LARGE-AMPLITUDE TWISTING MOTIONS OF AN INTERDOMAIN HINGE - A DISULFIDE TRAPPING STUDY OF THE GALACTOSE-GLUCOSE BINDING-PROTEIN
LARGE-AMPLITUDE TWISTING MOTIONS OF AN INTERDOMAIN HINGE - A DISULFIDE TRAPPING STUDY OF THE GALACTOSE-GLUCOSE BINDING-PROTEIN
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DOI：
10.1021/bi00009a036
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发表时间：
1995-03-07
期刊：
影响因子：
2.9
通讯作者：
FALKE, JJ
中科院分区：
文献类型：
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作者：
CAREAGA, CL;SUTHERLAND, J;FALKE, JJ
半乳糖-葡萄糖结合蛋白具有两个结构域，与配体结合间隙接壤，三个多肽链作为连接两个结构域的柔性铰链。众所周知，铰链可以弯曲，使裂缝以至少18度的角度打开。在这里，通过将工程半胱氨酸对放置在裂缝周围以产生六种稳定的二半胱氨酸蛋白来检查铰链的扭曲运动。每对半胱氨酸都将活性巯基引入到间隙的两个边缘，一个在n端结构域，另一个在c端结构域。不同区域的巯基之间的碰撞被二硫化物形成捕获，产生了振幅较大的区域旋转的敏感检测。当配体d -葡萄糖占据间隙时，检测到逆时针的铰链扭曲旋转，振幅高达36度，频率范围为10(1)到10(3)次碰撞s（-1）。从间隙中去除配体增加了3倍的扭转角范围和高达10(2)倍的运动频率。因此，在这个具有代表性的铰链裂蛋白中，在生物学相关的时间尺度上发生了大幅度的铰链扭转运动。讨论了这些运动的功能含义。
The galactose-glucose binding protein possesses two structural domains bordering a ligand binding cleft, with three polypeptide strands serving as a flexible hinge connecting the two domains. The hinge is known to bend, enabling the cleft to open by an angle of at least 18 degrees. Here the twisting motions of the hinge were examined by placing pairs of engineered cysteines on the perimeter of the cleft to generate six stable di-cysteine proteins. Each cysteine pair introduced reactive sulfhydryls into both rims of the cleft, one in the N-terminal domain and the other in the C-terminal domain. Collisions between sulfhydryls in different domains were trapped by disulfide formation, yielding sensitive detection of large amplitude domain rotations. When the cleft was occupied by the ligand D-glucose, counterclockwise hinge twist rotations were detected with amplitudes up to 36 degrees, and frequencies ranging from 10(1) to 10(3) collisions s(-1). Removal of ligand from the cleft increased the range of twist angles 3-fold and the frequency of motions up to 10(2)-fold. Thus, in this representative hinged cleft protein, large amplitude hinge twist motions occur on biologically relevant timescales. The functional implications of such motions are discussed.