Kinetics and motional dynamics of spin-labeled yeast iso-1-cytochrome c: 1. Stopped-flow electron paramagnetic resonance as a probe for protein folding/unfolding of the C-terminal helix spin-labeled at cysteine 102.
Kinetics and motional dynamics of spin-labeled yeast iso-1-cytochrome c: 1. Stopped-flow electron paramagnetic resonance as a probe for protein folding/unfolding of the C-terminal helix spin-labeled at cysteine 102.
复制标题
自旋标记酵母 iso-1-细胞色素 c 的动力学和运动动力学： 1. 停流电子顺磁共振作为在半胱氨酸 102 自旋标记的 C 末端螺旋的蛋白质折叠/展开的探针。
DOI：
10.1021/bi962155i
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Fetrow,JS
中科院分区：
文献类型：
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作者：
Qu,K;Vaughn,JL;Sienkiewicz,A;Scholes,CP;Fetrow,JS
用停流电子顺磁共振（EPR）技术研究了自旋标记酵母异1-细胞色素的化学诱导折叠和去折叠过程的动力学。基于新的介质谐振器结构的停流EPR [Sienkiewicz，A.，Qu，K.，& Scholes，C. P.（1994）Rev. Sci. Instrum.65，68−74]，给出了一种新的时间分量来探测纳秒级分子翻滚运动，这些运动由需要毫秒到秒的时间分辨率的大分子过程调制。在这项工作中提出的停流EPR技术是一种动力学技术，以前没有使用这样的时间分辨率自旋标记系统，它有可能应用于许多自旋标记的网站在这个和其他蛋白质。半胱氨酸特异性自旋标记，甲硫基磺酸自旋标记（MTSSL），被附加到酵母异-1-细胞色素在单一的天然存在的半胱氨酸102，这项工作的重点是在这个二硫键连接的自旋标记的原型。该探针具有反映蛋白质三级折叠的优点，如最近T4溶菌酶的系统性定点自旋标记所示[Mchaourab，H. S. Lietzow，M.一、Hideg，K.，& Hubbell，W. L.（1996）Biochemistry 35，7692 - 7704]和蛋白质骨架动力学，如也通过模型肽研究所示[托德，A. P.，& Millhauser，G. L.（1991）Biochemistry 30，5515 - 5523]。在C-末端的细胞色素螺旋的标签是连接被认为是关键的蛋白质折叠和展开的初始步骤。停流EPR在pH 6.5下解析了单指数胍诱导的去折叠过程，时间常数为20 ms;该实验需要不到150 μL的80 μM自旋标记蛋白。我们观察到，当胍变性剂浓度从0.6 M增加到2.0 M时，从1 s范围到20 ms时间范围，该解折叠时间减少了2.50倍。我们标记的细胞色素的更复杂的复性动力学研究在pH 5.0和6.5的停流EPR。自旋探针显示出一个快速的动力学过程，与氢/氘酰胺保护指示螺旋形成的时间范围相容;该过程在pH 5.0下是单指数的。在pH 6.5时，有证据表明，通过停流EPR和血红素连接敏感的UV−维斯，可以解决另外一个较慢的动力学阶段，这表明可能涉及血红素错连接的较慢折叠。由于二硫键连接的探针已报告在其他系统中的折叠和骨架动力学，这意味着我们的动力学实验是直接传感事件的C-末端螺旋形成和可能的N-和C-末端螺旋相互作用。半胱氨酸标记的蛋白质也进行了研究，在平衡条件下，以表征探针的流动性和对蛋白质热力学的影响的探针。折叠和变性蛋白质之间的自旋探针迁移率的差异是显着的，并在折叠的蛋白质，探针的运动是各向异性限制。折叠蛋白质中连接的氮氧自由基的运动似乎受到碳和硫键的限制，这些碳和硫键将其束缚在半胱氨酸上。半胱氨酸硫连接的起始点在其与N-末端螺旋的界面附近的C-末端螺旋内距离血红素铁约11 μ m，但低温EPR自旋探针线宽显示探针距离血红素铁更远（>15 μ m）。实验结果表明，半胱氨酸102标记的蛋白质折叠，但自旋探针扰动了蛋白质的堆积，降低了蛋白质的热熔融温度、折叠自由能、折叠过程中的胍离子浓度。
The kinetics of chemically induced folding and unfolding processes in spin-labeled yeast iso-1-cytochromecwere measured by stopped-flow electron paramagnetic resonance (EPR). Stopped-flow EPR, based on a new dielectric resonator structure [Sienkiewicz, A., Qu, K., & Scholes, C. P. (1994)Rev. Sci. Instrum.65, 68−74], gives a new temporal component to probing nanosecond molecular tumbling motions that are modulated by macromolecular processes requiring time resolution of milliseconds to seconds. The stopped-flow EPR technique presented in this work is a kinetic technique that has not been previously used with such a time resolution on spin-labeled systems, and it has the potential for application to numerous spin-labeled sites in this and other proteins. The cysteine-specific spin-label, methanethiosulfonate spin-label (MTSSL), was attached to yeast iso-1-cytochromecat the single naturally occurring cysteine102, and the emphasis for this work was on this disulfide-attached spin-labeled prototype. This probe has the advantage of reflecting the protein tertiary fold, as shown by recent, systematic site-directed spin labeling of T4 lysozyme [Mchaourab, H. S. Lietzow, M. A., Hideg, K., & Hubbell, W. L. (1996)Biochemistry35, 7692−7704], and protein backbone dynamics, as also shown by model peptide studies [Todd, A. P., & Millhauser, G. L. (1991)Biochemistry30, 5515−5523]. The C-terminal cytochromechelix where the label is attached is thought to be critical in the initial steps of protein folding and unfolding. Stopped-flow EPR resolved the monoexponential, guanidinium-induced unfolding process at pH 6.5 with an ∼20 ms time constant; this experiment required less than 150 μL of 80 μM spin-labeled protein. We observed an ∼50-fold decrease of this unfolding time from the 1 s range to the 20 ms time range as the guanidinium denaturant concentration was increased from 0.6 to 2.0 M. The more complex refolding kinetics of our labeled cytochrome were studied by stopped-flow EPR at pH 5.0 and 6.5. The spin probe showed a fast kinetic process compatible with the time range over which hydrogen/deuterium amide protection indicates helix formation; this process was monoexponential at pH 5.0. At pH 6.5, there was evidence of an additional slower kinetic phase resolved by stopped-flow EPR and by heme-ligation-sensitive UV−Vis that indicated a slower folding where heme misligation may be involved. Since the disulfide-attached probe has reported folding and backbone dynamics in other systems, the implication is that our kinetic experiments were directly sensing events of the C-terminal helix formation and possibly the N- and C-terminal helical interaction. The cysteine-labeled protein was also studied under equilibrium conditions to characterize probe mobility and the effect of the probe on protein thermodynamics. The difference in spin probe mobility between folded and denatured protein was marked, and in the folded protein, the motion of the probe was anisotropically restricted. The motion of the attached nitroxide in the folded protein appears to be restricted about the carbon and sulfur bonds which tether it to the cysteine. The original point of cysteine sulfur attachment is ∼11 Å from the heme iron within the C-terminal helix near its interface with the N-terminal helix, but the low-temperature EPR spin probe line width showed that the probe lies more distant (>15 Å) from the heme iron. By all physical evidence, the protein labeled at cysteine102folded, but the spin probe in this prototype system perturbed packing which lowered the thermal melting temperature, the free energy of folding, the guanidinium concentration at the …