title: DNA-binding interactions and conformational fluctuations of Tc3 transposase DNA binding domain examined with single molecule fluorescence spectroscopy.
abstract: The fluorescent dye tetramethylrhodamine (TMR) was conjugated to a synthetic peptide containing the sequence-specific DNA binding domain of Tc3 transposase. Steady-state and single molecule fluorescence spectroscopy was used to investigate protein conformational fluctuations and the thermodynamics of binding interactions. Evidence is presented to show that the TMR-Tc3 conjugate exists in at least two conformational states. The most stable conformation is one in which the TMR fluorescence is quenched. Upon binding to DNA, the total fluorescence from TMR-Tc3 increases by three- to fourfold. Single molecule measurements of TMR-Tc3 bound to DNA shows that this complex also fluctuates between a fluorescent and quenched form. The fluorescent form of the conjugate is stabilized when bound to DNA, and this accounts for part of the increase in total fluorescence. In addition, the inherent photodynamics of the dye itself is also altered (e.g., fluorescent lifetime or triplet yield) in such a way that the total fluorescence from the conjugate bound to DNA is enhanced relative to the unbound form.
authors.affiliation: Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287-1604, USA.
authors.forename: Douglas C
authors.initials: DC
authors.lastname: Daniel
authors.forename: Martin
authors.initials: M
authors.lastname: Thompson
authors.forename: Neal W
authors.initials: NW
authors.lastname: Woodbury
mesh_terms: D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D017173:Caenorhabditis elegans; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012235:Rhodamines / Q000494:pharmacology*; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D019895:Transposases / Q000737:chemistry*
chemical_list: D004268:DNA-Binding Proteins; D010455:Peptides; D012235:Rhodamines; C005358:tetramethylrhodamine; D004247:DNA; D019895:Transposases