Self-association of a small heat shock protein

Human Hsp27 oligomerizes in vivo in a phosphorylation-dependent manner that regulates the functional activity of the protein. We have studied the self-association of wild-type Hsp27 by both sedimentation velocity and sedimentation equilibrium analysis and established that the protein forms an equilibrium mixture of monomers/dimers, tetramers, 12-mers and 16-mers (20 mM Tris-HCl (pH 8.4), 100 mM NaCl, 20 degreesC). Corresponding analysis of the S15D/S78D/S82D triple variant, which is believed to mimic the behavior of phosphorylated Hsp27, establishes that this form of the protein forms primarily monomers and dimers but also forms a small fraction of very large oligomers. Variants in which critical N-terminal sequences have been deleted exhibit oligomerization behavior that is intermediate between that of the triple variant and the wild-type protein. On the other hand a C-terminal sequence deletion variant forms larger oligomers than does the wild-type protein, but also exhibits a greater fraction of smaller oligomers. Notably, the presence of an N-terminal His(6)-tag induces formation of much larger oligomers than observed for any other form of the protein. The results of this work establish that the wildtype protein forms smaller oligomers than previously believed, define the roles played by various structural domains in Hsp27 oligomerization, and provide improved molecular probes with better-defined properties for the design of future experiments. (C) 2004 Elsevier Ltd. All rights reserved.