Guha S, Bhattacharyya B
Department of Biochemistry, Bose Institute, Centenary Building, Calcutta 700 054, India.
Biochemistry. 1997 Oct 28;36(43):13208-13. doi: 10.1021/bi970993m.
Tubulin unfolding in urea proceeds via the formation of a partially unfolded intermediate state, stable in 2 M urea, that unfolds further in higher urea concentrations. The intermediate state had spectroscopic properties reminiscent of a molten globule and negligible colchicine binding activity. Refolding of totally unfolded tubulin in 8 M urea yielded an intermediatelike state characterized by partial burial of tryptophans and partial recovery of secondary and tertiary structures, although colchicine-binding activity of the protein was not regained. Further folding of this intermediatelike state, toward the native conformation, with respect to both structural and functional parameters did not occur. However, a significant percentage of colchicine binding activity and nativelike tertiary structure was recovered when refolding was initiated from partially denatured protein samples, viz., from <1.2 M urea. Thus, although high concentration of urea induced loss of structure and activity was irreversible, the conformational changes induced in restricted regions of tubulin by lower concentrations of urea, which are probably crucial for its various functional properties, could be reversed.
微管蛋白在尿素中展开是通过形成一种部分展开的中间状态进行的，该中间状态在2 M尿素中稳定，在更高的尿素浓度下会进一步展开。这种中间状态具有类似于熔球态的光谱特性，秋水仙碱结合活性可忽略不计。在8 M尿素中完全展开的微管蛋白复性产生了一种类似中间态的状态，其特征是色氨酸部分埋藏以及二级和三级结构部分恢复，尽管该蛋白质的秋水仙碱结合活性没有恢复。这种类似中间态的状态，就结构和功能参数而言，并没有进一步折叠成天然构象。然而，当从部分变性的蛋白质样品（即<1.2 M尿素）开始复性时，可恢复相当比例的秋水仙碱结合活性和类似天然的三级结构。因此，尽管高浓度尿素诱导的结构和活性丧失是不可逆的，但较低浓度尿素在微管蛋白受限区域诱导的构象变化（这可能对其各种功能特性至关重要）是可以逆转的。
