Rate and Equilibrium Constants for Protein Unfolding and Refolding Determined by Hydrogen Exchange-Mass Spectrometry

Deng, Yuzhong; Smith, David L.

doi:10.1006/abio.1999.4347

Cited by 47 publications

(56 citation statements)

References 39 publications

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“…To determine which mechanism was responsible for the observed increases in exchange, ␣ 1 -AT was incubated (at 45 or 25°C) for 5000 s in H 2 O buffer and then briefly pulse-labeled in D 2 O buffer. This type of pulse labeling experiment is well established as a means for detecting local and global protein unfolding (18). When corrected for temperature, we observed no significant difference in deuterium uptake at 25 and 45°C (Fig.…”

Section: Structure and Dynamics Of The Monomeric Intermediate Pre-mentioning

confidence: 63%

The Structural Basis of Serpin Polymerization Studied by Hydrogen/Deuterium Exchange and Mass Spectrometry

Tsutsui

Kuri

Sengupta

et al. 2008

Journal of Biological Chemistry

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The serpinopathies are a group of inherited disorders that share as their molecular basis the misfolding and polymerization of serpins, an important class of protease inhibitors. Depending on the identity of the serpin, conditions arising from polymerization include emphysema, thrombosis, and dementia. The structure of serpin polymers is thus of considerable medical interest. Wild-type ␣ 1 -antitrypsin will form polymers upon incubation at moderate temperatures and has been widely used as a model system for studying serpin polymerization. Using hydrogen/deuterium exchange and mass spectrometry, we have obtained molecular level structural information on the ␣ 1 -antitrypsin polymer. We found that the flexible reactive center loop becomes strongly protected upon polymerization. We also found significant increases in protection in the center of ␤-sheet A and in helix F. These results support a model in which linkage between serpins is achieved through insertion of the reactive center loop of one serpin into ␤-sheet A of another. We have also examined the heat-induced conformational changes preceding polymerization. We found that polymerization is preceded by significant destabilization of ␤-sheet C. On the basis of our results, we propose a mechanism for polymerization in which ␤-strand 1C is displaced from the rest of ␤-sheet C through a binary serpin/serpin interaction. Displacement of strand 1C triggers further conformational changes, including the opening of ␤-sheet A, and allows for subsequent polymerization.2 is a member of the serpin class of protease inhibitors. Serpins inhibit their target proteases via a unique mechanism ( Fig. 1) (1). Cleavage of a serpin's reactive center loop (RCL) by a protease triggers a massive conformational change in which the RCL inserts into the central ␤-sheet A, becoming a sixth strand. This process disrupts the protease active site and traps the acyl-enzyme intermediate, rendering the protease inactive. The active, loop-expelled form of ␣ 1 -AT is significantly less stable than the loop-inserted form, and as a result of this metastability, the serpin structure is readily disrupted by mutations that result in inappropriate conformational changes. It has been established that a number of serious diseases associated with serpin mutations are caused by the formation and accumulation of serpin polymers (2). The details of the polymerization mechanism are therefore of considerable interest.Serpin polymerization has been studied extensively by a variety of methods, including circular dichroism, fluorescence spectroscopy, electron microscopy, and x-ray crystallography (3-5). The most generally accepted model of pathological serpin polymerization is one in which the RCL of one serpin anneals between ␤-strands 3 and 5A of another (1). Two major lines of evidence support this model. The first is that RCLmimicking peptides have been shown to anneal between strands 3 and 5A and have also been shown to block polymerization in vitro (6). The second comes from fluorescence spectroscopy. Dist...

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Section: Structure and Dynamics Of The Monomeric Intermediate Pre-mentioning

confidence: 63%

The Structural Basis of Serpin Polymerization Studied by Hydrogen/Deuterium Exchange and Mass Spectrometry

Tsutsui

Kuri

Sengupta

et al. 2008

Journal of Biological Chemistry

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“…Recent studies have demonstrated that hydrogen exchange coupled with electrospray ionization MS can qualitatively distinguish native-like proteins from unfolded polypeptides in partially purified samples (9) and can be used to study the kinetics and thermodynamics of folding (8,10). In contrast, the experiments described here use MALDI MS to detect hydrogen exchange, an approach previously described by Komives and coworkers (11).…”

mentioning

confidence: 99%

A quantitative, high-throughput screen for protein stability

Ghaemmaghami

Fitzgerald

Oas

2000

Proc. Natl. Acad. Sci. U.S.A.

159

197

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In proteomic research, it is often necessary to screen a large number of polypeptides for the presence of stable structure. Described here is a technique (referred to as SUPREX, stability of unpurified proteins from rates of H͞D exchange) for measuring the stability of proteins in a rapid, high-throughput fashion. The method uses hydrogen exchange to estimate the stability of microgram quantities of unpurified protein extracts by using matrix-assisted laser desorption͞ionization MS. T he function of a protein is contingent on the stability of its native conformation. Consequently, in the field of protein biochemistry, stability measurements frequently are performed to establish a polypeptide as a stably folded protein and to study the physical forces that lead to its folding (1). Stability measurements also provide important biological information; a decrease in stability can be a sign of misfolding, which in some proteins leads to disease (2), whereas an increase in stability can be indicative of ligand binding (3). Despite their utility, stability measurements currently necessitate time-consuming experiments with pure protein samples. In proteomic experiments (4), where a large number of polypeptides often need to be analyzed, stability measurements are not practical. We have developed the SUPREX (stability of unpurified proteins from rates of H͞D exchange) technique to rapidly screen a large number of protein samples for the presence of stable structure. The method uses hydrogen exchange coupled with matrix-assisted laser desorption͞ionization (MALDI) MS to obtain quantitative measurements of stability from crude extracts of recombinant Escherichia coli cultures grown in 96-well microtiter plates.Within a polypeptide, certain labile hydrogen atoms can exchange freely with the surrounding solvent. Native structure protects a subset of these hydrogens from exchange (5), and some of these protected protons exchange only if the protein globally unfolds (6). The stability of a protein can be analyzed by monitoring the exchange rates of these ''globally protected'' hydrogens (7). Protein hydrogen exchange rates typically are measured by allowing labile protons to exchange with D 2 O. The proton͞deuteron exchange reaction can be monitored by NMR (6) or MS (8). MS is experimentally more convenient than NMR for several reasons: it requires less protein, is faster and simpler to use, does not require complicated spectral assignments, and does not require pure protein samples. These advantages come at the expense of the residue-specific information NMR affords, but that information is not necessary for assessing global stability.Recent studies have demonstrated that hydrogen exchange coupled with electrospray ionization MS can qualitatively distinguish native-like proteins from unfolded polypeptides in partially purified samples (9) and can be used to study the kinetics and thermodynamics of folding (8, 10). In contrast, the experiments described here use MALDI MS to detect hydrogen exchange, an approach previously desc...

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“…In particular, MS has been applied to evaluate the solvent-exposed regions of proteins by following the hydrogen/deuterium exchange rate of protein amide hydrogens with those of the solvent. This has been used for observing structural events during proteinprotein interactions (1) and conformational events during protein folding/unfolding (2). There is also a high potential for this approach to understanding the role of water or side chain hydration in enzyme catalysis.…”

Section: Mass Spectrometry (Ms)mentioning

confidence: 99%

Peptide Mapping and Disulfide Bond Analysis of the Cytoplasmic Region of an Intrinsic Membrane Protein by Mass Spectrometry

Mundt

Cuillel

Forest

et al. 2001

Analytical Biochemistry

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Rate and Equilibrium Constants for Protein Unfolding and Refolding Determined by Hydrogen Exchange-Mass Spectrometry

Cited by 47 publications

References 39 publications

The Structural Basis of Serpin Polymerization Studied by Hydrogen/Deuterium Exchange and Mass Spectrometry

The Structural Basis of Serpin Polymerization Studied by Hydrogen/Deuterium Exchange and Mass Spectrometry

A quantitative, high-throughput screen for protein stability

Peptide Mapping and Disulfide Bond Analysis of the Cytoplasmic Region of an Intrinsic Membrane Protein by Mass Spectrometry

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