Shao X, Matthews C R
Department of Chemistry, Center for Biomolecular Structure and Function, The Pennsylvania State University, University Park 16802, USA.
Biochemistry. 1998 May 26;37(21):7850-8. doi: 10.1021/bi973171y.
A monomeric version of the dimeric tryptophan repressor from Escherichia coli, L39E TR, has previously been shown to resemble a transient intermediate that appears in the first few milliseconds of folding [Shao, X., Hensley, P., and Matthews, C. R. (1997) Biochemistry 36, 9941-9949]. In the present study, the optical properties of the two intrinsic tryptophans were used to compare the structure and dynamics of the monomeric form with those of the native, dimeric form. The urea-induced unfolding equilibria of Trp19/L39E TR (Trp99 replaced with Phe) and Trp99/L39E TR (Trp19 replaced with Phe) mutants were monitored by circular dichroism and fluorescence spectroscopies at pH 7.6 and 25 degrees C. Coincident normalized transitions show that the urea denaturation process for each single-tryptophan mutant follows a two-state model involving monomeric native and unfolded forms. The free energies at standard state in the absence of denaturant for Trp19/L39E TR and Trp99/L39E TR are less than that for L39E TR, indicating that both tryptophans are involved in stabilizing the monomer. Fluorescence and near-UV circular dichroism spectroscopies indicate that the tryptophan side chains in monomeric Trp19/L39E TR and Trp99/L39E TR occupy hydrophobic, well-structured environments that are distinctively different from those found in their dimeric counterparts. Acrylamide quenching experiments show that both Trp19 and Trp99 are partially exposed to solvent in the native state, with Trp99 having a slightly greater degree of exposure. Measurements of the steady-state anisotropies of Trp19/L39E and Trp99/L39E TR demonstrate that the motions of both tryptophan side chains are restricted in the folded conformation. On the basis of these data, it can be concluded that this monomeric form of the tryptophan repressor adopts a well-folded, stable conformation with nonnative tertiary structure. When combined with previous results, the current findings demonstrate that the development of higher order structure during the folding of this intertwined dimer does not follow a simple hierarchical model.
来自大肠杆菌的二聚体色氨酸阻遏蛋白的单体形式L39E TR，此前已被证明类似于在折叠最初几毫秒出现的瞬态中间体[Shao, X., Hensley, P., and Matthews, C. R. (1997) Biochemistry 36, 9941 - 9949]。在本研究中，利用两个内在色氨酸的光学性质，将单体形式的结构和动力学与天然二聚体形式的进行比较。通过圆二色光谱和荧光光谱在pH 7.6和25℃下监测Trp19/L39E TR（Trp99被苯丙氨酸取代）和Trp99/L39E TR（Trp19被苯丙氨酸取代）突变体的尿素诱导的去折叠平衡。重合的归一化转变表明，每个单色氨酸突变体的尿素变性过程遵循涉及单体天然形式和未折叠形式的两态模型。在没有变性剂的标准状态下，Trp19/L39E TR和Trp99/L39E TR的自由能低于L39E TR的自由能，表明两个色氨酸都参与稳定单体。荧光光谱和近紫外圆二色光谱表明，单体Trp19/L39E TR和Trp99/L39E TR中的色氨酸侧链占据疏水、结构良好的环境，这与它们二聚体对应物中的环境明显不同。丙烯酰胺猝灭实验表明，Trp19和Trp99在天然状态下都部分暴露于溶剂中，Trp99的暴露程度略高。Trp19/L39E和Trp99/L39E TR的稳态各向异性测量表明，两个色氨酸侧链的运动在折叠构象中受到限制。基于这些数据，可以得出结论，这种色氨酸阻遏蛋白的单体形式采用具有非天然三级结构的折叠良好、稳定的构象。结合先前的结果，当前的发现表明，这种交织二聚体折叠过程中高阶结构的形成并不遵循简单的层次模型。
