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J. Biol. Chem., Vol. 280, Issue 15, 14485-14491, April 15, 2005

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Subunit Exchange of Polydisperse Proteins

MASS SPECTROMETRY REVEALS CONSEQUENCES OF A-CRYSTALLIN TRUNCATION^*

J. Andrew Aquilina¶, Justin L. P. Benesch¶||, Lin Lin Ding**, Orna Yaron**, Joseph Horwitz**, and Carol V. Robinson

From the Department of Chemistry, University of Cambridge, Cambridge, CB2 1EW, United Kingdom and the ^**Jules Stein Eye Institute, UCLA School of Medicine, Los Angeles, California 90095-7008

The small heat shock protein, -crystallin, plays a key role in maintaining lens transparency by chaperoning structurally compromised proteins. This is of particular importance in the human lens, where proteins are exposed to post-translational modifications over the life-time of an individual. Here, we examine the structural and functional consequences of one particular modification of A-crystallin involving the truncation of 5 C-terminal residues (A_1–168). Using novel mass spectrometry approaches and established biophysical techniques, we show that A_1–168 forms oligomeric assemblies with a lower average molecular mass than wild-type A-crystallin (A_WT). Also apparent from the mass spectra of both A_WT and A_1–168 assemblies is the predominance of oligomers containing even numbers of subunits; interestingly, this preference is more marked for A_1–168. To examine the rate of exchange of subunits between assemblies, we mixed B crystallin with either A_WT or A_1–168 and monitored in a real-time mass spectrometry experiment the formation of heteroligomers. The results show that there is a significant decrease in the rate of exchange when A_1–168 is involved. These reduced exchange kinetics, however, have no effect upon chaperone efficiency, which is found to be closely similar for both A_WT and A_1–168. Overall, therefore, our results allow us to conclude that, in contrast to mechanisms established for analogous proteins from plants, yeast, and bacteria, the rate of subunit exchange is not the critical parameter in determining efficient chaperone behavior for mammalian A-crystallin.

Received for publication, January 5, 2005

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

A Royal Society Howard Florey Postdoctoral Fellow. Present address: Dept. of Chemistry, University of Wollongong, Wollongong, NSW 2522, Australia.

^¶ Both authors contributed equally to this work.

^|| Supported by the Engineering and Physical Sciences Research Council.

Supported by National Institutes of Health Grant EY-3897.

Supported by the Royal Society. To whom correspondence should be addressed. Tel.: 44-1223-763-846; Fax: 44-1223-336-362; E-mail: cvr24{at}cam.ac.uk.

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