title: Investigation of the role of individual tryptophan residues in the binding of Escherichia coli single-stranded DNA binding protein to single-stranded polynucleotides. A study by optical detection of magnetic resonance and site-selected mutagenesis.
abstract: Fluorescence and optical detection of triplet state magnetic resonance (ODMR) spectroscopy have been employed to study the complexes formed between single-stranded polynucleotides and Escherichia coli ssb gene products (SSB) in which tryptophans 40, 54, and 88 are selectively, one residue at a time, replaced by phenylalanine using site-specific oligonucleotide mutagenesis. Fluorescence titrations and ODMR results indicate that tryptophans 40 and 54 are the only tryptophan residues in E. coli single-stranded DNA binding protein that are involved in stabilizing the protein-nucleic acid complexes via stacking interactions. Wavelength-selected ODMR measurements on E. coli SSB reveal the presence of two spectrally distinct tryptophan sites (Khamis, M. I., Casas-Finet, J. R., and Maki, A. H. (1987) J. Biol. Chem. 262, 1725-1733). Our present results indicate that tryptophan 54 belongs to the blue-shifted site, while tryptophan 40 belongs to the red-shifted site of the protein.
authors.forename: M I
authors.initials: MI
authors.lastname: Khamis
authors.forename: J R
authors.initials: JR
authors.lastname: Casas-Finet
authors.forename: A H
authors.initials: AH
authors.lastname: Maki
authors.forename: J B
authors.initials: JB
authors.lastname: Murphy
authors.forename: J W
authors.initials: JW
authors.lastname: Chase
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000032:analysis*; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D011071:Poly T / Q000378:metabolism; D011072:Poly U / Q000031:analogs & derivatives / Q000378:metabolism; D011119:Polynucleotides / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
chemical_list: D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D011119:Polynucleotides; D011071:Poly T; D011072:Poly U; C031089:poly-5-bromouridylic acid; C053361:poly-5-mercuriuridylic acid; D014364:Tryptophan