Cystathionine beta-synthase plays a key role in the intracellular disposal of homocysteine and is the single most common locus of mutations associated with homocystinuria. Elevated levels of homocysteine are correlated with heart disease, Alzheimer's and Parkinson's diseases, and neural tube defects. Cystathionine beta-synthase is modular and subjected to complex regulation, but insights into the structural basis of this regulation are lacking. We have employed hydrogen exchange mass spectrometry to map peptides whose motions are correlated with transmission of intrasteric inhibition and allosteric activation. The mass spectrometric data provide an excellent correlation between kinetically and conformationally distinguishable states of the enzyme. We also demonstrate that a pathogenic regulatory domain mutant, D444N, is conformationally locked in one of two states sampled by the wild type enzyme. Our hydrogen exchange data identify surfaces that are potentially involved in the juxtaposition of the regulatory and catalytic domains and form the basis of a docked structural model for the full-length enzyme.
The ␣-crystallins, ␣A and ␣B, are major lens structural proteins with chaperone-like activity and sequence homology to small heat-shock proteins. As yet, their crystal structures have not been determined because of the large size and heterogeneity of the assemblies they form in solution. Because ␣-crystallin chaperone activity increases with temperature, understanding structural changes of ␣-crystallin as it is heated may help elucidate the mechanism of chaperone activity. Although a variety of techniques have been used to probe changes in heat-stressed ␣-crystallin, the results have not yet yielded a clear understanding of chaperone activity. We report examination of native assemblies of human lens ␣-crystallin using hydrogen/deuterium exchange in conjunction with enzymatic digestion and analysis by mass spectrometry. This technique has the advantage of sensing structural changes along much of the protein backbone and being able to detect changes specific to ␣A and ␣B in the native assembly. The reactivity of the amide linkages to hydrogen/ deuterium exchange was determined for 92% of the sequence of ␣A and 99% of ␣B. The behavior of ␣A and ␣B is remarkably similar. At low temperatures, there are regions at the beginning of the ␣-crystallin domains in both ␣A and ␣B that have high protection to isotope exchange, whereas the C termini offer little protection. The N terminus of ␣A also has low protection. With increasing temperatures, both proteins show gradual unfolding. The maximum percent change in exposure with increasing temperatures was found in ␣A 72-75 and ␣B 76-79, two regions considered critical for chaperone activity.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.