Eva Illes-Toth, Don L Rempel, Michael L Gross
Alpha-synuclein (aS) forms toxic intermediates ranging from small oligomers and protofibrils to large amyloid fibrils. Understanding the time course of aS fibril formation and the role played by its regions is critical for therapeutic intervention. Here, we used pulsed hydrogen-deuterium-exchange and mass spectrometry (HDX-MS) for the first time to probe kinetic intermediates of the full aS aggregation in vitro, achieving kinetic snapshots containing spatially resolved protein information about critical stages...
March 30, 2018: ACS Chemical Neuroscience
Dokyong Kim, Harsimran Singh, Yuyuan Dai, Guangchao Dong, Laura S Busenlehner, Franklin Wayne Outten, Patrick A Frantom
In the Suf Fe-S cluster assembly pathway, the activity of the cysteine desulfurase, SufS, is regulated by interactions with the accessory sulfotransferase protein, SufE. SufE has been shown to stimulate SufS activity, likely through inducing conformational changes in the SufS active site that promote the desulfurase step and by acting as an efficient persulfide acceptor in the transpersulfuration step. Previous results point toward an additional level of regulation through a 'half-sites' mechanism that affects the stoichiometry and affinity for SufE as the dimeric SufS shifts between desulfurase and transpersulfuration activities...
March 28, 2018: Biochemistry
Andrea L Wang, Ying Zhou, Michael J Palmieri, Gang G Hao
Recombinant human erythropoietin (EPO) is a therapeutic glycoprotein widely used for treating anemia. EPO glycans carry extensive sialylation and the level of the modification is known to affect receptor binding, protein stability and pharmacokinetics. Nonetheless, a detailed understanding of the effects of sialylation on EPO conformation and dynamics is still lacking. Here we investigate the changes to EPO dynamics following enzymatic trimming of terminal sialic acid by amide hydrogen deuterium exchange mass spectrometry (HDX-MS)...
March 16, 2018: Journal of Pharmaceutical and Biomedical Analysis
Martin Lorenz Eisinger, Laiyin Nie, Aline Ricarda Dörrbaum, Julian David Langer, Hartmut Michel
Multidrug resistance (MDR) in bacterial pathogens has become a severe threat to public health. Membrane transporters of the multidrug and toxic compound extrusion (MATE) family contribute critically to MDR, making them promising drug targets. Despite recent advances, structures in different conformations and the mechanistic details of their antiport cycle are still elusive. Here we studied NorM_PS, a representative MATE transporter from Pseudomonas stutzeri, using biochemical assays in combination with hydrogen/deuterium exchange-mass spectrometry (HDX-MS)...
March 16, 2018: Journal of Molecular Biology
Ravi Kant, Aida Llauró, Vamseedhar Rayaprolu, Shefah Qazi, Pedro J de Pablo, Trevor Douglas, Brian Bothner
The capsid of P22 bacteriophage undergoes a series of structural transitions during maturation that guide it from spherical to icosahedral morphology. The transitions include the release of scaffold proteins and capsid expansion. Although P22 maturation has been investigated for decades, a unified model that incorporates thermodynamic and biophysical analyses is not available. A general and specific model of icosahedral capsid maturation are of significant interest to theoreticians searching for fundamental principles as well as virologists and material scientists seeking to alter maturation to their advantage...
March 14, 2018: Biochimica et Biophysica Acta
Laura J Byrnes, Yingrong Xu, Xiayang Qiu, Justin D Hall, Graham M West
Cystic Fibrosis (CF) is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). Mutations associated with CF cause loss-of-function in CFTR leading to salt imbalance in epithelial tissues. Kalydeco (also called VX-770 or ivacaftor) was approved for CF treatment in 2012 but little is known regarding the compound's interactions with CFTR including the site of binding or mechanisms of action. In this study we use hydrogen/deuterium exchange (HDX) coupled with mass spectrometry to assess the conformational dynamics of a thermostabilized form of CFTR in apo and ligand-bound states...
March 16, 2018: Scientific Reports
Kinga Nyíri, Haydyn D T Mertens, Borbála Tihanyi, Gergely N Nagy, Bianka Kőhegyi, Judit Matejka, Matthew J Harris, Judit E Szabó, Veronika Papp-Kádár, Veronika Németh-Pongrácz, Olivér Ozohanics, Károly Vékey, Dmitri I Svergun, Antoni J Borysik, Beáta G Vértessy
Human deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase), essential for DNA integrity, acts as a survival factor for tumor cells and is a target for cancer chemotherapy. Here we report that the Staphylococcal repressor protein StlSaPIBov1 (Stl) forms strong complex with human dUTPase. Functional analysis reveals that this interaction results in significant reduction of both dUTPase enzymatic activity and DNA binding capability of Stl. We conducted structural studies to understand the mechanism of this mutual inhibition...
March 12, 2018: Scientific Reports
Tamar Shamai Yamin, Hagit Prihed, Avital Shifrovitch, Shai Dagan, Avi Weissberg
A novel analytical technique for the structural elucidation of compounds bearing a tertiary amine side chain via "in vial" instantaneous oxidation and liquid chromatography-mass spectrometry (LC-MS) was developed. A series of lidocaine homologues and benzimidazole derivatives with a major/single amine-representative base peak in both their EI-MS and ESI-MS/MS spectra were subjected to oxidation by a 0.1% solution of hydrogen peroxide )including several16 O/18 O exchange experiments(, followed by LC-ESI-MS/MS analysis...
March 9, 2018: Journal of Mass Spectrometry: JMS
Guang Xu, Jacek Stupak, Li Yang, Luokai Hu, Bo Guo, Jianjun Li
Mass spectrometry (MS) has played a vital role across a broad range of fields and applications in proteomics. The development of high-resolution MS for the past decades has significantly advanced biology, such as protein structure, function, post translational modification and global protein dynamics. In MS, two ionization techniques are often used-electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). ESI mass spectra are generally crowded by signals of multiple charge values for each molecular mass and an isotopic cluster for each nominal mass-to-charge (m/z) value...
March 8, 2018: Rapid Communications in Mass Spectrometry: RCM
Yoshitomo Hamuro, Sook Yen E
The technological goal of hydrogen/deuterium exchange-mass spectrometry (HDX-MS) is to determine backbone amide hydrogen exchange rates. The most critical challenge to achieve this goal is obtaining the deuterium incorporation in single-amide resolution, and gas-phase fragmentation may provide a universal solution. The gas-phase fragmentation may generate the daughter ions which differ by a single amino acid and the difference in deuterium incorporations in the two analogous ions can yield the deuterium incorporation at the sub-localized site...
March 2, 2018: Journal of the American Society for Mass Spectrometry
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