title: Modulation of the affinity of the single-stranded DNA-binding protein of Escherichia coli (E. coli SSB) to poly(dT) by site-directed mutagenesis.
abstract: A vector for site-directed mutagenesis and overproduction of the Escherichia coli single-stranded-DNA-binding protein (E. coli SSB) was constructed. An E. coli strain carrying this vector produces up to 400 mg pure protein from 25 g wet cells. The vector was used to mutate specifically the Phe60 residue of E. coli SSB. Phe60 had been proposed to be located near the single-stranded-DNA-binding site. Substitution of the Phe60 residue by Val, Ser, Leu, His, Tyr and Trp gave proteins with no or only minor conformational changes, as detected by NMR spectroscopy. The affinity of the mutant E. coli SSB proteins for single-stranded DNA decreased in the order Trp greater than Phe (wild-type) greater than Tyr greater than Leu greater than His greater than Val greater than Ser, leading to the conclusion that position 60 is a site of hydrophobic interaction of the protein with DNA.
authors.affiliation: Zentrum Biochemie, Medizinische Hochschule Hannover, Federal Republic of Germany.
authors.forename: I
authors.initials: I
authors.lastname: Bayer
authors.forename: A
authors.initials: A
authors.lastname: Fliess
authors.forename: J
authors.initials: J
authors.lastname: Greipel
authors.forename: C
authors.initials: C
authors.lastname: Urbanke
authors.forename: G
authors.initials: G
authors.lastname: Maass
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D011071:Poly T / Q000378:metabolism*; D011089:Polydeoxyribonucleotides / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
chemical_list: D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D011089:Polydeoxyribonucleotides; D011071:Poly T