title: Genetic mapping of starch- and lambda-receptor sites in maltoporin: identification of substitutions causing direct and indirect effects on binding sites by cysteine mutagenesis.
abstract: Cysteine mutagenesis was used to test the proximity of 16 residues to protein-ligand interaction sites in maltoporin (LamB protein). LamB protein with additional cysteines was incorporated into the outer membrane of Escherichia coli except with a Ser-30----Cys substitution. Phage Lambda and starch binding was assayed before and after incubation of mutants with six thiol-specific reagents. Four categories of mutation were recognized on the basis of phenotype and modification for each of the Lambda- and starch binding sites. The thiol modification experiments helped to clarify whether the phenotype of a mutation was due to a substitution at the binding site or an indirect perturbation of the structure. This study suggests that the cysteine mutagenesis/thiol modification approach may be usefully applied to the operational mapping of surface-accessible binding sites or epitopes.
authors.affiliation: Department of Microbiology, University of Sydney, New South Wales, Australia.
authors.forename: G
authors.initials: G
authors.lastname: Francis
authors.forename: L
authors.initials: L
authors.lastname: Brennan
authors.forename: S
authors.initials: S
authors.lastname: Stretton
authors.forename: T
authors.initials: T
authors.lastname: Ferenci
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics; D010582:Bacteriophage lambda; D001665:Binding Sites / Q000235:genetics*; D002874:Chromosome Mapping; D003545:Cysteine / Q000737:chemistry; D000939:Epitopes / Q000235:genetics*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D018272:Porins; D011487:Protein Conformation; D011991:Receptors, Virus / Q000737:chemistry / Q000235:genetics*; D013213:Starch; D013438:Sulfhydryl Compounds / Q000737:chemistry
chemical_list: D001425:Bacterial Outer Membrane Proteins; D000939:Epitopes; D018272:Porins; D011991:Receptors, Virus; D013438:Sulfhydryl Compounds; C016532:maltoporins; D013213:Starch; D003545:Cysteine