Browse
Submit Data
Databases
API
Help

Dataset Information

22 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Hydrogen-deuterium exchange strategy for delineation of contact sites in protein complexes.

ABSTRACT: We use NMR spectra to determine protein-protein contact sites by observing differences in amide proton hydrogen-deuterium exchange in the complex compared to the free protein in solution. Aprotic organic solvents are used to preserve H/D labeling patterns that would be scrambled in water solutions. The binding site between the mammalian co-chaperone Aha1 with the middle domain of the chaperone Hsp90 obtained by our H/D exchange method corresponds well with that in the X-ray crystal structure of the homologous complex from yeast, even to the observation of a secondary binding site. This method can potentially provide data for complexes with unknown structure and for large or dynamic complexes inaccessible via NMR and X-ray methods.

SUBMITTER: Dyson HJ

PROVIDER: S-EPMC2440508 | biostudies-literature | 2008 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Hydrogen-deuterium exchange strategy for delineation of contact sites in protein complexes.

Dyson H Jane HJ Kostic Milka M Liu Jeff J Martinez-Yamout Maria A MA

FEBS letters 20080407 10

We use NMR spectra to determine protein-protein contact sites by observing differences in amide proton hydrogen-deuterium exchange in the complex compared to the free protein in solution. Aprotic organic solvents are used to preserve H/D labeling patterns that would be scrambled in water solutions. The binding site between the mammalian co-chaperone Aha1 with the middle domain of the chaperone Hsp90 obtained by our H/D exchange method corresponds well with that in the X-ray crystal structure of ...[more]

PMID: 18396165

Similar Datasets

Tracking hydrogen/deuterium exchange at glycan sites in glycoproteins by mass spectrometry.

Project description:Hydrogen/deuterium exchange coupled to mass spectrometry (HDX-MS) has emerged as a technique for studying glycoproteins, which are often refractory to classical methods. Glycan chains are generally assumed to exchange protons very rapidly, making them invisible to this technique. Here, we show that under conditions commonly used for HDX-MS, acetamido groups within glycan chains retain a significant amount of deuterium. Using mono- and polysaccharide standards along with glycopeptides from a panel of glycoproteins, we demonstrate that N-acetyl hexosamines, along with modified Asn side chains, are responsible for this effect. Model compounds for sialic acid also displayed similar exchange kinetics, but terminal sialic acids in the context of an entire glycan chain did not contribute to deuterium retention. Furthermore, the presence of sialic acid appears to enhance the exchange rate of the nearby N-acetyl glucosamines. The ability to detect deuterium exchange at the glycan level opens the possibility of applying HDX-MS to monitor glycan interactions and dynamics.

| S-EPMC3184328 | biostudies-literature

Synthesis, spectroscopy, and hydrogen/deuterium exchange in high-spin iron(II) hydride complexes.

Project description:Very few hydride complexes are known in which the metals have a high-spin electronic configuration. We describe the characterization of several high-spin iron(II) hydride/deuteride isotopologues and their exchange reactions with one another and with H2/D2. Though the hydride/deuteride signal is not observable in NMR spectra, the choice of isotope has an influence on the chemical shifts of distant protons in the dimers through the paramagnetic isotope effect on chemical shift. This provides the first way to monitor the exchange of H and D in the bridging positions of these hydride complexes. The rate of exchange depends on the size of the supporting ligand, and this is consistent with the idea that H2/D2 exchange into the hydrides occurs through the dimeric complexes rather than through a transient monomer. The understanding of H/D exchange mechanisms in these high-spin iron hydride complexes may be relevant to postulated nitrogenase mechanisms.

| S-EPMC3993920 | biostudies-literature

Probing conserved helical modules of portal complexes by mass spectrometry-based hydrogen/deuterium exchange.

Project description:The Double-stranded DNA bacteriophage P22 has a ring-shaped dodecameric complex composed of the 84 kDa portal protein subunit that forms the central channel of the phage DNA packaging motor. The overall morphology of the P22 portal complex is similar to that of the portal complexes of Phi29, SPP1, T3, T7 phages and herpes simplex virus. Secondary structure prediction of P22 portal protein and its threading onto the crystal structure of the Phi29 portal complexes suggested that the P22 portal protein complex shares conserved helical modules that were found in the dodecameric interfaces of the Phi29 portal complex. To identify the amino acids involved in intersubunit contacts in the P22 portal ring complexes and validate the threading model, we performed comparative hydrogen/deuterium exchange analysis of monomeric and in vitro assembled portal proteins of P22 and the dodecameric Phi29 portal. Hydrogen/deuterium exchange experiments provided evidence of intersubunit interactions in the P22 portal complex similar to those in the Phi29 portal that map to the regions predicted to be conserved helical modules.

| S-EPMC2593103 | biostudies-literature

Differential hydrogen/deuterium exchange mass spectrometry analysis of protein-ligand interactions.

Project description:Functional regulation of ligand-activated receptors is driven by alterations in the conformational dynamics of the protein upon ligand binding. Differential hydrogen/deuterium exchange (HDX) coupled with mass spectrometry has emerged as a rapid and sensitive approach for characterization of perturbations in conformational dynamics of proteins following ligand binding. While this technique is sensitive to detecting ligand interactions and alterations in receptor dynamics, it also can provide important mechanistic insights into ligand regulation. For example, HDX has been used to determine a novel mechanism of ligand activation of the nuclear receptor peroxisome proliferator activated receptor-?, perform detailed analyses of binding modes of ligands within the ligand-binding pocket of two estrogen receptor isoforms, providing insight into selectivity, and helped classify different types of estrogen receptor-? ligands by correlating their pharmacology with the way they interact with the receptor based solely on hierarchical clustering of receptor HDX signatures. Beyond small-molecule-receptor interactions, this technique has also been applied to study protein-protein complexes, such as mapping antibody-antigen interactions. In this article, we summarize the current state of the differential HDX approaches and the future outlook. We summarize how HDX analysis of protein-ligand interactions has had an impact on biology and drug discovery.

| S-EPMC3113475 | biostudies-literature

Hydrogen-deuterium exchange mass spectrometry reveals folding and allostery in protein-protein interactions.

Project description: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 performance 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. In this review, we focus on studies that have used a combination of approaches to reveal (re)folding of proteins as well as on long-distance allosteric changes upon interaction.

| S-EPMC6051914 | biostudies-literature

Rapid Hydrogen-Deuterium Exchange in Liquid Droplets.

Project description:The rate of hydrogen-deuterium exchange (HDX) in aqueous droplets of phenethylamine has been determined with submillisecond temporal resolution by mass spectrometry using nanoelectrospray ionization with a theta-capillary. The average speed of the microdroplets is measured using microparticle image velocimetry. The droplet travel time is varied from 20 to 320 ?s by changing the distance between the emitter and the heated inlet to the mass spectrometer and the voltage applied to the emitter source. The droplets were found to accelerate by ?30% during their observable travel time. Our droplet imaging shows that the theta-capillary produces two Taylor cone-jets (one per channel), causing mixing to take place from droplet fusion in the Taylor spray zone. Phenethylamine (?CH2CH2NH2) was chosen to study because it has only one functional group (-NH2) that undergoes rapid HDX. We model the HDX with a system of ordinary differential equations. The rate constant for the formation of -NH2D+ from -NH3+ is 3660 ± 290 s-1, and the rate constant for the formation of -NHD2+ from -NH2D+ is 3330 ± 270 s-1. The observed rates are about 3 times faster than what has been reported for rapidly exchangeable peptide side-chain groups in bulk measurements using stopped-flow kinetics and NMR spectroscopy. We also applied this technique to determine the HDX rates for a small 10-residue peptide, angiotensin I, in aqueous droplets, from which we found a 7-fold acceleration of HDX in the droplet compared to that in bulk solution.

| S-EPMC6700750 | biostudies-literature

Hydrogen-Deuterium Exchange Profiles of Polyubiquitin Fibrils.

Project description:Ubiquitin and its polymeric forms are conjugated to intracellular proteins to regulate diverse intracellular processes. Intriguingly, polyubiquitin has also been identified as a component of pathological protein aggregates associated with Alzheimer's disease and other neurodegenerative disorders. We recently found that polyubiquitin can form amyloid-like fibrils, and that these fibrillar aggregates can be degraded by macroautophagy. Although the structural properties appear to function in recognition of the fibrils, no structural information on polyubiquitin fibrils has been reported so far. Here, we identify the core of M1-linked diubiquitin fibrils from hydrogen-deuterium exchange experiments using solution nuclear magnetic resonance (NMR) spectroscopy. Intriguingly, intrinsically flexible regions became highly solvent-protected in the fibril structure. These results indicate that polyubiquitin fibrils are formed by inter-molecular interactions between relatively flexible structural components, including the loops and edges of secondary structure elements.

| S-EPMC6414897 | biostudies-literature

Understanding Curli Amyloid-Protein Aggregation by Hydrogen-Deuterium Exchange and Mass Spectrometry.

Project description:Bacteria within Curli biofilms are protected from environmental pressures (e.g., disinfectants, antibiotics), and this is responsible for intractable infections. Understanding aggregation of the major protein component of Curli, CsgA, may uncover disease-associated amyloidogenesis mechanisms. Here, we report the application of pulsed hydrogen-deuterium exchange and mass spectrometry (HDX-MS) to study CsgA aggregation, thereby obtaining region-specific information. By following time-dependent peptide signal depletion, presumably a result of insoluble fibril formation, we acquired sigmoidal profiles that are specific for regions (region-specific) of the protein. These signal-depletion profiles not only provide an alternative aggregation measurement, but also give insight on soluble species in the aggregation. The HDX data present as bimodal isotopic distributions, one representing a highly disordered species whereas the other a well-structured one. Although the extents of deuterium uptake of the two species remain the same with time, the relative abundance of the lower mass, less-exchanged species increases in a region-specific manner. The same region-specific aggregation properties also pertain to different aggregation conditions. Although CsgA is an intrinsically disordered protein, within the fibril it is thought to consist of five imperfect β-strand repeating units (labeled R1-R5). We found that the exterior repeating units R1 and R5 have higher aggregation propensities than do the interior units R2, R3, and R4. We also employed TEM to obtain complementary information of the well-structured species. The results provide insight on aggregation and a new approach for further application of HDX-MS to unravel aggregation mechanisms of amyloid proteins.

| S-EPMC5644351 | biostudies-literature

Hydrogen Deuterium Exchange of the CERT1 protein

Project description:HDX-MS of CERT1 to determine secondary structure characteristics and compare the WT and G243R mutant

2023-04-04 | PXD027356 | Pride

Using hydrogen deuterium exchange mass spectrometry to engineer optimized constructs for crystallization of protein complexes: Case study of PI4KIIIβ with Rab11.

Project description:The ability of proteins to bind and interact with protein partners plays fundamental roles in many cellular contexts. X-ray crystallography has been a powerful approach to understand protein-protein interactions; however, a challenge in the crystallization of proteins and their complexes is the presence of intrinsically disordered regions. In this article, we describe an application of hydrogen deuterium exchange mass spectrometry (HDX-MS) to identify dynamic regions within type III phosphatidylinositol 4 kinase beta (PI4KIIIβ) in complex with the GTPase Rab11. This information was then used to design deletions that allowed for the production of diffraction quality crystals. Importantly, we also used HDX-MS to verify that the new construct was properly folded, consistent with it being catalytically and functionally active. Structures of PI4KIIIβ in an Apo state and bound to the potent inhibitor BQR695 in complex with both GTPγS and GDP loaded Rab11 were determined. This hybrid HDX-MS/crystallographic strategy revealed novel aspects of the PI4KIIIβ-Rab11 complex, as well as the molecular mechanism of potency of a PI4K specific inhibitor (BQR695). This approach is widely applicable to protein-protein complexes, and is an excellent strategy to optimize constructs for high-resolution structural approaches.

| S-EPMC4832280 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data