EPR-detected folding kinetics of externally located cysteine-directed spin-labeled mutants of iso-1-cytochrome c.
EPR-detected folding kinetics of externally located cysteine-directed spin-labeled mutants of iso-1-cytochrome c.
复制标题
EPR 检测的 iso-1-细胞色素 c 外部半胱氨酸定向自旋标记突变体的折叠动力学。
DOI：
10.1021/bi011414n
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发表时间：
2001
期刊：
影响因子：
2.9
通讯作者：
Scholes,CP
中科院分区：
文献类型：
--
作者：
DeWeerd,K;Grigoryants,V;Sun,Y;Fetrow,JS;Scholes,CP
我们报告了我们新开发的基于介电共振器的流动和停流动力学EPR系统地探测蛋白质折叠在酵母iso-1-cytochromecat半胱氨酸定向自旋标记的位置的应用。研究的位置以前没有被其他技术直接探测过，我们在从50 μs到秒的时间尺度上观察它们。基于晶体结构和同源性信息，选择了以下突变耐受性、位于外部的半胱氨酸标记位点（在螺旋中，T8 C、E66 C和N92 C;在环中，E21 C、V28 C、H39 C、D50 C和K79 C），并且在这些位点处的标记不会去稳定。稀释变性剂用于诱导折叠，从而引起自旋标记EPR信号的变化，因为折叠改变了自旋标记的运动。在折叠条件下，包括咪唑的存在，以消除动力学捕获由于血红素错连接，在20−30 ms的时间尺度上的折叠阶段被发现，这一阶段不仅发生在T8 C和N92 C标记位点的N-和C-末端螺旋，这样的阶段已经与这些螺旋折叠，但在整个蛋白质的标记位点。在没有咪唑的情况下，20 - 30 ms的相位消失，另一个时间尺度为1 s的相位出现在整个蛋白质中。在所有条件下都有证据表明，在蛋白质序列中间的标记位置V28 C、H39 C、D50 C、E66 C和K79 C处发生了规模小于几毫秒的爆发期。在自旋标记的D50 C处，快速混合流EPR表明一个非常短的约50 μs阶段，可能与D50所属环的预折叠或压缩有关。自旋标签已被批评为扰动的现象，他们的措施，但我们的自旋标签策略报告了共同的动力学主题，而不是扰动，断开动力学事件。
We report the application of our newly developed dielectric resonator-based flow and stopped-flow kinetic EPR systematically to probe protein folding in yeast iso-1-cytochromecat cysteine-directed spin-labeled locations. The locations studied have not been previously directly probed by other techniques, and we observe them on a time scale stretching from 50 μs to seconds. On the basis of crystal structure and homology information, the following mutation-tolerant, externally located cysteine labeling sites were chosen (in helices, T8C, E66C, and N92C; in loops, E21C, V28C, H39C, D50C, and K79C), and labeling at these sites was not destabilizing. Dilution of denaturant was used to induce folding and thereby to cause a change in the spin label EPR signal as folding altered the motion of the spin label. Under folding conditions, including the presence of imidazole to eliminate kinetic trapping due to heme misligation, a phase of folding on the 20−30 ms time scale was found. This phase occurred not only at the T8C and N92C labeling sites in the N- and C-terminal helices, where such a phase has been associated with folding in these helices, but overall at labeling sites throughout the protein. In the absence of imidazole the 20−30 ms phase disappeared, and another phase having the time scale of 1 s appeared throughout the protein. There was evidence under all conditions for a burst phase on a scale of less than several milliseconds which occurred at labeling positions V28C, H39C, D50C, E66C, and K79C in the middle of the protein sequence. At spin-labeled D50C rapid-mix flow EPR indicated a very short ∼50 μs phase possibly associated with the prefolding or compaction of the loop to which D50 belongs. Spin labels have been criticized as perturbing the phenomena which they measure, but our spin labeling strategy has reported common kinetic themes and not perturbed, disconnected kinetic events.