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hydrogen deuterium exchange

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
Longhui Gao, Serge Perato, Sébastien Garcia-Argote, Céline Taglang, Luis Miguel Martínez-Prieto, Céline Chollet, David-Alexandre Buisson, Vincent Dauvois, Philippe Lesot, Bruno Chaudret, Bernard Rousseau, Sophie Feuillastre, Grégory Pieters
We present here the first example of C(sp3 )-H activation directed by a sulfur atom. Based on this transformation catalyzed by Ru/C, we have developed a hydrogen isotope exchange reaction for the deuterium and tritium labelling of thioether substructures in complex molecules.
March 5, 2018: Chemical Communications: Chem Comm
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
Estefania Fernandez, Nurgun Kose, Melissa A Edeling, Jagat Adhikari, Gopal Sapparapu, Susana M Lazarte, Christopher A Nelson, Jennifer Govero, Michael L Gross, Daved H Fremont, James E Crowe, Michael S Diamond
Japanese encephalitis virus (JEV) remains a leading cause of viral encephalitis worldwide. Although JEV-specific antibodies have been described, an assessment of their ability to neutralize multiple genotypes of JEV has been limited. Here, we describe the development of a panel of mouse and human neutralizing monoclonal antibodies (MAbs) that inhibit infection in cell culture of four different JEV genotypes tested. Mechanism-of-action studies showed that many of these MAbs inhibited infection at a postattachment step, including blockade of virus fusion...
February 27, 2018: MBio
Ravi G Kurumbail, Graham M West, Venkatasubramanian Dharmarajan, Kris A Borzilleri, Jane M Withka, Jessica Ward, Allan R Reyes, Francis Rajamohan, Patrick R Griffin, Matthew F Calabrese
Protein-ligand interactions can be evaluated by a number of different biophysical methods. Here we describe some of the experimental methods that we have used to generate AMPK protein reagents and characterize its interactions with direct synthetic activators. Recombinant heterotrimeric AMPK complexes were generated using standard molecular biology methods by expression either in insect cells via infection with three different viruses or more routinely in Escherichia coli with a tricistronic expression vector...
2018: Methods in Molecular Biology
Shafqat Rasool, Naoto Soya, Luc Truong, Nathalie Croteau, Gergely L Lukacs, Jean-François Trempe
Mutations in PINK1 cause autosomal recessive Parkinson's disease (PD), a neurodegenerative movement disorder. PINK1 is a kinase that acts as a sensor of mitochondrial damage and initiates Parkin-mediated clearance of the damaged organelle. PINK1 phosphorylates Ser65 in both ubiquitin and the ubiquitin-like (Ubl) domain of Parkin, which stimulates its E3 ligase activity. Autophosphorylation of PINK1 is required for Parkin activation, but how this modulates the ubiquitin kinase activity is unclear. Here, we show that autophosphorylation of Tribolium castaneum PINK1 is required for substrate recognition...
February 23, 2018: EMBO Reports
Gavin R Owen, Doris Le, Stoyan Stoychev, Nichole M Cerutti, Maria Papathanasopoulos
CD4, a membrane glycoprotein expressed by specific leukocytes, plays a vital role in the human immune response and acts as a primary receptor for HIV entry. Of its four ecto-domains (D1-D4), D1, D2, and D4 each contain a distinctive disulfide bond. Whereas the disulfides of D1 and D4 are more traditional in nature, providing structural functions, that of D2 is referred to as an "allosteric" disulfide due to its high dihedral strain energy and relative ease of reduction that is thought to regulate CD4 structure and function by shuffling its redox state...
February 19, 2018: Biochemical and Biophysical Research Communications
Vincent S Shaw, Hossein Mohammadiarani, Harish Vashisth, Richard R Neubig
Small-molecule inhibitor selectivity may be influenced by variation in dynamics among members of a protein family. Regulator of G-protein Signaling (RGS) proteins are a family that plays a key role in G-Protein Coupled Receptor (GPCR) signaling by binding to active Gα subunits and accelerating GTP hydrolysis, thereby terminating activity. Thiadiazolidinones (TDZDs) inhibit the RGS-Gα interaction by covalent modification of cysteine residues in RGS proteins. Some differences in specificity may be explained by differences in the complement of cysteines among RGS proteins...
February 20, 2018: Journal of the American Chemical Society
Quinlin M Hanson, Jeffrey R Carley, Tyler J Gilbreath, Brian C Smith, Eric S Underbakke
Nitric oxide synthase (NOS) is the primary generator of nitric oxide signals controlling diverse physiological processes such as neurotransmission and vasodilation. NOS activation is contingent on Ca2+ /calmodulin binding at a linker between its oxygenase and reductase domains to induce large conformational changes that orchestrate inter-domain electron transfer. However, the structural dynamics underlying activation of full-length NOS remain ambiguous. Employing hydrogen-deuterium exchange mass spectrometry, we reveal mechanisms underlying neuronal NOS (nNOS) activation by calmodulin and regulation by phosphorylation...
February 16, 2018: Journal of Molecular Biology
Abdelrahim Zoued, Jean-Pierre Duneau, Eric Durand, Alexandre P España, Laure Journet, Françoise Guerlesquin, Eric Cascales
The Type VI secretion system (T6SS) is a multiprotein complex used by bacteria to deliver effectors into target cells. The T6SS comprises a bacteriophage-like contractile tail structure anchored to the cell envelope by a membrane complex constituted of the TssJ outer membrane lipoprotein and the TssL and TssM inner membrane proteins. TssJ establishes contact with the periplasmic domain of TssM whereas the transmembrane segments of TssM and its cytoplasmic domain interact with TssL. TssL protrudes in the cytoplasm but is anchored by a C-terminal transmembrane helix (TMH)...
February 16, 2018: Journal of Molecular Biology
Yirui Guo, Anthony T Iavarone, Matthew M Cooper, Michael A Marletta
Heme-nitric oxide/oxygen binding (H-NOX) proteins are a group of hemoproteins that bind diatomic gas ligands such as nitric oxide (NO) and oxygen (O2 ). H-NOX proteins typically regulate histidine kinases (HK) located within the same operon. It has been reported that NO-bound H-NOXs inhibit cognate histidine kinase autophosphorylation in bacterial H-NOX/HK complexes, however, a detailed mechanism of NO-mediated regulation of the H-NOX/HK activity remains unknown. In this study, the binding interface of Vibrio cholerae (Vc) H-NOX/HK complex was characterized by hydrogen/deuterium exchange mass spectrometry (HDX-MS) and further validated by mutagenesis, leading to a new model for NO-dependent kinase inhibition...
February 19, 2018: Biochemistry
Ke Sherry Li, Liuqing Shi, Michael L Gross
Assessment of protein structure and interaction is crucial for understanding protein structure/function relationships. Compared to high-resolution structural tools, including X-ray crystallography, nuclear magnetic resonance (NMR), and cryo-EM, and traditional low-resolution methods, such as circular dichroism, UV-vis, and florescence spectroscopy, mass spectrometry (MS)-based protein footprinting affords medium-to-high resolution (i.e., regional and residue-specific insights) by taking advantage of proteomics methods focused on the primary structure...
February 16, 2018: Accounts of Chemical Research
Jingjie Mo, Renzhe Jin, Qingrong Yan, Izabela Sokolowska, Michael J Lewis, Ping Hu
Glycation has been observed in antibody therapeutics manufactured by the fed-batch fermentation process. It not only increases the heterogeneity of antibodies, but also potentially affects product safety and efficacy. In this study, non-glycated and glycated fractions enriched from a monoclonal antibody (mAb1) as well as glucose-stressed mAb1 were characterized using a variety of biochemical, biophysical and biological assays to determine the effects of glycation on the structure and function of mAb1. Glycation was detected at multiple lysine residues and reduced the antigen binding activity of mAb1...
February 13, 2018: MAbs
Regina Adão, Letícia M Zanphorlin, Tatiani B Lima, Dev Sriranganadane, Käthe M Dahlström, Glaucia M S Pinheiro, Fabio C Gozzo, Leandro R S Barbosa, Carlos H I Ramos
Proteostasis is dependent on the Hsp70/Hsp90 system (the two chaperones and their co-chaperones). Of these, Hop (Hsp70/Hsp90 organizing protein), also known as Sti1, forms an important scaffold to simultaneously binding to both Hsp70 and Hsp90. Hop/Sti1 has been implicated in several disease states, for instance cancer and transmissible spongiform encephalopathies. Therefore, human and yeast homologous have been better studied and information on plant homologous is still limited, even though plants are continuously exposed to environmental stress...
February 6, 2018: Journal of Proteomics
Shawn Sternisha, Peilu Liu, Alan G Marshall, Brian G Miller
Human glucokinase (GCK) acts as the body's primary glucose sensor and plays a critical role in glucose homeostatic maintenance. Gain-of-function mutations in gck produce hyperactive enzyme variants that cause congenital hyperinsulinism. Prior biochemical and biophysical studies suggest that activated disease variants can be segregated into two mechanistically distinct classes, termed α-type and β-type. Steady-state viscosity variation studies indicate that the kcat values of wild-type GCK and an α-type variant are partially diffusion-limited, whereas the kcat value of a β-type variant is viscosity-independent...
February 9, 2018: Biochemistry
Tobias Kromann-Hansen, Eva Louise Lange, Ida K Lund, Gunilla Høyer-Hansen, Peter A Andreasen, Elizabeth A Komives
The catalytic activity of trypsin-like serine proteases is in many cases regulated by conformational changes initiated by binding of physiological modulators to exosites located distantly from the active site. A trypsin-like serine protease of particular interest is urokinase-type plasminogen activator (uPA), which is involved in extracellular tissue remodeling processes. Herein, we used hydrogen/deuterium exchange mass spectrometry (HDXMS) to study regulation of activity in the catalytic domain of the murine version of uPA (muPA) by two muPA specific monoclonal antibodies...
2018: PloS One
Rupesh Bommana, Qing Chai, Christian Schöneich, William F Weiss, Ranajoy Majumdar
This work compares the conformational stability, backbone flexibility, and aggregation propensity of monomer and dimer fractions of an IgG1 monoclonal antibody (mAb) generated upon UVA light exposure for up to 72 hours collected by preparative size-exclusion chromatography (SEC), compared to unstressed control. UVA light exposure induced covalent aggregation, and fragmentation as measured by SEC, sodium dodecyl sulfate polyacrylamide gel electrophoresis and extensive oxidation of specific methionine residues (Met 257, Met 433, and Met 109) in both size fractions identified by reverse phase chromatography coupled to mass spectrometry...
January 30, 2018: Journal of Pharmaceutical Sciences
Philipp Högel, Alexander Götz, Felix Kuhne, Maximilian Ebert, Walter Stelzer, Kasper D Rand, Christina Scharnagl, Dieter Langosch
Flexible transmembrane helices frequently support the conformational transitions between different functional states of membrane proteins. While proline is well known to distort and destabilize transmembrane helices, the role of glycine is still debated. Here, we systematically investigated the effect of glycine on transmembrane helix flexibility by placing it at different sites within the otherwise uniform leucine/valine repeat sequence of the LV16 model helix. We show that amide deuterium/hydrogen exchange kinetics are increased near glycine...
February 1, 2018: Biochemistry
Gennady Khirich, Michael J Holliday, Jasper C Lin, Aditya Nandy
One-dimensional heteronuclear relaxation dispersion NMR spectroscopy at 13C natural abundance successfully characterized the dynamics of the hydrogen-deuterium exchange reaction occurring at the Nε position in L-arginine by monitoring Cδ in varying amounts of D2O. A small equilibrium isotope effect was observed and quantified, corresponding to ΔG = ‒0.14 kcal mol-1. A bimolecular rate constant of kD = 5.1 × 109 s-1M-1 was determined from the pH*-dependence of kex (where pH* is the direct electrode reading of pH in 10% D2O, and kex is the nuclear spin exchange rate constant), consistent with diffusion-controlled kinetics...
January 27, 2018: Journal of Physical Chemistry. B
Xiaoji Cao, Xue Cai, Weimin Mo
RATIONALE: The comparative study of higher-energy collisional dissociation(HCD) and collision-induced dissociation(CID) mechanisms for protonated cyclic indolyl α-amino esters in quadrupole/orbitrap(Q/Orbitrap) and quadrupole time-of-flight(QTOF) mass spectrometers, respectively, is helpful to study the structures and properties of biologically active indole derivatives using tandem mass spectrometry(MS/MS) technology. METHODS: HCD and CID experiments were carried out using electrospray ionization Q/Orbitrap MS and QTOFMS in positive ion mode, respectively...
January 25, 2018: Rapid Communications in Mass Spectrometry: RCM
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