SITE-DIRECTED SPIN-LABELING STUDY OF THE STRUCTURE AND SUBUNIT INTERACTIONS ALONG A CONSERVED SEQUENCE IN THE ALPHA-CRYSTALLIN DOMAIN OF HEAT-SHOCK-PROTEIN .27. EVIDENCE OF A CONSERVED SUBUNIT INTERFACE

Site-directed spin-labeling (SDSL) was used to investigate the seconda ry structure, solvent accessibility, and tertiary and quaternary inter actions along the sequence located between residues 133 and 144 in the alpha-crystallin domain of human heat-shock protein 27 (HSP 27), The sequence is conserved among mammalian sHSP and shows similarity to the region of highest homology between alpha A- and alpha B-crystallins. Eleven sequential single cysteine mutants were prepared and reacted wi th a sulfhydryl-specific spin-label. The accessibilities of attached n itroxide side chains to a paramagnetic probe in the aqueous solution w ere determined. Spectral line shapes were analyzed in terms of side-ch ain mobility and spatial proximity to nearby nitroxides. The sequence- specific mobilities and accessibilities varied with a period of 2, con sistent with the presence of a beta-strand along the sequence. At even sites, the nitroxide environment is highly ordered with virtually no accessibility to the hydrophilic probe, indicating that one face of th e strand is buried. Furthermore, spin-spin interactions between nitrox ides in the oligomeric structure strongly suggest that equivalent stra nds from different subunits are in close spatial proximity. These stru ctural characteristics are remarkably similar to those of the equivale nt sequence in alpha A-crystallin [Berengian, A. R., Bova, M. P., and Mchaourab, H. S. (1997) Biochemistry, 36, 9951-9957]. In both proteins , a beta-strand spans the sequence and is located at a subunit interfa ce, indicating that one set of interactions between subunits, and its associated symmetry, is conserved. This is the first report of sequenc e-specific structural similarity between alpha A-crystallin and HSP 27 and the first identification of a conserved secondary structural elem ent in the alpha-crystallin domain.