Evidence for coupling of folding and function in trp repressor: physical characterization of the superrepressor mutant AV77.
Evidence for coupling of folding and function in trp repressor: physical characterization of the superrepressor mutant AV77.
复制标题
trp 阻遏蛋白折叠和功能耦合的证据：超阻遏蛋白突变体 AV77 的物理表征。
DOI：
10.1006/jmbi.1995.0551
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发表时间：
1995
影响因子：
5.6
通讯作者：
Royer,CA
中科院分区：
文献类型：
--
作者：
Reedstrom,RJ;Royer,CA
在trprepressor系统中，基因表达的精细控制是通过涉及trprepressor蛋白（TR）、色氨酸（l-Trp）和DNA的多重平衡的热力学链接来实现的。我们已经对TR的超抑制突变体进行了研究，作为剖析抑制功能的耦合平衡的一种手段。与所有其他测试的超级抑制因子不同，与野生型TR DNA结合亲和力或化学量不同，AV77超级抑制因子（77位丙氨酸到缬氨酸的替代：AV77TR）与体外的tri无法区分。目前的研究使用各种生物物理测量比较TR和AV77TR提供了强有力的证据，证明apoTR的螺旋-转-螺旋（HTH）区域以部分折叠的构象存在。远紫外CD光谱显示，与apoTR相比，apoAV77TR的螺旋含量增加了10%。此外，尿素变性研究表明，apoAV77TR比apoTR对变性更稳定。ApoTR以高亲和力结合大量1,8- ans（一种用于检测蛋白质折叠中间体的疏水荧光探针），而apoAV77TR仅与该配体边缘结合。虽然两种蛋白的色氨酸亲和力通过滴定量热法测量和非常相似，但热力学特征是不同的，AV77TR的不利熵贡献大大减少。最后，通过AV77突变消除了l- trp对寡聚化的变构效应。综上所述，这些数据支持了之前的量热学研究，即TR的折叠和l- trp结合耦合。此外，它们与核磁共振观察结果一致，表明apoTR的HTH区部分紊乱。基于TR和AV77TR不同的生物物理特性，我们提出了一个模型，其中HTH区域的折叠伴随着TR中的配体结合。在该模型中，载脂蛋白和全息脂蛋白的不同蛋白质-蛋白质相互作用将这种构象变化与表观操作亲和联系起来，从而调节TR在体内的功能。
The fine-control of gene expression in thetrprepressor system is achieved through the thermodynamic linkage of multiple equilibria involving thetrprepressor protein (TR), tryptophan (l-Trp) and DNA. We have undertaken studies of superrepressor mutants of TR as a means of dissecting the coupled equilibria that contribute to repressor function. Unlike all the other tested super-repressors that exhibit differences from wild-type TR DNA binding affinity or stoichiometry, the AV77 superrepressor (an alanine to valine substitution at position 77: AV77TR) has been indistinguishable from TRin vitro. The present studies using a variety of biophysical measurements comparing TR and AV77TR provide strong evidence that the helix-turn-helix (HTH) region of apoTR exists in a partially folded conformation. Far UV CD spectra of the two proteins reveal a 10% increase in helical content for the apoAV77TR compared to apoTR. Moreover, urea denaturation studies demonstrate that apoAV77TR is more stable to denaturation than apoTR. ApoTR binds large amounts of 1,8-ANS, a hydrophobic fluorescence probe used to detect protein folding intermediates, with high affinity, where apoAV77TR exhibits only marginal binding of this ligand. While the tryptophan affinities of the two proteins as measured by titration calorimetry and quite similar, the thermodynamic signatures are distinct, with a much reduced unfavourable entropic contribution for AV77TR. Finally, the allosteric effect ofl-Trp on oligomerization is abolished by the AV77 mutation. Taken together these data support previous calorimetric studies implicating coupling of folding andl-Trp binding for TR. Moreover, they are consistent with NMR observations indicating partial disorder in the HTH region of apoTR. Based upon the distinct biophysical properties TR and AV77TR, we propose a model in which folding of the HTH region accompanies ligand binding in TR. In this model distinct protein–protein interactions of the apo- and holoTR link this conformational change to apparent operator affinities, thereby modulating TR functionin vivo.