The local electrostatic environment determines cysteine reactivity of tubulin
The local electrostatic environment determines cysteine reactivity of tubulin
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DOI：
10.1074/jbc.m204263200
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发表时间：
2002-08-09
影响因子：
4.8
通讯作者：
Wolff, J
中科院分区：
文献类型：
--
作者：
Britto, PJ;Knipling, L;Wolff, J
大鼠脑微管蛋白的20个半胱氨酸中，有的与巯基试剂反应迅速，有的反应缓慢。快速反应的半胱氨酸无法与 [C-14] 碘乙酰胺、N-[C-14] 乙基马来酰亚胺或 IAE-DANS（[5-((((2-碘乙酰基)氨基)乙基)氨基)萘-1-磺酸]) 区分开来，因为对摩尔比的修饰远小于 1 半胱氨酸/二聚体总是导致 6-7 的标记 半胱氨酸残基。通过质谱和测序，它们被鉴定为 Cys-305alpha、Cys-315alpha、Cys-316alpha、Cys-347alpha、Cys-376alpha、Cys-241beta 和 Cys-356beta。这种特异性的缺乏可归因于试剂反应性太强。只有相对不活跃的氯乙酰胺我们才能将 Cys-347alpha 鉴定为微管蛋白中最具反应性的半胱氨酸。使用3.5埃电子衍射结构，可以表明反应性半胱氨酸位于带正电荷的精氨酸和赖氨酸或芳香环的正边缘的6.5埃范围内，可能促进硫醇解离为硫醇盐阴离子。通过同样的推理，许多反应性较低的半胱氨酸的失活可归因于带负电荷的局部环境对修饰的抑制，即使对于一些表面暴露的半胱氨酸也是如此。我们得出的结论是，半胱氨酸的局部静电环境是其反应性的重要决定因素，尽管不一定是唯一的决定因素。
Of the 20 cysteines of rat brain tubulin, some react rapidly with sulfhydryl reagents, and some react slowly. The fast reacting cysteines cannot be distinguished with [C-14]iodoacetamide, N-[C-14]ethylmaleimide, or IAE-DANS ([5-((((2-iodoacetyl)amino)ethyl) amino) naphthalene-l-sulfonic acid]), since modification to mole ratios much less than 1 cysteine/dimer always leads to labeling of 6-7 cysteine residues. These have been identified as Cys-305alpha, Cys-315alpha, Cys-316alpha, Cys-347alpha, Cys-376alpha, Cys-241beta, and Cys-356beta by mass spectroscopy and sequencing. This lack of specificity can be ascribed to reagents that are too reactive; only with the relatively inactive chloroacetamide could we identify Cys-347alpha as the most reactive cysteine of tubulin. Using the 3.5-Angstrom electron diffraction structure, it could be shown that the reactive cysteines were within 6.5 Angstrom of positively charged arginines and lysines or the positive edges of aromatic rings, presumably promoting dissociation of the thiol to the thiolate anion. By the same reasoning the inactivity of a number of less reactive cysteines could be ascribed to inhibition of modification by negatively charged local environments, even with some surface-exposed cysteines. We conclude that the local electrostatic environment of cysteine is an important, although not necessarily the only, determinant of its reactivity.