title: Effect of folding on the export of ribose-binding protein studied with the genetically isolated suppressors for the signal sequence mutation.
abstract: Ribose-binding protein (RBP) has a bilobate structure and functions in the periplasm of Escherichia coli. Mutations that affect the folding of RBP were isolated as intragenic suppressors for the export-defective signal sequence mutation. Of 13 different mutational changes found in the mature region, 12 were located in the several peptides forming the N-domain, and one in the C-domain. Translocation kinetics of mutant proteins were analyzed by pulse-labeling and chase experiments, showing the recovery of precursor processing in the range of 42 to 70%. Folding properties of seven mutant RBPs purified were investigated in vitro by means of tyrosine fluorescence. The stability of the mutant proteins, estimated by equilibrium analysis in the presence of denaturant, were reduced by 2.1 to 5.1 kcal/mol of changes in free energy of unfolding. All the mutant proteins showed retardation in folding rate by 4.4 to 63-fold compared to wild-type while unfolding was little affected. The only exception was the L129Q that has a change in the C-domain resulting in unstability due to faster unfolding. Our approach took advantage of an involvement of the folding process in protein export, which was genetically employed to dissect the folding pathway of RBP. As a result, amino acid residues that are specifically involved in the folding pathway of RBP were identified. Most of them are concentrated in one of the subdomains, suggesting that the folding event in the N-domain of RBP is crucial in the rate-determining step.
authors.affiliation: Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Yusong-Ku, Taejon, Korea.
authors.forename: T
authors.initials: T
authors.lastname: Song
authors.forename: C
authors.initials: C
authors.lastname: Park
mesh_terms: D002151:Calorimetry; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003062:Codon / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D016153:Genes, Suppressor; D007438:Introns; D007700:Kinetics; D008958:Models, Molecular; D033902:Periplasmic Binding Proteins; D010957:Plasmids; D017354:Point Mutation; D017510:Protein Folding; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012266:Ribose / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D002352:Carrier Proteins; D003062:Codon; D029968:Escherichia coli Proteins; D033902:Periplasmic Binding Proteins; D021382:Protein Sorting Signals; C028003:RbsB protein, E coli; D011994:Recombinant Proteins; D012266:Ribose