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Protein Science (2004), 13:332-341. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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Thermal stability of human {alpha}-crystallins sensed by amide hydrogen exchange

Azeem Hasan, Jiong Yu, David L. Smith and Jean B. Smith

Department of Chemistry, University of Nebraska–Lincoln, Lincoln, Nebraska 68588-0304, USA

(RECEIVED May 1, 2003; FINAL REVISION August 25, 2003; ACCEPTED October 2, 2003)



Abstract

The {alpha}-crystallins, {alpha}A and {alpha}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 {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 {alpha}A and {alpha}B in the native assembly. The reactivity of the amide linkages to hydrogen/deuterium exchange was determined for 92% of the sequence of {alpha}A and 99% of {alpha}B. The behavior of {alpha}A and {alpha}B is remarkably similar. At low temperatures, there are regions at the beginning of the {alpha}-crystallin domains in both {alpha}A and {alpha}B that have high protection to isotope exchange, whereas the C termini offer little protection. The N terminus of {alpha}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 {alpha}A 72–75 and {alpha}B 76–79, two regions considered critical for chaperone activity.

Keywords: Human lens; {alpha}-crystallins; conformation; mass spectrometry; cataract

Reprint requests to: Jean B. Smith, Department of Chemistry, University of Nebraska–Lincoln, Lincoln, NE 68588-0304, USA; e-mail: jsmith8{at}unl.edu; fax: (402) 472-9402.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03180004.


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