A vector for site-directed mutagenesis and overproduction of the Escherichia coli single-stranded-DNAbinding 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.

The histidine at position 55 of the amino acid sequence of the Escherichia coli single-stranded DNA binding protein was replaced by tyrosine, glutamic acid, lysine, phenylalanine, and isoleucine. The properties of the mutant proteins were determined using analytical ultracentrifugation, NMR spectroscopy, gel filtration, and fluorimetric detection of their single-stranded DNA binding ability. While the phenylalanine and isoleucine substitutions did not change the properties of the protein measurably, tyrosine and lysine mutants dissociate into subunits and loose some of their binding affinity for poly (dT). For the lysine mutant we show by electron microscopy that the protein, although fully dissociated and possibly denatured in the free state, binds to poly(dT) as a tetramer indistinguishable from the wild-type protein. The process of tetramerization as observed via single-stranded DNA binding ability is composed of a variety of steps ranging in time from some milliseconds to several hours; it probably involves several forms of dissociated and non-native protein.In the living cell, the single-stranded DNA binding protein of Escherichia coli (EcoSSB) stabilizes the single-stranded conformation of the DNA which is necessary for subsequent reactions like replication, recombination or repair. Another important functional role of EcoSSB is the protection of single-stranded DNA against nucleolytic attack. For a review of biological and physico-chemical properties of EcoSSB seeThe amino acid sequence of EcoSSB (177 amino acids) has been deduced from its DNA sequence [3]. The protein has been crystallized [4, 51, but so far, no tertiary structure from X-ray crystallography is known. Under native conditions four identical EcoSSB protomers form a tetramer with a D2 symmetry [6]. Secondary structure prediction according to the algorithm of Chou and Fasman [7] suggests the presence of two structurally different regions in the protomer [3]. Approximately 100 amino-terminal residues form a structured region containing M helices and P sheets whereas the remaining carboxy-terminal part containing 19 glycine and 10 proline residues seems to have no definable secondary structure. It could be shown from partial proteolytic digests that removing 62 amino acids from the carboxy terminus did not diminish the DNA binding ability; it only slightly reduced the tetramerization of the protein [S].A variety of mutagenesis experiments has been carried out to elucidate the role of defined amino acids in the structure and function of EcoSSB. It could be shown that the tryptophan residues at positions 40 and 54 [9, 101 and the phenylalanine residue at position 60 [ll] are involved in DNA binding. 11, 21.We have previously shown that the phenylalanine residue at position 60 is directly involved in a hydrophobic interaction with the DNA [12]. From chemical mutagenesis a temperature-sensitive mutant strain of E. coli could be isolated in which histidine at position 55 was substituted by tyrosine (ssb-1) [13]. The temperature sensitivity of the ...