title: Design of highly stable functional GroEL minichaperones.
abstract: GroEL minichaperones have potential in the biotechnology industry for the refolding of recombinant proteins. With the aim of enhancing and widening their use, we have created two highly stable functional variants of minichaperone GroEL(193-345). A sequence alignment of 130 members of the chaperonin 60 (Cpn60) family was used to design 37 single mutations. Two small-to-large mutations, A223T, A223V and one similar-size mutation, M233L, all located in the hydrophobic core were found to stabilize the protein by more than 1 kcal mol(-1) each. Six stabilizing mutations were combined, yielding two multiple mutants that were 6.99 and 6.15 kcal mol(-1) more stable than wild-type protein. Even though some of the substituted residue pairs are close to each other in the protein structure, the energetic effects of mutation are approximately additive. In particular, the stabilizing substitution A223T is unexpected and would have been missed by purely structural analysis. In the light of previously reported successes employing similar methods with several other proteins, our results show that a homology based approach is a simple and efficient method of increasing the stability of a protein.
authors.affiliation: Cambridge Centre for Protein Engineering and Cambridge University Chemical Laboratory, United Kingdom.
authors.forename: Q
authors.initials: Q
authors.lastname: Wang
authors.forename: A M
authors.initials: AM
authors.lastname: Buckle
authors.forename: N W
authors.initials: NW
authors.lastname: Foster
authors.forename: C M
authors.initials: CM
authors.lastname: Johnson
authors.forename: A R
authors.initials: AR
authors.lastname: Fersht
mesh_terms: D018834:Chaperonin 60 / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000737:chemistry; D006860:Hydrogen Bonding; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D013816:Thermodynamics
chemical_list: D018834:Chaperonin 60; D018832:Molecular Chaperones