A possible tertiary structure change induced by acrylamide in the DNA-binding domain of the Tn10-encoded Tet repressor. A fluorescence study

Abstract

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.

Chemistry, Biochemistry, and Safety of Acrylamide. A Review

“…The quenching reaction involves physical contact between acrylamide and the excited indole ring of tryptophan. Because acrylamide is a neutral, highly polar molecule, it readily diffuses to and senses the microenvironment of fluorophores by nonpolar charge-transfer complex formation (115). Tryptophan quenching by acrylamide is therefore widely used in studies of protein structure and folding.…”

“…In this case, acrylamide interacts non-covalently with tryptophan and covalently with cysteine residues. Quenching of fluorescence by acrylamide has also been used to study DNA-binding domains (115,125). It is not known whether the charge-transfer effects associated with protein and DNA quenching are involved in the biological effects of acrylamide and whether such interactions will affect the digestibility and nutritional utilization of tryptophan in food proteins (126).…”