Guha S, Bhattacharyya B
Department of Biochemistry, Bose Institute, Calcutta, India.
Biochemistry. 1995 May 30;34(21):6925-31. doi: 10.1021/bi00021a003.
The unfolding reaction of the dimeric protein tubulin, isolated from goat brain, was studied using fluorescence and circular dichroism techniques. The unfolding of the tubulin dimer was found to be a two-step process at pH 7. The first step leads to the formation of an intermediate conformation, stable at around 1-2 M urea, followed by a second step that was due to unfolding of the intermediate state. At pH 3, the urea-induced biphasic unfolding profiles obtained at pH 7 became a one-step process indicating that a stable intermediate was also formed at this pH. The intermediate at pH 3 was more stable toward urea denaturation than that at pH 7. The intermediate state has about 60% secondary structure, partially exposed aromatic residues, and less tertiary structure as compared to the native states. Also, hydrophobic surfaces were more exposed in the intermediate than in the native or unfolded states. These results indicate that the intermediate state observed during tubulin unfolding is not only distinct from both the native and unfolded forms but also possesses some properties characteristic of a molten globule.
利用荧光和圆二色性技术研究了从山羊脑中分离出的二聚体蛋白微管蛋白的去折叠反应。发现在pH 7时，微管蛋白二聚体的去折叠是一个两步过程。第一步导致形成一种中间构象，在大约1 - 2 M尿素浓度下稳定，随后第二步是由于中间态的去折叠。在pH 3时，在pH 7获得的尿素诱导的双相去折叠曲线变成了一步过程，表明在这个pH值下也形成了一种稳定的中间体。与pH 7时的中间体相比，pH 3时的中间体对尿素变性更稳定。与天然状态相比，中间态具有约60%的二级结构、部分暴露的芳香族残基和较少的三级结构。此外，与天然态或去折叠态相比，中间体的疏水表面暴露得更多。这些结果表明，在微管蛋白去折叠过程中观察到的中间态不仅与天然态和去折叠态都不同，而且具有一些熔球态的特征性质。
