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 secondary structure, solvent accessibility, and tertiary and quaternary interactions 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 with a sulfhydryl-specific spin-label. The accessibilities of attached nitroxide side chains to a paramagnetic probe in the aqueous solution were determined. Spectral line shapes were analyzed in terms of side-chain mobility and spatial proximity to nearby nitroxides. The sequence-specific mobilities and accessibilities varied with a period of 2, consistent 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 the strand is buried. Furthermore, spin-spin interactions between nitroxides in the oligomeric structure strongly suggest that equivalent strands from different subunits are in close spatial proximity. These structural characteristics are remarkably similar to those of the equivalent 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 interface, indicating that one set of interactions between subunits, and its associated symmetry, is conserved. This is the first report of sequence-specific structural similarity between alpha A-crystallin and HSP 27 and the first identification of a conserved secondary structural element in the alpha-crystallin domain.