alpha-Crystallin is a major protein of the vertebrate lens once though
t to be highly specialized for conferring transparency. However, recen
t work has revealed a wide tissue distribution and a sequence homology
to small heat shock proteins, suggesting a more general role for the
protein. Like other molecular chaperons, alpha-crystallin is known to
bind to unfolded proteins and suppress nonspecific aggregation in vitr
o. In the present work, spin-labeled derivatives of the insulin B chai
n and melittin were used to investigate the state of these proteins bo
und to alpha-crystallin. Insulin was selected since unfolding can be t
riggered by reduction of the interchain disulfide bonds, a treatment t
hat does not affect alpha-crystallin. Upon reduction of insulin, the s
eparated B chains aggregate. In the presence of alpha-crystallin, the
B chains bind to alpha-crystallin and aggregation is suppressed. Melit
tin, a 26 amino acid peptide from bee venom, was selected for study si
nce it is a random coil under physiological conditions, and its intera
ction with alpha-crystallin can be directly studied. EPR analysis of t
he spin-labeled peptides shows that the nitroxide side chains are immo
bilized in a polar environment on alpha-crystallin and that they are s
eparated by 25 Angstrom or more in the complex, indicating that the bo
und proteins are not clustered. The bound B chains of insulin are not
in a fully extended conformation, and melittin does not appear to bind
to a hydrophobic surface in alpha-crystallin as an amphipathic helix,
as it does to membranes and some other proteins. Equilibrium binding
studies for melittin give a stoichiometry of approximately 1:1 melitti
n/alpha-crystallin monomer, with a dissociation constant of 7.3 mu M.