title: An arginine residue (Arg101), which is conserved in many GroEL homologues, is required for interactions between the two heptameric rings.
abstract: Homologous recombination was used to construct a series of hybrid chaperonin genes, containing various lengths of Escherichia coli groEL replaced by the equivalent region from the homologous cpn60-1 gene of Rhizobium leguminosarum. Analysis of proteins produced by these hybrids showed that many of them formed structures with properties consistent with their being single heptameric rings under some conditions, as opposed to the double ring form in which both the GroEL and the Cpn60-1 proteins are found. By determining precise cross-over points, two regions in Cpn60-1 were defined which appeared to be critical for ring-ring interactions. Within one of these regions is a highly conserved arginine residue (Arg101), which we hypothesised to interact with a residue or residues toward the C terminus of the protein, this contact being required for double rings to form. To test this hypothesis, we mutagenised this residue from arginine to threonine in chaperonin genes from two different species of Rhizobium. In both cases, proteins which ran on non-denaturing gels as single rings were produced. Conversion of Arg101 to serine also had the same effect, whereas conversion of Arg101 to lysine did not. Two different single rings created by homologous recombination could be converted back to double rings by changing the threonine, which naturally occurs at this position in E. coli GroEL, back to arginine. The in vivo properties of the proteins were investigated by complementation following deletion of the chromosomal copy of the groEL gene, and by monitoring the ability of cells expressing the hybrid proteins to plate bacteriophage. Most of the hybrid and mutant proteins were functional in these assays, despite their altered properties compared to wild-type GroEL.
authors.affiliation: School of Biological Sciences, University of Birmingham, Birmingham, B15 2TT, UK.
authors.forename: S
authors.initials: S
authors.lastname: Jones
authors.forename: E J
authors.initials: EJ
authors.lastname: Wallington
authors.forename: R
authors.initials: R
authors.lastname: George
authors.forename: P A
authors.initials: PA
authors.lastname: Lund
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001120:Arginine / Q000235:genetics*; D001435:Bacteriophages / Q000254:growth & development; D001483:Base Sequence; D018834:Chaperonin 60 / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017124:Conserved Sequence / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism / Q000821:virology; D017353:Gene Deletion; D005816:Genetic Complementation Test; D018832:Molecular Chaperones / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011485:Protein Binding; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D016961:Rhizobium leguminosarum / Q000235:genetics*; D013696:Temperature
chemical_list: D018834:Chaperonin 60; D018832:Molecular Chaperones; D011993:Recombinant Fusion Proteins; D001120:Arginine