A possible tertiary structure change induced by acrylamide in the DNA-binding domain of the Tn10-encoded Tet repressor. A fluorescence study.
A thorough investigation of the acrylamide fluorescence quenching of F75TetR, a mutant of the Tn10-encoded TetR repressor containing a single Trp residue at position 43, was carried out. The Trp-43 residue is located in a helix alpha-turn-helix alpha (H-t-H) motif involved in the specific binding of F75TetR to the operator site in specific DNA. Distinct Ranges of acrylamide concentration have been assumed. At acrylamide concentrations below 0.15-0.2 M (a usual range of values in fluorescence quenching studies) the observed limited tertiary structure change induced by acrylamide is consistent with a noncooperative local unfolding of the DNA-binding domain. It is suggested that penetration of the neutral quencher could cause the deletion of a hydrophobic tertiary structure contact, partly involving TrP-43, responsible for the anchoring of the H-t-H motif inside the three-helix protein bundle, characterizing the N-terminal part. Correspondingly, the affinity of the mutant repressor for the operator was shown to decrease substantially (about five orders of magnitude), seemingly losing its specificity. A subsequent phase, up to 0.8 M acrylamide, was observed in which the involved intermediate protein structure is not further perturbed, nor is DNA binding.
Bousquet
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JA
Université Louis Pasteur CNRS URA 491, Laboratoire de Biophysique, Faculté de Pharmacie de Strasbourg, Illkirch-Graffenstaden.
Ettner
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D020106:Acrylamide; D000178:Acrylamides / Q000737:chemistry*; D004268:DNA-Binding Proteins; D005454:Fluorescence Polarization; D019077:Helix-Turn-Helix Motifs; D007700:Kinetics; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry
D000178:Acrylamides; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein; D020106:Acrylamide; D014364:Tryptophan