title: Evidence for an essential function of the N terminus of a small heat shock protein in vivo, independent of in vitro chaperone activity.
abstract: To investigate the mechanism of small heat shock protein (sHsp) function, unbiased by current models of sHsp chaperone activity, we performed a screen for mutations of Synechocystis Hsp16.6 that reduced the ability of the protein to provide thermotolerance in vivo. Missense mutations at 17 positions throughout the protein and a C-terminal truncation of 5 aa were identified, representing the largest collection of sHsp mutants impaired in function in vivo. Ten mutant proteins were purified and tested for alterations in native oligomeric structure and in vitro chaperone activity. These biochemical assays separated the mutants into two groups. The C-terminal truncation and six mutations in the alpha-crystallin domain destabilized the sHsp oligomer and reduced in vitro chaperone activity. In contrast, the other three mutations had little effect on oligomer stability or chaperone activity in vitro. These mutations were clustered in the N terminus of Hsp16.6, pointing to a previously unrecognized, important function for this evolutionarily variable domain. Furthermore, the fact that the N-terminal mutations were impaired in function in vivo, but active as chaperones in vitro, indicates that current biochemical assays do not adequately measure essential features of the sHsp mechanism of action.
authors.affiliation: Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, AZ 85721, USA.
authors.forename: Kim C
authors.initials: KC
authors.lastname: Giese
authors.forename: Eman
authors.initials: E
authors.lastname: Basha
authors.forename: Belmund Y
authors.initials: BY
authors.lastname: Catague
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Vierling
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000502:physiology*; D002845:Chromatography; D003062:Codon; D003459:Crystallins / Q000737:chemistry; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism; D017353:Gene Deletion; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000502:physiology*; D006358:Hot Temperature; D008156:Luciferases / Q000378:metabolism; D018832:Molecular Chaperones / Q000737:chemistry; D008969:Molecular Sequence Data; D009154:Mutation; D020125:Mutation, Missense; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry; D046939:Synechocystis / Q000378:metabolism; D013696:Temperature; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D003062:Codon; D003459:Crystallins; D006360:Heat-Shock Proteins; C113725:HspA protein, bacteria; D018832:Molecular Chaperones; D011506:Proteins; D008156:Luciferases