Single-tryptophan mutants of monomeric tryptophan repressor: optical spectroscopy reveals nonnative structure in a model for an early folding intermediate.
Single-tryptophan mutants of monomeric tryptophan repressor: optical spectroscopy reveals nonnative structure in a model for an early folding intermediate.
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单体色氨酸阻遏物的单色氨酸突变体：光谱揭示了早期折叠中间体模型中的非天然结构。
DOI：
10.1021/bi973171y
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Matthews,CR
中科院分区：
文献类型：
--
作者：
Shao,X;Matthews,CR
先前已显示来自geophtheton ichiacoli的二聚色氨酸阻遏物的单体形式L39 E TR类似于在折叠的最初几毫秒中出现的瞬时中间体[Shao，X.，Hensley，P.，和马修斯，C. R.（1997）Biochemistry36，9941 - 9949]。在本研究中，两个内在的双光子晶体的光学性质被用来比较的结构和动力学的单体形式与那些本地，二聚体形式。在pH 7.6和25 °C下，通过圆二色谱和荧光光谱监测Trp 19/L39 E TR（Trp 99被Phe取代）和Trp 99/L39 E TR（Trp 19被Phe取代）突变体的尿素诱导的解折叠平衡。一致的归一化转换表明，尿素变性过程中，每个单色氨酸突变体遵循两个国家的模型，涉及单体的天然和未折叠的形式。在不存在变性剂的情况下，Trp 19/L39 E TR和Trp 99/L39 E TR在标准状态下的自由能小于L39 E TR的自由能，表明两种双链烷烃都参与稳定单体。荧光和近紫外圆二色光谱表明，色氨酸侧链在单体Trp 19/L39 E TR和Trp 99/L39 E TR占据疏水性，结构良好的环境，是显着不同的发现，在其二聚体的对应物。丙烯酰胺淬灭实验表明，Trp 19和Trp 99都部分暴露于天然状态下的溶剂，Trp 99的暴露程度略高。Trp 19/L39 E和Trp 99/L39 E TR的稳态各向异性的测量表明，两个色氨酸侧链的运动在折叠构象中受到限制。根据这些数据，可以得出结论，这种单体形式的色氨酸阻遏物采用了良好的折叠，稳定的构象与非天然的三级结构。当与以前的结果相结合，目前的研究结果表明，这种交织的二聚体的折叠过程中的高阶结构的发展并不遵循一个简单的层次模型。
A monomeric version of the dimeric tryptophan repressor fromEscherichiacoli, 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)Biochemistry36, 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 °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.
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DOI：
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发表时间：
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期刊：
総務省統計研修所 リサーチペーパー 第5号
影响因子：
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发表时间：
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