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hydrogen deuterium exchange

Marjaana Viljanto, Catarina Horro Pita, James Scarth, Christopher J Walker, Andrew T Kicman, Mark C Parkin
The effective analysis of anabolic-androgenic steroids in urine usually requires a suitable deconjugation method for the analysis of phase II metabolites such as sulfates and glucuronides. Acid hydrolysis using methanolysis is one adopted method of deconjugation that efficiently and rapidly cleaves both sulfates and glucuronides contemporaneously. The formation of artefactual by-products is a known disadvantage of this harsh method. However, the possible promotion of deuterium-hydrogen exchange of isotopically labelled internal standards has received little attention in the literature...
May 14, 2018: Drug Testing and Analysis
Cameron D Buchman, Sergio C Chai, Taosheng Chen
Pregnane X receptor (PXR) and the constitutive androstane receptor (CAR) are two members of the nuclear receptor superfamily that play major roles in the expression of various drug metabolism enzymes and are known for their ligand promiscuity. As with other nuclear receptors, PXR and CAR are each composed of a ligand-binding domain (LBD) and a DNA-binding domain (DBD) connected by a hinge region. Areas covered: This review focuses on the information obtained over the last 15+ years from X-ray crystallography studies of the structure of PXR and CAR...
May 14, 2018: Expert Opinion on Drug Metabolism & Toxicology
Patrick S Merkle, Kamil Gotfryd, Michel A Cuendet, Katrine Z Leth-Espensen, Ulrik Gether, Claus J Loland, Kasper D Rand
LeuT, a prokaryotic member of the neurotransmitter:sodium symporter (NSS) family, is an established structural model for mammalian NSS counterparts. We investigate the substrate translocation mechanism of LeuT by measuring the solution-phase structural dynamics of the transporter in distinct functional states by hydrogen/deuterium exchange mass spectrometry (HDX-MS). Our HDX-MS data pinpoint LeuT segments involved in substrate transport and reveal for the first time a comprehensive and detailed view of the dynamics associated with transition of the transporter between outward- and inward-facing configurations in a Na+ - and K+ -dependent manner...
May 2018: Science Advances
Ulrik H Mistarz, Kasper D Rand
Gas-phase hydrogen/deuterium exchange measured by mass spectrometry in a millisecond timeframe after ESI (gas-phase HDX-MS) is a fast and sensitive, yet unharnessed method to analyze the primary- and higher-order structure, intramolecular and intermolecular interactions, surface properties, and charge location of peptides and proteins. During a gas-phase HDX-MS experiment, heteroatom-bound non-amide hydrogens are made to exchange with deuterium during a millisecond timespan after electrospray ionization (ESI) by reaction with the highly basic reagent ND3 , enabling conformational analysis of protein states that are pertinent to the native solution-phase...
May 10, 2018: Methods: a Companion to Methods in Enzymology
Amberley Stephens, Nadezhda Nespovitaya, Maria Zacharopoulou, Clemens F Kaminski, Jonathan James Phillips, Gabriele S Kaminski Schierle
Understanding the mechanisms behind amyloid protein aggregation in diseases such as Parkinson's and Alzheimer's disease is often hampered by the reproducibility of in vitro assays. Yet, understanding the basic mechanisms of protein misfolding is essential for the development of novel therapeutic strategies. We show here, that for the amyloid protein alpha-synuclein (aSyn), a protein involved in Parkinson's disease (PD), chromatographic buffers and storage conditions can significantly interfere with the overall structure of the protein and thus affect protein aggregation kinetics...
May 11, 2018: Analytical Chemistry
Richard Y-C Huang, Steven R O'Neil, Daša Lipovšek, Guodong Chen
Higher-order structure (HOS) characterization of therapeutic protein-drug conjugates for comprehensive assessment of conjugation-induced protein conformational changes is an important consideration in the biopharmaceutical industry to ensure proper behavior of protein therapeutics. In this study, conformational dynamics of a small therapeutic protein, adnectin 1, together with its drug conjugate were characterized by hydrogen/deuterium exchange mass spectrometry (HDX-MS) with different spatial resolutions. Top-down HDX allows detailed assessment of the residue-level deuterium content in the payload conjugation region...
May 7, 2018: Journal of the American Society for Mass Spectrometry
Kyle W Anderson, Elyssia S Gallagher, Jeffrey W Hudgens
Membrane proteins are currently the most common targets for pharmaceuticals. However, characterization of their structural dynamics by hydrogen/deuterium exchange mass spectrometry (HDX-MS) is sparse due to insufficient automated methods to handle full-length membrane proteins in lipid bilayers. Additionally, membrane lipids used to mimic the membrane environment and to solubilize membrane proteins can impair chromatography performance and cause ion suppression in the mass spectrometer. The workflow discussed herein advances HDX-MS capabilities and other MS applications for membrane proteins by providing a fully automated method for HDX-MS analysis based on a phospholipid removal scheme compatible with robotic handling...
May 9, 2018: Analytical Chemistry
Luke Berry, Angela Patterson, Natasha Pence, John W Peters, Brian Bothner
The protocol detailed here describes a way to perform hydrogen deuterium exchange coupled to mass spectrometry (HDX-MS) on oxygen sensitive proteins. HDX-MS is a powerful tool for studying the protein structure-function relationship. Applying this technique to anaerobic proteins provides insight into the mechanism of proteins that perform oxygen sensitive chemistry. A problem when using HDX-MS to study anaerobic proteins is that there are many parts that require constant movement into and out of an anaerobic chamber...
March 20, 2018: Bio-protocol
Anne Sömmer, Sönke Behrends
Soluble guanylyl cyclase (sGC) is a heterodimeric enzyme consisting of one α and one β subunit. The α1 β1 (GC-1) and α2 β1 (GC-2) heterodimers are important for NO signaling in humans and catalyse the conversion from GTP to cGMP. Each sGC subunit consists of four domains. Several crystal structures of the isolated domains are available. However, crystals of full-length sGC have failed to materialise. In consequence, the detailed three dimensional structure of sGC remains unknown to date. Different techniques including stopped-flow spectroscopy, Förster-resonance energy transfer, direct fluorescence, analytical ultracentrifugation, chemical cross-linking, small-angle X-ray scattering, electron microscopy, hydrogen-deuterium exchange and protein thermal shift assays, were used to collect indirect information...
April 26, 2018: Nitric Oxide: Biology and Chemistry
Irina Oganesyan, Cristina Lento, Derek J Wilson
Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) emerged as a tool for biochemistry and structural biology around 25 years ago. It has since become a key approach for studying protein dynamics, protein-ligand interactions, membrane proteins and intrinsically disordered proteins (IDPs). In HDX labeling, proteins are exposed to deuterated solvent (usually D2 O) for a variable 'labeling time', resulting in isotope exchange of unprotected labile protons on the amide backbone and amino acid side chains. By comparing the levels of deuterium uptake in different regions of a protein, information on conformational and dynamic changes in the system can be acquired...
April 25, 2018: Methods: a Companion to Methods in Enzymology
Stefan Etzl, Robert Lindner, Matthew D Nelson, Andreas Winkler
Genetically targeting biological systems to control cellular processes with light is the concept of optogenetics. Despite impressive developments in this field, underlying molecular mechanisms of signal transduction of the employed photoreceptor modules are frequently not sufficiently understood to rationally design new optogenetic tools. Here, we investigate the requirements for functional coupling of red light sensing phytochromes with non-natural enzymatic effectors by creating a series of constructs featuring the Deinococcus radiodurans bacteriophytochrome linked to a Synechocystis guanylate/adenylate cyclase...
April 25, 2018: Journal of Biological Chemistry
Volker Derdau, Mégane Valero, Remo Weck, Jens Atzrodt, Stefan Güssregen
For the first time we describe the highly selective homogeneous iridium-catalyzed hydrogen isotope exchange (HIE) of unactivated C(sp3)-centers in aliphatic amides. Using the commercially available Kerr-catalyst, the HIE reaction on a series of common antibody-drug-conjugates (ADC) linker side chains proceeds with high yields, high regioselectivity and with deuterium incorporation of up to 99%. The method is fully translatable to the specific requirements of tritium chemistry and its effectiveness was demonstrated by direct tritium labelling of a maytansinoid...
April 25, 2018: Angewandte Chemie
M Rafalik, M Spodzieja, A S Kołodziejczyk, S Rodziewicz-Motowidło, A Szymańska, A Grubb, P Czaplewska
Human cystatin C (hCC) is a cysteine proteinase inhibitor involved in pathophysiological processes of dimerization and amyloid formation. These processes are directly associated with a number of neurodegenerative disorders such as Alzheimer disease or hereditary cystatin C amyloid angiopathy (HCCAA). One of the ideas on how to prevent amyloid formation is to use immunotherapy. HCC3 is one of a group of antibodies binding to hCC and reducing the in vitro formation of cystatin C dimers. Therefore, identification of the binding sites in the hCC-HCC3 complex may facilitate a search of effective drugs against HCCAA as well as understanding the mechanisms of neurodegenerative disorders...
April 20, 2018: Journal of Proteomics
Darryl Aucoin, Yongjie Xia, Theint Theint, Philippe S Nadaud, Krystyna Surewicz, Witold K Surewicz, Christopher P Jaroniec
The C-terminally truncated Y145Stop variant of prion protein (PrP23-144), which is associated with heritable PrP cerebral amyloid angiopathy in humans and also capable of triggering a transmissible prion disease in mice, serves as a useful in vitro model for investigating the molecular and structural basis of amyloid strains and cross-seeding specificities. Here, we determine the protein-solvent interfaces in human PrP23-144 amyloid fibrils generated from recombinant 13 C,15 N-enriched protein and incubated in aqueous solution containing paramagnetic Cu(II)-EDTA, by measuring residue-specific 15 N longitudinal paramagnetic relaxation enhancements using two-dimensional magic-angle spinning solid-state NMR spectroscopy...
April 18, 2018: Journal of Structural Biology
Devrishi Goswami, Jun Zhang, Pavel V Bondarenko, Zhongqi Zhang
Mass spectrometry (MS)-based methods for analyzing protein higher order structures have gained increasing application in the field of biopharmaceutical development. The predominant methods used in this area include native MS, hydrogen deuterium exchange-MS, covalent labeling, cross-linking and limited proteolysis. These MS-based methods will be briefly described in this article, followed by a discussion on how these methods contribute at different stages of discovery and development of protein therapeutics.
April 17, 2018: Methods: a Companion to Methods in Enzymology
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
Estella A Newcombe, Kiersten M Ruff, Ashish Sethi, Angelique R Ormsby, Yasmin M Ramdzan, Archa Fox, Anthony W Purcell, Paul R Gooley, Rohit V Pappu, Danny M Hatters
Soluble huntingtin exon 1 (Httex1) with expanded polyglutamine (polyQ) engenders neurotoxicity in Huntington's disease. To uncover the physical basis of this toxicity, we performed structural studies of soluble Httex1 for wild type and mutant polyQ lengths. Nuclear magnetic resonance experiments show evidence for conformational rigidity across the polyQ region. In contrast, hydrogen-deuterium exchange shows absence of backbone amide protection, suggesting negligible persistence of hydrogen bonds. The seemingly conflicting results are explained by all-atom simulations, which show that Httex1 adopts tadpole-like structures with a globular head encompassing the N-terminal amphipathic and polyQ regions and the tail encompassing the C-terminal proline-rich region...
April 5, 2018: Journal of Molecular Biology
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
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