Lassalle Michael W, Kondou Shinobu
iCLA (International College of Liberal Arts), Yamanashi Gakuin University, 2-4-5 Sakaori, Kofu-city, Yamanashi-ken, 400-8575 Japan.
Ehime Prefectural Police HQ, Forensic Science Laboratory, Japan.
Biochim Open. 2015 Nov 30;2:1-8. doi: 10.1016/j.biopen.2015.11.004. eCollection 2016 Jun.
Recently, it has been recognized that, much like an electric current in an electric circuit, dynamic disruptions from flexible, unstructured regions distal to the active region are transferred through the contact network to the active site and influence protein stability and/or function. As transmembrane proteins frequently possess the β-barrel structure, studies of proteins with this topology are required. The unstructured lid segments of the β-barrel GFP protein are conserved and could play a role in the backbone stabilization required for chromophore function. A study of the disordered C-terminus and the function within the lid is necessary. In this study, we entirely truncated the flexible C-terminal tail and investigated the N-terminal Strep-tagged GFP by fluorescence spectroscopy, and the temperature- and GdnHCl-induced unfolding by circular dichroism. The introduction of the unstructured Strep-tag itself changed the unfolding pathway. Truncating the entire flexible tail did not decrease the fluorescence intensity to a large extent; however, the protein stability changed dramatically. The temperature for half-denaturation T changed significantly from 79 °C for the wild-type to 72.8 °C for the mutant. Unfolding kinetics at different temperatures have been induced by 4 M GdnHCl, and the apparent Arrhenius activation energy decreased by 40% as compared to the wild-type.
最近，人们认识到，就像电路中的电流一样，来自活性区域远端的柔性、非结构化区域的动态干扰会通过接触网络传递到活性位点，并影响蛋白质的稳定性和/或功能。由于跨膜蛋白通常具有β-桶状结构，因此需要对具有这种拓扑结构的蛋白质进行研究。β-桶状绿色荧光蛋白（GFP）的非结构化帽状片段是保守的，可能在发色团功能所需的主链稳定中发挥作用。对无序的C末端及其在帽状结构内的功能进行研究是必要的。在本研究中，我们完全截短了柔性C末端尾巴，并通过荧光光谱研究了N末端带有链霉亲和素标签的GFP，以及通过圆二色性研究了温度和盐酸胍诱导的去折叠。引入非结构化的链霉亲和素标签本身改变了去折叠途径。截短整个柔性尾巴并没有在很大程度上降低荧光强度；然而，蛋白质稳定性发生了显著变化。半变性温度T从野生型的79℃显著变化到突变体的72.8℃。用4M盐酸胍诱导了不同温度下的去折叠动力学，与野生型相比，表观阿累尼乌斯活化能降低了40%。
