Folding pattern of the alpha-crystallin domain in alpha A-crystallin determined by site-directed spin labeling

The folding pattern of the alpha-crystallin domain, a conserved protein mod ule encoding the molecular determinants of structure and function in the sm all heat-shock protein superfamily, was determined in the context of the le ns protein alpha A-crystallin by systematic application of site-directed sp in labeling. The sequence-specific secondary structure was assigned primari ly from nitroxide scanning experiments in which the solvent accessibility a nd mobility of a nitroxide probe were measured as a function of residue num ber. Seven beta-strands were identified and their orientation relative to t he aqueous solvent determined, thus defining the residues Lining the hydrop hobic core. The pairwise packing of adjacent strands in the primary structu re was deduced from patterns of proximities in nitroxide pairs with one mem ber on the exposed surface of each strand. In addition to identifying super secondary structures, these proximities revealed that the seven strands are arranged in two beta-sheets. The overall packing of the two sheets was det ermined by application of the general rules of protein structure and from p roximities in nitroxide pairs designed to distinguish between known all bet a-sheet folds. Our data are consistent with an immunoglobulin-like fold con sisting of two aligned beta-sheets. Comparison of this folding pattern to t hat of the evolutionary distant alpha-crystallin domain in Methanococcus ja nnaschii heat-shock protein 16.5 reveals a conserved core structure with th e differences sequestered at one edge of the beta-sandwich. A beta-strand d eletion in alpha A-crystallin disrupts a subunit interface and allows for a different dimerization motif. Putative substrate binding regions appear to include a buried loop and a buried turn, suggesting that the chaperone fun ction involves a disassembly of the oligomer. (C) 1999 Academic Press.