Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Effect of benzyl alcohol on recombinant human interleukin-1 receptor antagonist structure and hydrogen-deuterium exchange.

ABSTRACT: Benzyl alcohol, a preservative commonly added to multidose therapeutic protein formulations, can accelerate aggregation of recombinant human interleukin-1 receptor antagonist (rhIL-1ra). To investigate the interactions between benzyl alcohol and rhIL-1ra, we used nuclear magnetic resonance to observe the effect of benzyl alcohol on the chemical shifts of amide resonances of rhIL-1ra and to measure hydrogen-deuterium exchange rates of individual rhIL-1ra residues. Addition of 0.9% benzyl alcohol caused significant chemical shifts of amide resonances for residues 90-97, suggesting that these solvent-exposed residues participate in the binding of benzyl alcohol. In contrast, little perturbation of exchange rates was observed in the presence of either sucrose or benzyl alcohol.

SUBMITTER: Alford JR

PROVIDER: S-EPMC6261337 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Effect of benzyl alcohol on recombinant human interleukin-1 receptor antagonist structure and hydrogen-deuterium exchange.

Alford John R JR Fowler Andrew C AC Wuttke Deborah S DS Kerwin Bruce A BA Latypov Ramil F RF Carpenter John F JF Randolph Theodore W TW

Journal of pharmaceutical sciences 20110505 10

Benzyl alcohol, a preservative commonly added to multidose therapeutic protein formulations, can accelerate aggregation of recombinant human interleukin-1 receptor antagonist (rhIL-1ra). To investigate the interactions between benzyl alcohol and rhIL-1ra, we used nuclear magnetic resonance to observe the effect of benzyl alcohol on the chemical shifts of amide resonances of rhIL-1ra and to measure hydrogen-deuterium exchange rates of individual rhIL-1ra residues. Addition of 0.9% benzyl alcohol ...[more]

PMID: 21557223

Similar Datasets

Analysis of agonist and antagonist effects on thyroid hormone receptor conformation by hydrogen/deuterium exchange.

Project description:Thyroid hormone receptors (TRs) are ligand-gated transcription factors with critical roles in development and metabolism. Although x-ray structures of TR ligand-binding domains (LBDs) with agonists are available, comparable structures without ligand (apo-TR) or with antagonists are not. It remains important to understand apo-LBD conformation and the way that it rearranges with ligands to develop better TR pharmaceuticals. In this study, we conducted hydrogen/deuterium exchange on TR LBDs with or without agonist (T(3)) or antagonist (NH3). Both ligands reduce deuterium incorporation into LBD amide hydrogens, implying tighter overall folding of the domain. As predicted, mass spectroscopic analysis of individual proteolytic peptides after hydrogen/deuterium exchange reveals that ligand increases the degree of solvent protection of regions close to the buried ligand-binding pocket. However, there is also extensive ligand protection of other regions, including the dimer surface at H10-H11, providing evidence for allosteric communication between the ligand-binding pocket and distant interaction surfaces. Surprisingly, C-terminal activation helix H12, which is known to alter position with ligand, remains relatively protected from solvent in all conditions suggesting that it is packed against the LBD irrespective of the presence or type of ligand. T(3), but not NH3, increases accessibility of the upper part of H3-H5 to solvent, and we propose that TR H12 interacts with this region in apo-TR and that this interaction is blocked by T(3) but not NH3. We present data from site-directed mutagenesis experiments and molecular dynamics simulations that lend support to this structural model of apo-TR and its ligand-dependent conformational changes.

| S-EPMC3033054 | 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

Protein structure change studied by hydrogen-deuterium exchange, functional labeling, and mass spectrometry.

Project description:An automated high-throughput, high-resolution deuterium exchange HPLC-MS method (DXMS) was used to extend previous hydrogen exchange studies on the position and energetic role of regulatory structure changes in hemoglobin. The results match earlier highly accurate but much more limited tritium exchange results, extend the analysis to the entire sequence of both hemoglobin subunits, and identify some energetically important changes. Allosterically sensitive amide hydrogens located at near amino acid resolution help to confirm the reality of local unfolding reactions and their use to evaluate resolved structure changes in terms of allosteric free energy.

| S-EPMC165829 | biostudies-literature

Dissecting structure of prion amyloid fibrils by hydrogen-deuterium exchange ultraviolet Raman spectroscopy.

Project description:The molecular mechanisms underlying structural diversity of amyloid fibrils or prion strains formed within the same primary structure is considered to be one of the most enigmatic questions in prion biology. We report here on the direct characterization of amyloid structures using a novel spectroscopic technique, hydrogen-deuterium exchange ultraviolet Raman spectroscopy. This method enables us to assess the structural differences within highly ordered cross-?-cores of two amyloid states produced within the same amino acid sequence of full-length mammalian prion protein. We found that while both amyloid states consisted of ?-structures, their cross-?-cores exhibited hydrogen bonding of different strengths. Moreover, Raman spectroscopy revealed that both amyloid states displayed equally narrow crystalline-like bands, suggesting uniform structures of cross-?-cores within each state. Taken together, these data suggest that highly polymorphous fibrils can display highly uniform structures of their cross-?-core and belong to the same prion strain.

| S-EPMC3490051 | biostudies-literature

Vibrio cholerae toxin-coregulated pilus structure analyzed by hydrogen/deuterium exchange mass spectrometry.

Project description:The bacterial pathogen Vibrio cholerae uses toxin-coregulated pili (TCP) to colonize the human intestine, causing the severe diarrheal disease cholera. TCP are long, thin, flexible homopolymers of the TcpA subunit that self-associate to hold cells together in microcolonies and serve as the receptor for the cholera toxin phage. To better understand TCP's roles in pathogenesis, we characterized its structure using hydrogen/deuterium exchange mass spectrometry and computational modeling. We show that the pilin subunits are held together by tight packing of the N-terminal alpha helices, but loose packing of the C-terminal globular domains leaves substantial gaps on the filament surface. These gaps expose a glycine-rich, amphipathic segment of the N-terminal alpha-helix, contradicting the consensus view that this region is buried in the filament core. Our results explain extreme filament flexibility, suggest a molecular basis for pilus-pilus interactions, and reveal a previously unrecognized therapeutic target for V. cholerae and other enteric pathogens.

| S-EPMC2238685 | biostudies-literature

Rapid analysis of protein structure and dynamics by hydrogen/deuterium exchange mass spectrometry.

Project description:An automated approach for the rapid analysis of protein structure has been developed and used to study acid-induced conformational changes in human growth hormone. The labeling approach involves hydrogen/deuterium exchange (H/D-Ex) of protein backbone amide hydrogens with rapid and sensitive detection by mass spectrometry (MS). Briefly, the protein is incubated for defined intervals in a deuterated environment. After rapid quenching of the exchange reaction, the partially deuterated protein is enzymatically digested and the resulting peptide fragments are analyzed by liquid chromatography mass spectrometry (LC-MS). The deuterium buildup curve measured for each fragment yields an average amide exchange rate that reflects the environment of the peptide in the intact protein. Additional analyses allow mapping of the free energy of folding on localized segments along the protein sequence affording unique dynamic and structural information. While amide H/D-Ex coupled with MS is recognized as a powerful technique for studying protein structure and protein-ligand interactions, it has remained a labor-intensive task. The improvements in the amide H/D-Ex methodology described here include solid phase proteolysis, automated liquid handling and sample preparation, and integrated data reduction software that together improve sequence coverage and resolution, while achieving a sample throughput nearly 10-fold higher than the commonly used manual methods.

| S-EPMC2279949 | biostudies-literature

Changes in protein structure monitored by use of gas-phase hydrogen/deuterium exchange.

Project description:The study of protein conformation by solution-phase hydrogen/deuterium exchange (HDX) coupled to MS is well documented. This involves monitoring the exchange of backbone amide protons with deuterium and provides details concerning the protein's tertiary structure. However, undesired back-exchange during post-HDX analyses can be difficult to control. Here, gas-phase HDX-MS, during which labile hydrogens on amino acid side chains are exchanged in sub-millisecond time scales, has been employed to probe changes within protein structures. Addition of the solvent 2,2,2-trifluoroethanol to a protein in solution can affect the structure of the protein, resulting in an increase in secondary and/or tertiary structure which is detected using circular dichroism. Using a Synapt G2-S ESI-mass spectrometer modified to allow deuterated ammonia into the transfer ion guide (situated between the ion mobility cell and the TOF analyser), gas-phase HDX-MS is shown to reflect minor structural changes experienced by the proteins ?-lactoglobulin and ubiquitin, as observed by the reduction in the level of deuterium incorporation. Additionally, the use of gas-phase HDX-MS to distinguish between co-populated proteins conformers within a solution is demonstrated with the disordered protein calmodulin; the gas-phase HDX-MS results correspond directly with complementary data obtained by use of ion mobility spectrometry-MS.

| S-EPMC4973844 | biostudies-literature

Residual Structure of Unfolded Ubiquitin as Revealed by Hydrogen/Deuterium-Exchange 2D NMR.

Project description:The characterization of residual structures persistent in unfolded proteins in concentrated denaturant solution is currently an important issue in studies of protein folding because the residual structure present, if any, in the unfolded state may form a folding initiation site and guide the subsequent folding reactions. Here, we studied the hydrogen/deuterium (H/D)-exchange behavior of unfolded human ubiquitin in 6 M guanidinium chloride. We employed a dimethylsulfoxide (DMSO)-quenched H/D-exchange NMR technique with the use of spin desalting columns, which allowed us to perform a quick medium exchange from 6 M guanidinium chloride to a quenching DMSO solution. Based on the backbone resonance assignment of ubiquitin in the DMSO solution, we successfully investigated the H/D-exchange kinetics of 60 identified peptide amide groups in the ubiquitin sequence. Although a majority of these amide groups were not protected, certain amide groups involved in a middle helix (residues 23-34) and an N-terminal β-hairpin (residues 2-16) were significantly protected with a protection factor of 2.1-4.2, indicating that there were residual structures in unfolded ubiquitin and that these amide groups were more than 52% hydrogen bonded in the residual structures. We show that the hydrogen-bonded residual structures in the α-helix and the β-hairpin are formed even in 6 M guanidinium chloride, suggesting that these residual structures may function as a folding initiation site to guide the subsequent folding reactions of ubiquitin.

| S-EPMC7732725 | biostudies-literature

Construct optimization for protein NMR structure analysis using amide hydrogen/deuterium exchange mass spectrometry.

Project description:Disordered or unstructured regions of proteins, while often very important biologically, can pose significant challenges for resonance assignment and three-dimensional structure determination of the ordered regions of proteins by NMR methods. In this article, we demonstrate the application of (1)H/(2)H exchange mass spectrometry (DXMS) for the rapid identification of disordered segments of proteins and design of protein constructs that are more suitable for structural analysis by NMR. In this benchmark study, DXMS is applied to five NMR protein targets chosen from the Northeast Structural Genomics project. These data were then used to design optimized constructs for three partially disordered proteins. Truncated proteins obtained by deletion of disordered N- and C-terminal tails were evaluated using (1)H-(15)N HSQC and (1)H-(15)N heteronuclear NOE NMR experiments to assess their structural integrity. These constructs provide significantly improved NMR spectra, with minimal structural perturbations to the ordered regions of the protein structure. As a representative example, we compare the solution structures of the full length and DXMS-based truncated construct for a 77-residue partially disordered DUF896 family protein YnzC from Bacillus subtilis, where deletion of the disordered residues (ca. 40% of the protein) does not affect the native structure. In addition, we demonstrate that throughput of the DXMS process can be increased by analyzing mixtures of up to four proteins without reducing the sequence coverage for each protein. Our results demonstrate that DXMS can serve as a central component of a process for optimizing protein constructs for NMR structure determination.

| S-EPMC2739808 | 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