Isolation and characterisation of mutants of GroEL that are fully functional as single rings.
A key aspect of the reaction mechanism for the molecular chaperone GroEL is the transmission of an allosteric signal between the two rings of the GroEL complex. Thus, the single-ring mutant SR1 is unable to act as a chaperone as it cannot release bound substrate or GroES. We used a simple selection procedure to identify mutants of SR1 that restored chaperone activity in vivo. A large number of single amino acid changes, mapping at diverse positions throughout the protein, enabled SR1 to regain its ability to act as a chaperone while remaining as a single ring. In vivo assays were used to identify the proteins that had regained maximal activity. In some cases, no difference could be detected between strains expressing wild-type GroEL and those expressing the mutated proteins. Three of the most active proteins where the mutations were in distinct parts of the protein were purified to homogeneity and characterised in vitro. All were capable of acting efficiently as chaperones for two different GroES-dependent substrates. All three proteins bound nucleotide as effectively as did GroEL, but the binding of GroES in the presence of ATP or ADP was reduced significantly relative to the wild-type. These active single rings should provide a useful tool for studying the nature of the allosteric changes that occur in the GroEL reaction cycle.
Sun
Zhe
Z
School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.
Scott
David J
DJ
Lund
Peter A
PA
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000494:Allosteric Regulation; D018834:Chaperonin 60 / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002950:Citrate (si)-Synthase / Q000378:metabolism; D008291:Malate Dehydrogenase / Q000378:metabolism; D009154:Mutation; D011487:Protein Conformation; D017510:Protein Folding
D018834:Chaperonin 60; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D008291:Malate Dehydrogenase; D002950:Citrate (si)-Synthase