Thermodynamics of specific and nonspecific DNA binding by two DNA-binding domains conjugated to fluorescent probes.
The complexes designed in this work combine the sequence-specific binding properties of helix-turn-helix DNA-binding motifs with intercalating cyanine dyes. Thermodynamics of the Hin recombinase and Tc3 transposase DNA-binding domains with and without the conjugated dyes were studied by fluorescence techniques to determine the contributions to specific and nonspecific binding in terms of the polyelectrolyte and hydrophobic effects. The roles of the electrostatic interactions in binding to the cognate and noncognate sequences indicate that nonspecific binding is more sensitive to changes in salt concentration, whereas the change in the heat capacity shows a greater sensitivity to temperature for the sequence-specific complexes in each case. The conjugated dyes affect the Hin DNA-binding domain by acting to anchor a short stretch of amino acids at the N-terminal end into the minor groove. In contrast, the N-terminal end of the Tc3 DNA-binding domain is bound in a well-ordered fashion to the DNA even in the absence of the conjugated dye. The conjugated dye and the DNA-binding domain portions of each conjugate bind noncooperatively to the DNA. The characteristic thermodynamic parameters of specific and nonspecific DNA binding by each of the DNA-binding domains and their respective conjugates are presented.
Thompson
M
M
Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287-1604, USA.
Woodbury
N W
NW
D000595:Amino Acid Sequence; D001483:Base Sequence; D002232:Carbocyanines / Q000378:metabolism; D016384:Consensus Sequence; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004254:DNA Nucleotidyltransferases / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D004573:Electrolytes / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism*; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013816:Thermodynamics; D019895:Transposases / Q000737:chemistry / Q000378:metabolism
D002232:Carbocyanines; D004268:DNA-Binding Proteins; D004573:Electrolytes; D005456:Fluorescent Dyes; D009841:Oligonucleotides; D010446:Peptide Fragments; D004247:DNA; D004254:DNA Nucleotidyltransferases; C059997:Hin recombinase; D019895:Transposases