The stability of the molecular chaperonin cpn60 is affected by site-directed replacement of cysteine 518.
Cysteine 518 of the molecular chaperonin cpn60 (groEL) has been replaced with serine (C518S) by site-directed mutagenesis. The resulting mutant chaperonin protein is still functional and it can: (a) arrest the spontaneous folding of rhodanese in the absence of GroES and ATP, (b) assist refolding of the enzyme rhodanese in the presence of GroES and ATP/Mg2+, and (c) permit the urea-induced release and refolding of rhodanese from its complex with cpn60. ATP/Mg2+, alone, could discharge active rhodanese from cpn60 complexes formed with either wild type or C518S. In contrast with wild type cpn60, C518S has: (a) reduced stability of its quaternary structure, (b) reduced ability to reassemble tetradecamers after dissociation by urea; (c) reduced ATPase activity; and (d) more easily exposed hydrophobic surfaces. The data suggest that replacement of Cys-518 with Ser in cpn60 destabilizes its oligomeric structure, but there is no significant effect on cpn60 function or the stability of the monomers formed in urea.
Luo
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GX
Department of Biochemistry, University of Texas Health Science Center at San Antonio 78240-7760.
Horowitz
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PM
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate; D000817:Anilino Naphthalenesulfonates; D001483:Base Sequence; D018834:Chaperonin 60 / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes; D008274:Magnesium; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D013884:Thiosulfate Sulfurtransferase / Q000378:metabolism; D014508:Urea
D000817:Anilino Naphthalenesulfonates; D018834:Chaperonin 60; D017931:DNA Primers; D005456:Fluorescent Dyes; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D000255:Adenosine Triphosphate; D014508:Urea; D013884:Thiosulfate Sulfurtransferase; D000251:Adenosine Triphosphatases; D008274:Magnesium; D003545:Cysteine