Optically detected magnetic resonance of tryptophan residues in complexes formed between a bacterial single-stranded DNA binding protein and heavy atom modified poly(uridylic acid).
Optically detected magnetic resonance (ODMR) methods were employed to study three single-stranded DNA binding (SSB) proteins encoded by plasmids of enteric bacteria: pIP71a, R64, and F. Equilibrium binding isotherms obtained by fluorescence titrations reveal that the complexes of the plasmid SSB proteins with heavy atom modified polynucleotides are readily disrupted by salt. Since all the plasmid SSB proteins show limited solubility at low ionic strength (pIP71a greater than R64 greater than F), we were able to bind only the pIP71a protein to mercurated poly(uridylic acid) [poly(5-HgU)] and brominated poly(uridylic acid) [poly(5-BrU)]. ODMR results reveal the existence of at least one heavy atom perturbed, red-shifted, stacked Trp residue in these complexes. Amplitude-modulated phosphorescence microwave double resonance spectra display selectively the phosphorescence associated with Hg-perturbed Trp residue(s) in the pIP71a SSB protein-poly(5-HgU) complex, which has a broad, red-shifted 0,0-band. Our results suggest that Trp-135 in Escherichia coli SSB, which is absent in the plasmid-encoded SSB proteins, is located in a polar environment and is not involved in stacking interactions with the nucleotide bases. Phosphorescence spectra and lifetime measurements of the pIP71a SSB protein-poly (5-BrU) complex show that at least one Trp residue in the complex does not undergo stacking. This sets a higher limit of two stacking interactions of Trp residues with nucleotide bases in complexes of pIP71a SSB with single-stranded polynucleotides.
Khamis
M I
MI
Chemistry Department, University of California, Davis 95616.
Casas-Finet
J R
JR
Maki
A H
AH
Ruvolo
P P
PP
Chase
J W
JW
D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D008163:Luminescent Measurements; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D009994:Osmolar Concentration; D010957:Plasmids; D011072:Poly U / Q000031:analogs & derivatives* / Q000378:metabolism; D014364:Tryptophan
D004268:DNA-Binding Proteins; D011072:Poly U; C031089:poly-5-bromouridylic acid; D014364:Tryptophan