A THERMOSTABLE 35-RESIDUE SUBDOMAIN WITHIN VILLIN HEADPIECE

The actin-bundling protein villin contains, at its extreme C terminus, a compact f-actin binding domain called ''headpiece'' This 76-amino a cid domain from chicken is highly thermostable. Here, we show that the stable folded structure in headpiece is localized to a subdomain form ed by the C-terminal 35 residues. The subdomain, denoted HP-35, is mon omeric and retains high thermostability, with a T-m of 70 (+/-1) degre es C at pH 7.0. There are no cysteine residues in HP-35 and its foldin g is not dependent on the binding of metals or other ligands. HP-35 is not a molten globule, but instead, has properties expected for a full y folded protein with a unique structure. In particular, the slowly ex changing amide protons in HP-35 have protection factors that are sligh tly larger than those predicted if exchange occurred only from globall y unfolded molecules. NMR studies indicate that the headpiece subdomai n contains three short alpha-helices, and that these same helices are present in the corresponding regions of intact headpiece. HP-35 is the smallest monomeric polypeptide characterized consisting of only natur ally occurring amino acids that autonomously folds into a unique and t hermostable structure without disulfide bonds or ligand binding. (C) 1 996 Academic Press Limited