Small heat-shock protein structures reveal a continuum from symmetric to variable assemblies

The small heat-shock proteins (sHSPs) form a diverse family of proteins tha t are produced in all organisms. They function as chaperone-like proteins i n that they bind unfolded polypeptides and prevent uncontrolled protein agg regation. Here, we present parallel cryo-electron microscopy studies of fiv e different sHSP assemblies: Methanococcus jannaschii HSP16.5, human alpha B-crystallin, human HSP27, bovine native alpha-crystallin, and the complex of alpha B-crystallin and unfolded alpha-lactalbumin. Gel-filtration chroma tography indicated that HSP16.5 is the most monodisperse, while HSP27 and t he alpha-crystallin assemblies are more polydisperse. Particle images revea led a similar trend showing mostly regular and symmetric assemblies for HSP 16.5 particles and the most irregular assemblies with a wide range of diame ters for HSP27. A symmetry test on the particle images indicated stronger o ctahedral symmetry for HSP16.5 than for HSP27 or the a-crystallin assemblie s. A single particle reconstruction of HSP16.5, based on 5772 particle imag es with imposed octahedral symmetry, resulted in a structure that closely m atched the crystal structure. In addition, the cryo-EM reconstruction revea led internal density presumably corresponding to the flexible 32 N-terminal residues that were not observed in the crystal structure. The N termini we re found to partially fill the central cavity making it unlikely that HSP16 .5 sequesters denatured proteins in the cavity. A reconstruction calculated without imposed symmetry confirmed the presence of at least loose octahedr al symmetry for HSP16.5 in contrast to the other sHSPs examined, which disp layed no clear overall symmetry. Asymmetric reconstructions for the alpha-c rystallin assemblies, with an additional mass selection step during image p rocessing, resulted in lower resolution structures. We interpret the alpha- crystallin reconstructions to be average representations of variable assemb lies and suggest that the resolutions achieved indicate the degree of varia bility. Quaternary structural information derived from cryo-electron micros copy is related to recent EPR studies of the alpha-crystallin domain fold a nd dimer interface of alpha A-crystallin. (C) 2000 Academic Press.