A type II DNA-binding protein genetically engineered for fluorescence spectroscopy: the "arm" of transcription factor 1 binds in the DNA grooves.
We examined the fluorescence properties of a mutant TF1 protein (transcription factor 1; a member of the type II class of DNA-binding proteins, DBPII) containing tryptophan in place of phenylalanine (TF1-W61) at position 61 in the "arms" of the protein dimer. The time-resolved fluorescence (excited at 295 nm) of Trp61 decays as a double exponential with lifetimes and amplitudes that are comparable to those found in other tryptophan-containing proteins and peptides, and the time-resolved fluorescence polarization decay indicates that the tryptophan residue possesses considerable internal flexibility, in agreement with crystal studies of the homologous HU protein. The tryptophan emission is quenched when TF1-W61 binds to DNA, and equilibrium studies based on fluorescence show that the nonspecific binding affinity of the TF1-W61 mutant to DNA is similar to that of wild-type TF1. Comparison of the time-resolved fluorescence decay and steady-state fluorescence intensity reveals at least two general classes of Trp61 in the DNA complexes. One class of tryptophans is partially quenched, and the extent of quenching in the complexes with various natural DNAs and synthetic double-stranded polynucleotides correlates with the spectral overlap between tryptophan emission and DNA absorption, indicating that through-space excitation energy transfer contributes to the observed quenching. Comparisons between experimentally determined energy transfer rates and model calculations suggest that the Trp61 is located in one of the DNA grooves at a distance of less than 7.5 A from the DNA helix axis. The second class of Trp61 is "totally" quenched, and we attribute this to tryptophan residues that are in direct contact with the DNA bases.(ABSTRACT TRUNCATED AT 250 WORDS)
Härd
T
T
Department of Chemistry, University of California, San Diego, La Jolla 92093-0342.
Sayre
M H
MH
Geiduschek
E P
EP
Kearns
D R
DR
D003062:Codon; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D009690:Nucleic Acid Conformation; D010957:Plasmids; D011089:Polydeoxyribonucleotides; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014364:Tryptophan
D003062:Codon; D004268:DNA-Binding Proteins; D011089:Polydeoxyribonucleotides; D014157:Transcription Factors; D014364:Tryptophan; D004247:DNA