Parallel Post-Translational Modification Scanning Enhancing Hydrogen-Deuterium Exchange-Mass Spectrometry Coverage of Key Structural Regions.
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ABSTRACT:
SUBMITTER: Liu H
PROVIDER: S-EPMC9270701 | biostudies-literature |
REPOSITORIES: biostudies-literature
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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.
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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.
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Project description:Human catechol O-methyltransferase (COMT) has emerged as a model for understanding enzyme-catalyzed methyl transfer from S-adenosylmethionine (AdoMet) to small-molecule catecholate acceptors. Mutation of a single residue (tyrosine 68) behind the methyl-bearing sulfonium of AdoMet was previously shown to impair COMT activity by interfering with methyl donor-acceptor compaction within the activated ground state of the wild type enzyme [J. Zhang, H. J. Kulik, T. J. Martinez, J. P. Klinman, Proc. Natl. Acad. Sci. U.S.A. 112, 7954-7959 (2015)]. This predicts the involvement of spatially defined protein dynamical effects that further tune the donor/acceptor distance and geometry as well as the electrostatics of the reactants. Here, we present a hydrogen/deuterium exchange (HDX)-mass spectrometric study of wild type and mutant COMT, comparing temperature dependences of HDX against corresponding kinetic and cofactor binding parameters. The data show that the impaired Tyr68Ala mutant displays similar breaks in Arrhenius plots of both kinetic and HDX properties that are absent in the wild type enzyme. The spatial resolution of HDX below a break point of 15-20 °C indicates changes in flexibility across ?40% of the protein structure that is confined primarily to the periphery of the AdoMet binding site. Above 20 °C, Tyr68Ala behaves more like WT in HDX, but its rate and enthalpic barrier remain significantly altered. The impairment of catalysis by Tyr68Ala can be understood in the context of a mutationally induced alteration in protein motions that becomes manifest along and perpendicular to the primary group transfer coordinate.
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Project description:Cystic fibrosis (CF) is one of the most common, lethal autosomal recessive diseases in Caucasians with a life expectancy of 37-47 years. The CF transmembrane conductance regulator (CFTR) is a plasma membrane ion channel, confined to apical membrane of epithelia, and ensures transepithelial water and solute movement across secretory epithelia in several organs. Numerous CF mutations, including the most prevalent deletion of F508 (ΔF508) in the nucleotide binding domain 1 (NBD1) leads to CFTR global misfolding and premature intracellular degradation at the endoplasmic reticulum (ER). To better understand the misfolding mechanism caused by CF-causing point mutations in the NBD1, which is poorly understood, differential scanning fluorimetry (DSF) and hydrogen deuterium exchange coupled with mass spectrometry (HDX-MS) are the choice of techniques. These established methods can measure the conformational dynamics of the NBD1 globally and at peptide resolution level by monitoring backbone amide HDX, respectively, and will be instrumental to evaluate the mechanism of action of CF mutations and folding correctors that rescue CFTR folding defects via stabilizing the mutant NBD1.
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Project description:This project consisted of three HDX-MS experiments. First, we compared the dimeric PDK1(SKD-PIF) to monomeric PDK1(SKD) and mapped the differences in deuterium incorporation onto the dimer model. We then compared the deuterium incorporation kinetics for the kinase (PDK1(SKD)) and PH (PDK1(PH) domains of PDK1 with full-length PDK1 (PDK1(FL)) in pairwise experiments.
2022-05-20 | PXD027401 | Pride
Project description:Hydrogen/deuterium exchange mass spectrometric (H/DXMS) methods for protein structural analysis are conventionally performed in solution. We present Tissue Deuterium Exchange Mass Spectrometry (TDXMS), a method to directly monitor deuterium uptake on tissue, as a means to better approximate the deuterium exchange behavior of proteins in their native microenvironment. Using this method, a difference in deuterium uptake behavior was observed when the same proteins were monitored in solution and on tissue. The higher maximum deuterium uptake at equilibrium for all proteins analyzed in solution suggests a more open conformation in the absence of interacting partners normally observed on tissue. We also demonstrate a difference in the deuterium uptake behavior of a few proteins across different morphological regions of the same tissue section. Modifications of the total number of hydrogens exchanged, as well as the kinetics of exchange, were both observed. These results provide information on the implication of protein interactions with partners as well as on the conformational changes related to these interactions, and illustrate the importance of examining protein deuterium exchange behavior in the presence of its specific microenvironment directly at the level of tissues.
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