Spectroscopic investigation of Tet repressor tryptophan-43 upon specific and nonspecific DNA binding.
The F75 Tet repressor mutant (F75 TetR) contains a single tryptophan residue located at position 43 in the operator recognition alpha-helix. Previous studies [Hansen, D., & Hillen, W. (1987) J. Biol. Chem. 262, 12269-12274] have shown that the fluorescence intensity of this residue is dramatically reduced upon operator binding. In order to determine the origin of this quenching and the role of Trp-43 in the binding mechanism, we have investigated its fluorescence properties upon F75 TetR binding to a tet operator containing 76 bp DNA fragment (specific binding) and to sheared calf thymus DNA (nonspecific binding). Trp-43 steady-state fluorescence intensity was quenched by 72% upon specific binding and by 45% upon nonspecific binding. These fluorescence intensity decreases were not accounted for by similar decreases in the respective fluorescence lifetimes. The apparent quenching calculated from the average lifetimes was about 0.33 in both binding modes. This shows the presence of a static quenching process, clearly favored upon specific binding as compared to nonspecific binding. This is consistent with stacking interactions between Trp-43 and the DNA bases, as suggested by molecular graphics [Baumeister, R., Helbl, V., & Hillen, W. (1992) J. Mol. Biol. 226, 1257-1270]. The equilibrium constant between nonfluorescent and fluorescent tryptophan residues was 5 times higher upon binding to specific DNA than to nonspecific DNA. The preferential static quenching of Trp-43 in the specific complex suggests that stacking interactions might contribute to the specific binding mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)
Peviani
C
C
Dipartimento di Fisica, Università degli Studi di Milano, Italy.
Hillen
W
W
Ettner
N
N
Lami
H
H
Doglia
S M
SM
Piémont
E
E
Ellouze
C
C
Chabbert
M
M
D016880:Anisotropy; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D066298:In Vitro Techniques; D009875:Operator Regions, Genetic; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan / Q000737:chemistry
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein; D014364:Tryptophan; D004247:DNA