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J Biol Chem, Vol. 275, Issue 2, 1035-1042, January 14, 2000
From the Jules Stein Eye Institute, UCLA School of Medicine,
Los Angeles, California 90095
Subunit Exchange of Small Heat Shock Proteins
ANALYSIS OF OLIGOMER FORMATION OF 
A-CRYSTALLIN AND Hsp27 BY
FLUORESCENCE RESONANCE ENERGY TRANSFER AND SITE-DIRECTED
TRUNCATIONS*
,A-Crystallin, a member of the small heat shock
protein (sHsp) family, is a large multimeric protein composed of 30-40
identical subunits. Its quaternary structure is highly dynamic, with
subunits capable of freely and rapidly exchanging between oligomers. We report here the development of a fluorescence resonance energy transfer
method for measuring structural compatibility between A-crystallin
and other proteins. We found that Hsp27 and B-crystallin readily
exchanged with fluorescence-labeled A-crystallin, but not with other
proteins structurally unrelated to sHsps. Truncation of 19 residues
from the N terminus or 10 residues from the C terminus of
A-crystallin did not significantly change its subunit organization or exchange rate constant. In contrast, removal of the first 56 or more
residues converts A-crystallin into a predominantly small multimeric
form consisting of three or four subunits, with a concomitant loss of
exchange activity. These findings suggest residues 20-56 are essential
for the formation of large oligomers and the exchange of subunits.
Similar results were obtained with truncated Hsp27 lacking the first 87 residues. We further showed that the exchange rate is independent of
A-crystallin concentration, suggesting subunit dissociation may be
the rate-limiting step in the exchange reaction. Our findings reveal a
quarternary structure of A-crystallin, consisting of small multimers
of A-crystallin subunits in a dynamic equilibrium with the
oligomeric complex.
*
This work was supported in part by Grants EY05895 (to
B. K.-K. F.) and EY12018 (to H. S. M) from NEI, National
Institutes of Health and by a grant from the Wong Fund (to B. K.-K. F).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Recipient of Predoctoral Training Grant EY07026 from NEI, National
Institutes of Health, as well as a postdoctoral fellowship from the
Bank of America Giannini Foundation.
§
To whom all correspondence should be addressed. Tel.: 310-825-9541;
Fax: 310-794-2144.
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