THERMAL MOTIONS OF SURFACE ALPHA-HELICES IN THE D-GALACTOSE CHEMOSENSORY RECEPTOR - DETECTION BY DISULFIDE TRAPPING
THERMAL MOTIONS OF SURFACE ALPHA-HELICES IN THE D-GALACTOSE CHEMOSENSORY RECEPTOR - DETECTION BY DISULFIDE TRAPPING
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DOI：
10.1016/0022-2836(92)91063-u
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发表时间：
1992-08-20
影响因子：
5.6
通讯作者：
FALKE, JJ
中科院分区：
文献类型：
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作者：
CAREAGA, CL;FALKE, JJ
大肠杆菌的d-半乳糖化学感受受体是一个32 kDa的球状蛋白，具有两个不同的结构域，每个结构域以α/β折叠基序组织。螺旋I和X位于靠近铰链区的N-末端结构域表面上相邻的大致平行位置。为了分析这两个螺旋的相对热运动，本研究利用了一种可推广的二硫键捕获方法：首先，定点诱变用于在蛋白质表面上的感兴趣的位置处的一对半胱氨酸残基，然后二硫键形成用于捕获分子内半胱氨酸-半胱氨酸碰撞导致的热运动。具体地，已经构建了四种工程化的双半胱氨酸受体，每种受体在螺旋I上的位置26处具有一个半胱氨酸，并且在螺旋X上的不同位置处具有第二个半胱氨酸。第五个控制受体在26位具有一个半胱氨酸，在分子的相对表面上具有第二个半胱氨酸。这些表面的半胱氨酸取代有很小或没有影响的可测量的受体参数作为判断的配体结合平衡和动力学，蛋白质的稳定性，and19 F核磁共振，表明工程受体是有用的探针天然backbone dynamics.Spatial和动力学特征的骨干运动已被调查通过测量分子内二硫键形成率半胱氨酸对完全配体受体。所得到的速率随着晶体结构中半胱氨酸之间的距离的增加而单调降低，而对于对照对没有观察到二硫键形成，除非分子被解折叠。观测到的脊柱运动的最小平移振幅范围为4·5 ~ 15·2 π，最小旋转振幅可达35°。对于每一种运动，分子内巯基-巯基碰撞的速率已经从测量的二硫键形成速率中估算出来：4·5和15·2 π平移分别产生π 4和π 4碰撞s− 1 molecule −1。碰撞率，这是比配体解离快，可能低估了实际的运动频率，因为只有一个不确定的部分，总运动产生碰撞。能够产生这种碰撞的最简单的合理轨迹是一个或两个螺旋沿着其长轴的限速平移，再加上较小的螺旋旋转。当糖从受体中除去时，观察到主链动力学的大幅增加，表明存在新的长程主链轨迹。总的来说，结果表明，蛋白质的内部运动可能比以前观察到的幅度更大。
Thed-galactose chemosensory receptor ofEscherichia coliis a 32 k Da globular protein possessing two distinct structural domains, each organized in an α/β folding motif. Helices I and X lie at adjacent approximately parallel positions on the surface of the N-terminal domain, near the hinge region. In order to analyze the relative thermal motions of these two helices, the present study utilizes a generalizable disulfide trapping approach: first, site-directed mutagenesis is used to plase a pair of cysteine residues at locations of interest on the protein surface, then disulfide bond formation is used to trap intramolecular cysteine-cysteine collisions resulting from thermal motions. Specifically, four engineered di-cysteine receptors have been constructed, each possessing one cysteine at position 26 on helix I, and a second cysteine at varying positions on helix X. A fifth control receptor possesses one cysteine at position 26, and a second on the opposite surface of the molecule. These surface cysteine substitutions have little or no effect on the measurable receptor parameters as judged by ligand binding equilibria and kinetics, protein stability, and19F nuclear magnetic resonance, indicating that the engineered receptors are useful probes of native backbone dynamics.Spatial and kinetic features of backbone motions have been investigated by measuring intramolecular disulfide formation rates for cysteine pairs in the fully liganded receptor. The resulting rates decrease monotonically with increasing distance between cysteines in the crystal structures, while no disulfide formation is observed for the control pair unless the molecule is unfolded. The minimum translational amplitudes of the observed backbone motions range from 4·5 to 15·2Å, and the minimum rotational amplitudes are as long as 35°. For each motion the rate of intramolecular sulfhydryl-sulfhydryl collisions has been estimated from the measured rate of disulfide formation: the 4·5 and 15·2Åtranslations yield ∼4and ∼ collisions s−1molecule−1, respectively. The collisions rates, which are faster than ligand dissociation, likely underestimate the actual motional frequencies since only an undetermined fraction of the total motions yield collisions. The simplest plausible trajectory capable of producing such collisions is a rate-limiting translation of one or both helices along their long axes, coupled with minor helix rotations. When sugar is removed from the receptor, a substantial increase in backbone dynamics is observed, indicating the presence of new long-range backbone trajectories. Overall, the results suggest that internal motions in proteins may have larger amplitudes than previously observed.