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Two tetracycline repressor (TetR) sequence variants sharing 63%identical aminoacidswere investigated in terms of their recognition specificity for tetracycline and anhydrotetra-cycline. Thermodynamic complexstabilities determined by urea-dependent unfolding reveal that tetracycline stabilizes bothvariants toa similar extentbut that anhydrotetracycline discriminates between them significantly. Isofunctional TetR hybrid proteins of these sequence variants were constructed and their denaturation profiles identified resi-dues 57 and 61 as the complexstability determinant | Eur. J. Biochem. 271 2144-2152 2004 FEBS 2004 doi 10.1111 j.1432-1033.2004.04130.x Tet repressor residues indirectly recognizing anhydrotetracycline Peter Schubert Klaus Pfleiderer and Wolfgang Hillen Lehrstuhlfur Mikrobiologie Institut fur Mikrobiologie Biochemie und Genetik Friedrich-Alexander-Universitat Erlangen Germany Two tetracycline repressor TetR sequence variants sharing 63 identical amino acids were investigated in terms of their recognition specificity for tetracycline and anhydrotetracycline. Thermodynamic complex ttabiiities determined by urea-dependent unfolding reveal that tetracycline stabilizes both variants to a similar extent but that anhydrotetracycline discriminates between them significantly. Isofunctional TetR hybrid proteins of these sequence variants were constructed and their denaturation profiles identified residues 57 and 61 as the complex ab-t - determ ínant. Association kinetics reveal different recognition of these TetR variants by anhydrotetracycline but the binding constants indicate similar stabilization. The identified residues connect to an internal water network which suggests that the discrepancy in the observed thermodynamics may be caused by an entropy effect. Exchange of these interacting residues between the two TetR variants appears to influence the flexibility of this water organization demonstrating the importance of buried structural water molecules for ligand recognition and protein function. Therefore this structural module seems to be a key requisite for the plasticity of the multiple ligand binding protein TetR. Keywords Tet repressor gene regulation protein stability ligand binding antibiotic resistance. The biological function of many proteins is triggered and modulated by binding of effector molecules or a variety of extrinsic cofactors that greatly expand the repertoire of cellular processes executed by polypeptides DNA or small proteins 1 . Therefore molecular recognition is a fundamental process in all living
