Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555–1055, USA.
Biophys Chem. 2011 Nov;159(1):210-6. doi: 10.1016/j.bpc.2011.06.015. Epub 2011 Jul 3.
The communication mechanism(s) responsible for the allosteric behavior of E.coli cAMP binding receptor protein, CRP, is still a subject of intense investigation. As a tool to explore the communication mechanism, the mutations at various positions in the cAMP-binding (K52N, D53H, S62F and T127L) or the DNA- binding (H159L) domain or both (K52N/H159L) were generated. The sites and specific nature of side chain substitutions were defined by earlier genetic studies, the results of which show that these mutants have a similar phenotype i.e. they are activated without exogenous cAMP. Presently, no significant changes in the structures of WT and mutant CRPs have been observed. Hence, the pressing issue is to identify a physical parameter that reflects the effects of mutations. In this study, the stability of these various CRP species in the presence of GuHCl was monitored by three spectroscopic techniques, namely, CD, tryptophan fluorescence and FT-IR which could provide data on the stability of α-helices and β-strands separately. Results of this study led to the following conclusions: 1. The α-helices can be grouped into two families with different stabilities. Mutations exert a differential effect on the stability of helices as demonstrated by a biphasic unfolding curve for the helices. 2. Regardless of the locations of mutations, the effects can be communicated to the other domain resulting in a perturbation of the stability of both domains, although the effects are more significantly expressed in the stability of the helices. 3. Although in an earlier study [Gekko, et al. Biochemistry 43 (2004) 3844] we showed that cooperativity of cAMP binding is generally correlated to the global dynamics of the protein and DNA binding affinity, in this study we found that generally there is no clear correlation between functional energetics and stability of secondary structures. Thus, results of this study imply that modulation of allostery in CRP is entropic in nature.
负责大肠杆菌 cAMP 结合受体蛋白 CRP 变构行为的通讯机制仍然是一个研究热点。作为探索通讯机制的工具，在 cAMP 结合（K52N、D53H、S62F 和 T127L）或 DNA 结合（H159L）域或两者（K52N/H159L）的各个位置生成突变。这些突变体的侧链取代的位置和特定性质是由早期的遗传研究定义的，结果表明这些突变体在没有外源 cAMP 的情况下被激活。目前，尚未观察到 WT 和突变 CRP 结构的显著变化。因此，当务之急是确定反映突变影响的物理参数。在这项研究中，通过三种光谱技术，即 CD、色氨酸荧光和 FT-IR，监测了这些不同 CRP 物种在 GuHCl 存在下的稳定性，这些技术可以分别提供关于α-螺旋和β-链稳定性的数据。这项研究的结果得出以下结论：1.α-螺旋可以分为两组，具有不同的稳定性。突变对螺旋的稳定性产生了不同的影响，这表现为双相展开曲线。2.无论突变的位置如何，都可以将影响传递到另一个结构域，导致两个结构域的稳定性受到干扰，尽管影响在螺旋的稳定性上更为显著。3.尽管在早期的研究中[Gekko 等人，生物化学 43（2004）3844]我们表明 cAMP 结合的协同作用通常与蛋白质和 DNA 结合亲和力的整体动力学相关，但在这项研究中，我们发现一般来说，功能能量学和二级结构稳定性之间没有明显的相关性。因此，这项研究的结果表明，CRP 变构的调节本质上是熵驱动的。
