STRUCTURE AND FUNCTION OF THE CONSERVED DOMAIN IN ALPHA-A-CRYSTALLIN - SITE-DIRECTED SPIN-LABELING IDENTIFIES A BETA-STRAND LOCATED NEAR A SUBUNIT INTERFACE

Twelve sequential single cysteine mutants of alpha A-crystallin extend ing between amino acids Y109 and L120 were prepared and reacted with a sulfhydryl specific spin label in order to investigate the role of th is sequence in the assembly of the alpha A-crystallin quaternary struc ture and its chaperone-like function. The sequence is located in the r egion of highest homology in the alpha-crystallin domain, a stretch of 100 amino acids conserved among lens alpha-crystallins and small heat -shock proteins (sHSPs). Analysis of the solvent accessibility and mob ility of the attached nitroxides reveals that the sequence, as a whole , is relatively sequestered from the aqueous solvent. Furthermore, as the nitroxide is scanned across the sequence, both mobility and access ibility vary with a periodicity of 2, demonstrating that the backbone conformation is that of a beta-strand. One face of the strand, contain ing the highly conserved residues R112 and R116, is buried with virtua lly no accessibility to the aqueous solvent. Equivalent strands from d ifferent subunits are in close spatial proximity, as inferred from spi n-spin interactions between identical residues along the strand. Taken together, our results are consistent with the hypothesis that the alp ha-crystallin domain is a building block of the alpha-crystallins quat ernary structure and suggest that the charge conservation observed in the alpha-crystallins evolution might be important for the assembly of the oligomer. This work reports the first use of SDSL to identify a b eta-strand in an unknown structure and demonstrates the feasibility of using this technique to investigate the oligomeric structure of the a lpha-crystallins and sHSPs.