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(Received for publication, July 18, 1997, and in revised form, September 18, 1997)
From the Jules Stein Eye Institute, University of California School
of Medicine, Los Angeles, California 90095
Volume 272, Number 47,
Issue of November 21, 1997
pp. 29511-29517
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
Subunit Exchange of
A-Crystallin
-Crystallin, the major protein in the
mammalian lens, is a molecular chaperone that can bind denaturing
proteins and prevent their aggregation. Like other structurally related
small heat shock proteins, each -crystallin molecule is composed of
an average of 40 subunits that can undergo extensive reorganization. In
this study we used fluorescence resonance energy transfer to monitor the rapid exchange of recombinant -crystallin subunits. We labeled A-crystallin with stilbene iodoacetamide
(4-acetamido-4
-((iodoacetyl)amino)stilbene-2,2-disulfonic acid),
which serves as an energy donor and with lucifer yellow iodoacetamide,
which serves as an energy acceptor. Upon mixing the two populations of
labeled A-crystallin, we observed a reversible, time-dependent decrease in stilbene iodoacetamide emission
intensity and a concomitant increase in lucifer yellow iodoacetamide
fluorescence. This result is indicative of an exchange reaction that
brings the fluorescent A-crystallin subunits close to each other. We further showed that the exchange reaction is strongly dependent on
temperature, with a rate constant of 0.075 min
1 at
37 °C and an activation energy of 60 kcal/mol. The subunit exchange
is independent of pH and calcium concentration but decreases at low and
high ionic strength, suggesting the involvement of both ionic and
hydrophobic interactions. It is also markedly reduced by the binding of
large denatured proteins. The degree of inhibition is directly
proportional to the molecular mass and the amount of bound polypeptide,
suggesting an interaction of several A-crystallin subunits with
multiple binding sites of the denaturing protein. Our findings reveal a
dynamic organization of A-crystallin subunits, which may be a key
factor in preventing protein aggregation during denaturation.
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