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hydrogen deuterium exchange

Rane A Harrison, Jia Lu, Martin Carrasco, John Hunter, Anuj Manandhar, Sudershan Gondi, Kenneth D Westover, John R Engen
Structural dynamics of Ras proteins contribute to their activity in signal transduction cascades. Directly targeting Ras proteins with small molecules may rely on movement of a conserved structural motif, switch II. To understand Ras signaling and advance Ras targeting strategies, experimental methods to measure Ras dynamics are required. Here we demonstrate the utility of hydrogen-deuterium exchange mass spectrometry to measure Ras dynamics by studying representatives from two branches of the Ras superfamily, Ras and Rho...
October 14, 2016: Journal of Molecular Biology
Tycho E T Mevissen, Yogesh Kulathu, Monique P C Mulder, Paul P Geurink, Sarah L Maslen, Malte Gersch, Paul R Elliott, John E Burke, Bianca D M van Tol, Masato Akutsu, Farid El Oualid, Masato Kawasaki, Stefan M V Freund, Huib Ovaa, David Komander
The post-translational modification of proteins with polyubiquitin regulates virtually all aspects of cell biology. Eight distinct chain linkage types co-exist in polyubiquitin and are independently regulated in cells. This 'ubiquitin code' determines the fate of the modified protein. Deubiquitinating enzymes of the ovarian tumour (OTU) family regulate cellular signalling by targeting distinct linkage types within polyubiquitin, and understanding their mechanisms of linkage specificity gives fundamental insights into the ubiquitin system...
October 12, 2016: Nature
Ursula Schulze-Gahmen, Ignacia Echeverria, Goran Stjepanovic, Yun Bai, Huasong Lu, Dina Schneidman-Duhovny, Jennifer A Doudna, Qiang Zhou, Andrej Sali, James H Hurley
HIV-1 Tat hijacks the human superelongation complex (SEC) to promote proviral transcription. Here we report the 5.9 Å structure of HIV-1 TAR in complex with HIV-1 Tat and human AFF4, CDK9, and CycT1. The TAR central loop contacts the CycT1 Tat-TAR recognition motif (TRM) and the second Tat Zn(2+)-binding loop. Hydrogen-deuterium exchange (HDX) shows that AFF4 helix 2 is stabilized in the TAR complex despite not touching the RNA, explaining how it enhances TAR binding to the SEC 50-fold. RNA SHAPE and SAXS data were used to help model the extended (Tat Arginine-Rich Motif) ARM, which enters the TAR major groove between the bulge and the central loop...
October 12, 2016: ELife
In-Kang Song, Jae-Jin Lee, Jin-Hwan Cho, Jihye Jeong, Dong-Hae Shin, Kong-Joo Lee
Reactive oxygen species (ROS) are key molecules regulating various cellular processes. However, what the cellular targets of ROS are and how their functions are regulated is unclear. This study explored the cellular proteomic changes in response to oxidative stress using H2O2 in dose- and recovery time-dependent ways. We found discernible changes in 76 proteins appearing as 103 spots on 2D-PAGE. Of these, Prxs, DJ-1, UCH-L3 and Rla0 are readily oxidized in response to mild H2O2 stress, and then degraded and active proteins are newly synthesized during recovery...
October 5, 2016: Scientific Reports
Erika Darrah, AeRyon Kim, Xi Zhang, Tatiana Boronina, Robert N Cole, Andrea Fava, Jon T Giles, Clifton O Bingham Iii, Michael J Chalmers, Patrick R Griffin, Scheherazade Sadegh-Nasseri, Antony Rosen
Proteolysis of autoantigens can alter normal MHC class II antigen processing and has been implicated in the induction of autoimmune diseases. Many autoantigens are substrates for the protease granzyme B (GrB), but the mechanistic significance of this association is unknown. Peptidylarginine deiminase 4 (PAD4) is a frequent target of autoantibodies in patients with rheumatoid arthritis (RA) and a substrate for GrB. RA is strongly associated with specific MHC class II alleles, and elevated levels of GrB and PAD4 are found in the joints of RA patients, suggesting that GrB may alter presentation of PAD4 by RA-associated class II alleles...
October 4, 2016: Journal of Proteome Research
Silke von Horsten, Simon Straß, Nils Hellwig, Verena Gruth, Ramona Klasen, Andreas Mielcarek, Uwe Linne, Nina Morgner, Lars-Oliver Essen
Organisms developed different photoreceptors to be able to adapt to changing environmental light conditions. Phytochromes are red/far-red (r/fr) photochromic photoreceptors that belong to the classical photoreceptors along with cryptochromes and phototropins. They convert absorbed light into a biological signal by switching between two states in a light-dependent manner therefore enabling the light control downstream signalling. Their Pfr conformation is the biological active form in plants, but until now only a structure of the ground state (Pr) was solved...
October 3, 2016: Scientific Reports
Aiswarya Premchandar, Norbert Mücke, Jarosław Poznański, Tatjana Wedig, Magdalena Kaus-Drobek, Harald Herrmann, Michał Dadlez
Intermediate filaments (IF) are major constituents of the cytoskeleton of metazoan cells. They not only are responsible for the mechanical properties but also for various physiological activities in different cells and tissues. The building blocks of IFs are extended coiled-coil-forming proteins exhibiting a characteristic central α-helical domain (″rod″). The fundamental principles of the filament assembly mechanism and the network formation have been widely elucidated for the cytoplasmic IF protein vimentin...
September 30, 2016: Journal of Biological Chemistry
Wallace H Liu, Sarah C Roemer, Yeyun Zhou, Zih-Jie Shen, Briana K Dennehey, Jeremy L Balsbaugh, Jennifer C Liddle, Travis Nemkov, Natalie G Ahn, Kirk C Hansen, Jessica K Tyler, Mair Ea Churchill
The histone chaperone Chromatin Assembly Factor 1 (CAF-1) deposits tetrameric (H3/H4)2 histones onto newly-synthesized DNA during DNA replication. To understand the mechanism of the tri-subunit CAF-1 complex in this process, we investigated the protein-protein interactions within the CAF-1-H3/H4 architecture using biophysical and biochemical approaches. Hydrogen/deuterium exchange and chemical cross-linking coupled to mass spectrometry reveal interactions that are essential for CAF-1 function in budding yeast, and importantly indicate that the Cac1 subunit functions as a scaffold within the CAF-1-H3/H4 complex...
September 30, 2016: ELife
Alexey V Gribenko, Kevin Parris, Lidia Mosyak, Sheng Li, Luke Handke, Julio C Hawkins, Elena Severina, Yury V Matsuka, Annaliesa S Anderson
The Staphylococcus aureus manganese transporter protein MntC is under investigation as a component of a prophylactic S.aureus vaccine. Passive immunization with monoclonal antibodies mAB 305-78-7 and mAB 305-101-8 produced using MntC was shown to significantly reduce S. aureus burden in an infant rat model of infection. Earlier interference mapping suggested that a total of 23 monoclonal antibodies generated against MntC could be subdivided into three interference groups, representing three independent immunogenic regions...
September 2016: PLoS Pathogens
Binhan Yu, Daiwen Yang
Proteins are dynamic over a wide range of timescales, but determining the number of distinct dynamic processes and identifying functionally relevant dynamics are still challenging. Here we present the study on human intestinal fatty acid binding protein (hIFABP) using a novel analysis of (15)N relaxation dispersion (RD) and chemical shift saturation transfer (CEST) experiments. Through combined analysis of the two types of experiments, we found that hIFABP exists in a four-state equilibrium in which three minor states interconvert directly with the major state...
September 28, 2016: Scientific Reports
Jordi Redondo, Carlos Jaime, Albert Marqués
The use of deuterated drug bioisosteres to obtain superior pharmacokinetic properties or to investigate biotransformations at the molecular level is a growing field of pharmaceutical research. This work presents a NMR study on the deuteration of three structurally related antisecretory proton-pump inhibitors, the sodium salts of esomeprazole, 1, pantoprazole, 2, and rabeprazole, 3. It has been found that the methylene adjacent to the sulfinyl group displays stereoselective deuteration when the sodium salts of these products are dissolved at room temperature in D2O or CD3OD, a process that also occurs very efficiently in DMSO-d6 (a solvent considered non-deuterating) if a catalytic amount of NaOH is added...
November 30, 2016: Journal of Pharmaceutical and Biomedical Analysis
Bin Deng, Cristina Lento, Derek J Wilson
Protein therapeutics have emerged as a major class of biopharmaceuticals over the past several decades, a trend that has motivated the advancement of bioanalytical technologies for protein therapeutic characterization. Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful and sensitive technique that can probe the higher order structure of proteins and has been used in the assessment and development of monoclonal antibodies (mAbs), antibody-drug conjugates (ADCs) and biosimilar antibodies. It has also been used to quantify protein-ligand, protein-receptor and other protein-protein interactions involved in signaling pathways...
October 12, 2016: Analytica Chimica Acta
Rui Fang, Pawel J Grobelny, Robin H Bogner, Michael J Pikal
Lyophilized proteins are generally stored below their glass transition temperature (Tg) to maintain long-term stability. Some proteins in the (pure) solid state showed a distinct endotherm at a temperature well below the glass transition, designated as a pre-Tg endotherm. The pre-Tg endothermic event has been linked with a transition in protein internal mobility. The aim of this study was to investigate the internal dynamics of 2 proteins, insulin and human growth hormone (hGH), both of which exhibit the pre-Tg endothermic event with onsets at 50°C-60°C...
November 2016: Journal of Pharmaceutical Sciences
Yohei Ohashi, Nicolas Soler, Miguel García Ortegón, Lufei Zhang, Marie L Kirsten, Olga Perisic, Glenn R Masson, John E Burke, Arjen J Jakobi, Apostolos A Apostolakis, Christopher M Johnson, Maki Ohashi, Nicholas T Ktistakis, Carsten Sachse, Roger L Williams
The phosphatidylinositol 3-kinase Vps34 is part of several protein complexes. The structural organization of heterotetrameric complexes is starting to emerge, but little is known about organization of additional accessory subunits that interact with these assemblies. Combining hydrogen-deuterium exchange mass spectrometry (HDX-MS), X-ray crystallography and electron microscopy (EM), we have characterized Atg38 and its human ortholog NRBF2, accessory components of complex I consisting of Vps15-Vps34-Vps30/Atg6-Atg14 (yeast) and PIK3R4/VPS15-PIK3C3/VPS34-BECN1/Beclin 1-ATG14 (human)...
September 14, 2016: Autophagy
Monika Kijewska, Karolina Radziszewska, Marta Cal, Mateusz Waliczek, Piotr Stefanowicz, Zbigniew Szewczuk
The combination of deuterium-hydrogen exchange (DHX) and mass spectrometry (MS) can be used for studying a high pressure denaturation (HPD) of proteins. Herein we present the results of investigations of the influence of glycation on the HPD of ubiquitin. Application of various values of pressure causes different degrees of protein unfolding, resulting in molecules with a different number of protons available for exchange with deuterons. The dependence of this number on pressure gives information on the denaturation state of a protein...
October 2016: Bioscience Reports
Modupeola A Sowole, Sarah Simpson, Yulia V Skovpen, David R J Palmer, Lars Konermann
Dihydrodipicolinate synthase is a tetrameric enzyme of the diaminopimelate pathway in bacteria and plants. The protein catalyzes the condensation of pyruvate (Pyr) and aspartate semialdehyde en route to the end product lysine (Lys). Dihydrodipicolinate synthase from Campylobacter jejuni (CjDHDPS) is allosterically inhibited by Lys. CjDHDPS is a promising antibiotic target, as highlighted by the recent development of a potent bis-lysine (bisLys) inhibitor. The mechanism whereby Lys and bisLys allosterically inhibit CjDHDPS remains poorly understood...
September 27, 2016: Biochemistry
Jürgen Claesen, Tomasz Burzykowski
Hydrogen/Deuterium exchange (HDX) has been applied, since the 1930s, as an analytical tool to study the structure and dynamics of (small) biomolecules. The popularity of using HDX to study proteins increased drastically in the last two decades due to the successful combination with mass spectrometry (MS). Together with this growth in popularity, several technological advances have been made, such as improved quenching and fragmentation. As a consequence of these experimental improvements and the increased use of protein-HDXMS, large amounts of complex data are generated, which require appropriate analysis...
September 7, 2016: Mass Spectrometry Reviews
Liping Yang, David Broderick, Yan Campbell, Adrian F Gombart, Jan F Stevens, Yuan Jiang, Victor L Hsu, William H Bisson, Claudia S Maier
We report on the molecular interactions of the farnesoid X receptor (FXR) with prenylflavonoids, an emerging class of FXR modulators. FXR is an attractive therapeutic target for mitigating metabolic syndromes (MetS) because FXR activates the inhibitory nuclear receptor, small heterodimer partner (SHP), thereby inhibiting both gluconeogenesis and de novo lipogenesis. We and others have shown that xanthohumol (XN), the principal prenylflavonoid of the hop plant (Humulus lupulus L.), is a FXR agonist based on its ability to affect lipid and glucose metabolism in vivo and to induces FXR target genes in biliary carcinoma cells and HEK293 cells...
September 3, 2016: Biochimica et Biophysica Acta
Aleš Marek, Martin H F Pedersen, Stine B Vogensen, Rasmus P Clausen, Bente Frølund, Tomáš Elbert
3-Hydroxycyclopent-1-ene-1-carboxylic acid (HOCPCA (1)) is a potent ligand for high-affinity γ-hydroxybutyric acid binding sites in the central nervous system. Various approaches to the introduction of a hydrogen label onto the HOCPCA skeleton are reported. The outcomes of the feasible C─H activation of olefin carbon (C-2) by iridium catalyst are compared with the reduction of the carbonyl group (C-3) by freshly prepared borodeuterides. The most efficient iridium catalysts proved to be Kerr bulky phosphine N-heterocyclic species providing outstanding deuterium enrichment (up to 91%) in a short period of time...
October 2016: Journal of Labelled Compounds & Radiopharmaceuticals
Xueyan Chen, Ugur Uzuner, Man Li, Weibing Shi, Joshua S Yuan, Susie Y Dai
Endocrine disrupters include a broad spectrum of chemicals such as industrial chemicals, natural estrogens and androgens, synthetic estrogens and androgens. Phytoestrogens are widely present in diet and food supplements; mycoestrogens are frequently found in grains. As human beings and animals are commonly exposed to phytoestrogens and mycoestrogens in diet and environment, it is important to understand the potential beneficial or hazardous effects of estrogenic compounds. Many bioassays have been established to study the binding of estrogenic compounds with estrogen receptor (ER) and provided rich data in the literature...
2016: International Journal of Environmental Research and Public Health
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