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J. Biol. Chem., Vol. 277, Issue 43, 40557-40566, October 25, 2002
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A-CRYSTALLIN*
,
From the Department of Molecular Physiology and Biophysics,
Vanderbilt University, Nashville, Tennessee 37232
To elucidate the mechanism of A-crystallin
chaperone function, a detailed thermodynamic analysis of its binding to
destabilized, site-directed mutants of T4 lysozyme was carried out. The
selected mutants form a ladder of stabilities spanning the 5-10
kcal/mol range of free energy of unfolding. The crystal structures of
the majority of the mutants have been previously determined and found to be similar to that of the wild type with no evidence of static local
unfolding. Complex formation between A-crystallin and T4 lysozyme
was observed directly via the changes in the electron paramagnetic
resonance lineshape of a nitroxide introduced at a non-destabilizing,
solvent exposed site in T4 lysozyme. A-Crystallin differentially
interacts with the mutants, binding the more destabilized ones to a
larger extent despite the similar structure of their native states. Our
results suggest that the states recognized by A-crystallin are
non-native excited states distinct from the unfolded state. Stable
complexes are formed when the free energy of binding to A-crystallin
is on the order of the free energy associated with the transition from
the excited state to the native state. Biphasic binding isotherms
reveal two modes of interactions with distinct affinities and
stoichiometries. Highly destabilized mutants preferentially bind to the
high capacity mode, suggesting conformational preference in the
use of each mode. Furthermore, binding can be enhanced by increased
temperature and pH, which may be reflecting conformational changes in
A-crystallin oligomeric structure.
To whom correspondence should be addressed: Dept. of Molecular
Physiology and Biophysics, Vanderbilt University, 741 Light Hall,
Nashville, TN 37232. Tel.: 615-322-3307; Fax: 615-322-7236; E-mail: hassane.mchaourab@vanderbilt.edu.
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