Multiple binding modes of the single-stranded DNA binding protein from Escherichia coli as detected by tryptophan fluorescence and site-directed mutagenesis.
We have systematically substituted the four tryptophan residues of the single-stranded DNA binding protein from Escherichia coli (EcoSSB) by polar (serine or threonine) and aromatic (tyrosine or phenylalanine) amino acids. The resulting mutants with either single amino acid exchanges or triple substitutions are all active in ssDNA binding, though in some cases with reduced affinities. Measurements of the fluorescence of the mutated EcoSSBs show that there is no interaction between the four different tryptophan residues. We analyzed the ssDNA binding of the mutant proteins by fluorescence titrations. At 0.3 M NaCl ("high salt"), all singly substituted proteins bind to poly(dT) in a manner comparable to wild-type EcoSSB, covering 65 nucleotides with 1 EcoSSB tetramer. W54S mutant protein is an exception since even at 0.3 M NaCl it covers approximately 35 nucleotides, a behavior which is typical of salt concentrations below 10 mM NaCl ("low salt"). From this observation, it is inferred that tryptophan-54 is involved in a direct interaction with the ssDNA favoring the "high-salt" binding mode. All mutant proteins lacking tryptophan-54 but possessing tryptophan-88 at "low-salt" concentrations show a nonmonotonous behavior in the fluorescence titrations. This behavior can be interpreted assuming a model of cooperative binding of EcoSSB to poly(dT) with two different binding site sizes (n approximately 27 and n approximately 33) and different binding affinities. A quantitative treatment of the problem of multiple binding modes in the interaction of a multidentate ligand with a linear polymer is applied to these titrations.
Curth
U
U
Medizinische Hochschule, Abteilung Biophysikalische Chemie, Hannover, FRG.
Greipel
J
J
Urbanke
C
C
Maass
G
G
D000595:Amino Acid Sequence; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D011071:Poly T / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D008024:Ligands; D011994:Recombinant Proteins; D011071:Poly T; D014364:Tryptophan