title: Insertion mutagenesis of Escherichiacoli GroEL.
abstract: To gain insights into the in vivo folding and assembly of bacterial chaperonins, groEL was subjected to insertion mutagenesis using transposon ISlacZ/in. Four GroEL-LacZ fusions and the corresponding insertion mutants were obtained after residues 34, 90, 291, and 367. Apical domain insertion mutants GroEL291 and GroEL367 were degraded into monomeric 30- and 40-kDa fragments, respectively. Only the latter was fully soluble, suggesting that proper isomerization of an essentially complete apical domain is required for efficient protomer folding. Truncated variants were inactive as minichaperones as they failed to restore the growth of groEL140 cells at 43 degrees C whether or not GroES was co-expressed. A 31-residue insertion in equatorial helix D led to complete degradation of GroEL90. By contrast, extraneous amino acids were tolerated at equatorial position 34, indicating that this region is highly flexible. Nevertheless, GroEL34 did not fold as efficiently as authentic GroEL and reached only a heptameric conformation.
authors.affiliation: Department of Chemical Engineering, Box 351750, University of Washington, Seattle 98195-1750, USA.
authors.forename: Danielle
authors.initials: D
authors.lastname: Amatore
authors.forename: François
authors.initials: F
authors.lastname: Baneyx
mesh_terms: D000483:Alleles; D018834:Chaperonin 60 / Q000737:chemistry / Q000235:genetics*; D004251:DNA Transposable Elements / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D017353:Gene Deletion; D005816:Genetic Complementation Test; D016254:Mutagenesis, Insertional; D020836:Protein Structure, Quaternary; D013347:Subcellular Fractions
chemical_list: D018834:Chaperonin 60; D004251:DNA Transposable Elements