title: Chemical synthesis and characterisation of rat chaperonin 10: effect of chain length, ions, heat and N-terminal acetylation on unchaperoned folding into its heptameric form.
abstract: Recently, the sequence of mitochondrial chaperonin 10 from Rattus norvegicus (rat cpn10), with N-terminal acetylation, has been published. Two syntheses of rat cpn10 were performed, the first using a classical carbodiimide-mediated double coupling protocol (Method A) and the second a more efficient HBTU/HOBT/single coupling procedure (Method B). The latter also involved the application of a capping procedure, using N-(2-chlorobenzyloxycarbonyloxy)succinimide [Z(2-Cl)-OSu]. The crude protein from Method A was purified using a two-step isoelectric focusing/RP-HPLC scheme and found to contain a high proportion of a deletion peptide (less Gln60). Conversely, rat cpn10 from Method B was purified to homogeneity by one-step RP-HPLC, using a reversible lipophilic chromatographic probe. The proportion of biologically active heptameric structure was directly related to the purity of the protein and attained 84% with material from Method B. The addition of Ca/Mg ions, pH 7.2, or a heating/cooling cycle increased the proportion of heptamer for less pure protein. Shorter sequences were found not to fold into heptamers, suggesting that aggregation/folding motifs are located in 1-25 and 77-101 regions of rat cpn10. The heptameric cpn10 (Method B) bound correctly to GroEL from E. coli, demonstrating that N-terminal acetylation is not necessary for its folding and binding to bacterial cpn60.
authors.affiliation: Italfarmaco Research Centre, Milan, Italy.
authors.forename: H L
authors.initials: HL
authors.lastname: Ball
authors.forename: P
authors.initials: P
authors.lastname: Giuliani
authors.forename: P
authors.initials: P
authors.lastname: Lucietto
authors.forename: G
authors.initials: G
authors.lastname: Fossati
authors.forename: P
authors.initials: P
authors.lastname: Mascagni
mesh_terms: D000107:Acetylation; D000818:Animals; D018835:Chaperonin 10 / Q000138:chemical synthesis* / Q000737:chemistry*; D006358:Hot Temperature; D007477:Ions; D017510:Protein Folding; D051381:Rats
chemical_list: D018835:Chaperonin 10; D007477:Ions