Allosteric regulation of the Tet repressor (TetR) homodimer relies on tetracycline binding that abolishes the affinity for the DNA operator. Previously, interpretation of circular dichroism data called for unfolding of the α-helical DNA-binding domains in absence of binding to DNA or tetracycline. Our small angle X-ray scattering of TetR(D) in solution contradicts this unfolding as a physiological process. Instead, in the core domain crystal structures analyses show increased immobilisation of helix α9 and two C-terminal turns of helix α8 upon tetracycline binding. Tetracycline complexes of TetR(D) and four single-site alanine variants were characterised by isothermal titration calorimetry, fluorescence titration, X-ray crystal structures, and melting curves. Five crystal structures confirm that Thr103 is a key residue for the allosteric events of induction, with the T103A variant lacking induction by any tetracycline. The T103A variant shows anti-cooperative inducer binding, and a melting curve of the tetracycline complex different to TetR(D) and other variants. For the N82A variant inducer binding is clearly anti-cooperative but triggers the induced conformation.

中文翻译：

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四环素阻滞剂（TetR）在四环素结合后的热力学，协同性和稳定性。

Tet阻遏物（TetR）同二聚体的变构调节依赖于四环素结合，从而消除了对DNA操纵子的亲和力。以前，对圆二色性数据的解释要求在不与DNA或四环素结合的情况下展开α-螺旋DNA结合结构域。我们在溶液中TetR（D）的小角度X射线散射与生理过程中的这种展开矛盾。取而代之的是，在核心结构域中的晶体结构分析显示，在四环素结合后，螺旋α9的固定化增加，螺旋α8的两个C末端转位增加。TetR（D）和四个单点丙氨酸变体的四环素复合物通过等温滴定量热法，荧光滴定，X射线晶体结构和熔解曲线进行表征。五个晶体结构证实，Thr103是诱导变构事件的关键残基，T103A变体缺乏任何四环素的诱导。T103A变体显示出抗合作诱导剂结合，并且四环素复合物的熔解曲线不同于TetR（D）和其他变体。对于N82A变体，诱导剂结合显然是抗合作的，但会触发诱导的构象。