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Thermal stability of human α‐crystallins sensed by amide hydrogen exchange
Author(s) -
Hasan Azeem,
Yu Jiong,
Smith David L.,
Smith Jean B.
Publication year - 2004
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.03180004
Subject(s) - crystallin , chaperone (clinical) , chemistry , hydrogen–deuterium exchange , biophysics , heat shock protein , mass spectrometry , crystallography , amide , biochemistry , chromatography , biology , medicine , pathology , gene
The α‐crystallins, αA and αB, 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 α‐crystallin chaperone activity increases with temperature, understanding structural changes of α‐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 α‐crystallin, the results have not yet yielded a clear understanding of chaperone activity. We report examination of native assemblies of human lens α‐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 αA and αB in the native assembly. The reactivity of the amide linkages to hydrogen/deuterium exchange was determined for 92% of the sequence of αA and 99% of αB. The behavior of αA and αB is remarkably similar. At low temperatures, there are regions at the beginning of the α‐crystallin domains in both αA and αB that have high protection to isotope exchange, whereas the C termini offer little protection. The N terminus of αA also has low protection. With increasing temperatures, both proteins show gradual unfolding. The maximum percent change in exposure with increasing temperatures was found in αA 72–75 and αB 76–79, two regions considered critical for chaperone activity.