INTERACTION OF ALPHA-CRYSTALLIN WITH SPIN-LABELED PEPTIDES

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.