Thermal stability of human alpha-crystallins sensed by amide hydrogen exchange.
The alpha-crystallins, alphaA and alphaB, 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 alpha-crystallin chaperone activity increases with temperature, understanding structural changes of alpha-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 alpha-crystallin, the results have not yet yielded a clear understanding of chaperone activity. We report examination of native assemblies of human lens alpha-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 alphaA and alphaB in the native assembly. The reactivity of the amide linkages to hydrogen/deuterium exchange was determined for 92% of the sequence of alphaA and 99% of alphaB. The behavior of alphaA and alphaB is remarkably similar. At low temperatures, there are regions at the beginning of the alpha-crystallin domains in both alphaA and alphaB that have high protection to isotope exchange, whereas the C termini offer little protection. The N terminus of alphaA also has low protection. With increasing temperatures, both proteins show gradual unfolding. The maximum percent change in exposure with increasing temperatures was found in alphaA 72-75 and alphaB 76-79, two regions considered critical for chaperone activity.
Hasan
Azeem
A
Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE 68588-0304, USA.
Yu
Jiong
J
Smith
David L
DL
Smith
Jean B
JB
D000577:Amides / Q000737:chemistry*; D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003903:Deuterium / Q000737:chemistry; D041961:Deuterium Exchange Measurement; D006801:Humans; D006859:Hydrogen / Q000737:chemistry*; D007700:Kinetics; D008969:Molecular Sequence Data; D010434:Pepsin A / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors; D038201:alpha-Crystallins / Q000737:chemistry* / Q000378:metabolism
D000577:Amides; D010446:Peptide Fragments; D038201:alpha-Crystallins; D006859:Hydrogen; D003903:Deuterium; D010434:Pepsin A