Optically detected magnetic resonance of tryptophan residues in Escherichia coli ssb gene product and E. coli plasmid-encoded single-stranded DNA-binding proteins and their complexes with poly(deoxythymidylic) acid.
Optically detected magnetic resonance (ODMR) spectroscopy has been applied to several single-stranded DNA-binding (SSB) proteins encoded by conjugative plasmids of enteric bacteria. Fluorimetric equilibrium binding isotherms confirm their preferential binding to single-stranded DNA and polynucleotides and reveal a limited protein solubility at low ionic strength. The plasmid SSB-like proteins show the highest affinity for polydeoxythymidylic acid; these complexes are the least sensitive to disruption by salt. ODMR data on these complexes suggest the existence of stacking interactions between tryptophan residue(s) and thymine bases, as evidenced by spectral red shifts of the tryptophan phosphorescence 0,0 band, reduction of the magnitude of D zero field splitting parameter, and a dramatic reversal of the polarity of the ODMR signals. Wavelength-selected ODMR results point to the existence of two distinct tryptophan sites in these complexes. The triplet state properties of the red-shifted site are drastically altered by its interaction with the thymine bases. The chromosomal Escherichia coli SSB protein-poly(dT) complex shows an additional tryptophan site with zero field splitting parameters similar to those of the free protein. This site can be attributed to Trp-135, which is missing in each of the other plasmid SSB proteins, suggesting that this particular residue is not involved in the interaction with polynucleotides.
Casas-Finet
J R
JR
Khamis
M I
MI
Maki
A H
AH
Ruvolo
P P
PP
Chase
J W
JW
D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005796:Genes; D005798:Genes, Bacterial; D008163:Luminescent Measurements; D009682:Magnetic Resonance Spectroscopy; D010957:Plasmids; D011071:Poly T / Q000378:metabolism*; D011089:Polydeoxyribonucleotides / Q000378:metabolism*; D014364:Tryptophan
D004268:DNA-Binding Proteins; D011089:Polydeoxyribonucleotides; D011071:Poly T; D014364:Tryptophan