Modulation of the affinity of the single‐stranded DNA‐binding protein of Escherichia coli (E. coli SSB) to poly(dT) by site‐directed mutagenesis
Open Access
- 1 February 1989
- journal article
- research article
- Published by Wiley in European Journal of Biochemistry
- Vol. 179 (2), 399-404
- https://doi.org/10.1111/j.1432-1033.1989.tb14567.x
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 > Phe (wild‐type) > Tyr > Leu > His > Val > Ser, leading to the conclusion that position 60 is a site of hydrophobic interaction of the protein with DNA.This publication has 23 references indexed in Scilit:
- Equilibrium binding of Escherichia coli single-strand binding protein to single-stranded nucleic acids in the (SSB)65 binding mode. Cation and anion effects and polynucleotide specificityBiochemistry, 1988
- SINGLE-STRANDED DNA BINDING PROTEINS REQUIRED FOR DNA REPLICATIONAnnual Review of Biochemistry, 1986
- Large-scale overproduction and rapid purification of the Escherichia coli ssb gene product. Expression of the ssb gene under .lambda. PL controlBiochemistry, 1986
- Single-Stranded DNA Binding Proteins Required for DNA ReplicationAnnual Review of Biochemistry, 1986
- Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sitesGene, 1985
- Kinetics and mechanism of dissociation of cooperatively bound T4 gene 32 protein-single-stranded nucleic acid complexes. 1. Irreversible dissociation induced by sodium chloride concentration jumpsBiochemistry, 1984
- Novel crystal forms of a proteolytic core of the single‐stranded DNA‐binding protein (SSB) from E. coliFEBS Letters, 1984
- Crystallization of single-strand DNA-binding proteinJournal of Molecular Biology, 1983
- Crystals of Escherichia coli single-strand DNA-binding protein show that the tetramer has D2 symmetryJournal of Molecular Biology, 1983
- Nucleotide sequence of bacteriophage λ DNAJournal of Molecular Biology, 1982