Qu K, Vaughn J L, Sienkiewicz A, Scholes C P, Fetrow J S
Department of Chemistry, State University of New York at Albany, 12222, USA.
Biochemistry. 1997 Mar 11;36(10):2884-97. doi: 10.1021/bi962155i.
The kinetics of chemically induced folding and unfolding processes in spin-labeled yeast iso-1-cytochrome c were 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-cytochrome c at 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) Biochemistry 35, 7692-7704], and protein backbone dynamics, as also shown by model peptide studies [Todd, A. P., & Millhauser, G. L. (1991) Biochemistry 30, 5515-5523]. The C-terminal cytochrome c helix 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 approximately 20 ms time constant; this experiment required less than 150 microL of 80 microM spin-labeled protein. We observed an approximately 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 approximately 11 A 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 A) from the heme iron. By all physical evidence, the protein labeled at cysteine102 folded, 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 midpoint of the unfolding transition, the m parameter of the denaturant, and the helical CD signature. This study prepares the way for study of protein folding/unfolding kinetics using EPR spectroscopy of spin-labels placed at specific cysteine-mutated sites within
通过停流电子顺磁共振（EPR）测量了自旋标记的酵母异 - 1 - 细胞色素c中化学诱导的折叠和去折叠过程的动力学。基于一种新的介电谐振器结构的停流EPR [Sienkiewicz, A., Qu, K., & Scholes, C. P. (1994) Rev. Sci. Instrum. 65, 68 - 74]，为探测纳秒级分子翻滚运动提供了一个新的时间分量，这种运动受到需要毫秒到秒时间分辨率的大分子过程的调制。本文介绍的停流EPR技术是一种动力学技术，以前尚未在自旋标记系统上以如此高的时间分辨率使用过，它有可能应用于该蛋白及其他蛋白中的众多自旋标记位点。半胱氨酸特异性自旋标记物甲硫基磺酸盐自旋标记（MTSSL）在酵母异 - 1 - 细胞色素c唯一的天然半胱氨酸102位点处连接，这项工作的重点是这个通过二硫键连接的自旋标记原型。如最近对T4溶菌酶进行的系统定点自旋标记所示 [Mchaourab, H. S., Lietzow, M. A., Hideg, K., & Hubbell, W. L. (1996) Biochemistry 35, 7692 - 7704]，该探针具有反映蛋白质三级结构的优点，并且如模型肽研究所示 [Todd, A. P., & Millhauser, G. L. (1991) Biochemistry 30, 5515 - 5523]，还能反映蛋白质主链动力学。标记物所连接的细胞色素c的C末端螺旋被认为在蛋白质折叠和去折叠的初始步骤中至关重要。停流EPR解析了在pH 6.5时胍诱导的单指数去折叠过程，时间常数约为20毫秒；该实验所需的80 microM自旋标记蛋白少于150微升。当胍变性剂浓度从0.6 M增加到2.0 M时，我们观察到这种去折叠时间从1秒范围减少到20毫秒范围，下降了约50倍。我们通过在pH 5.0和6.5下的停流EPR研究了标记的细胞色素更复杂的重折叠动力学。自旋探针显示出一个与氢/氘酰胺保护表明螺旋形成的时间范围相符的快速动力学过程；该过程在pH 5.0时是单指数的。在pH 6.5时，有证据表明通过停流EPR和血红素连接敏感的紫外可见光谱解析出一个额外的较慢动力学阶段，这表明存在较慢的折叠过程，可能涉及血红素错配。由于通过二硫键连接的探针已在其他系统中报道了折叠和主链动力学，这意味着我们的动力学实验直接检测到了C末端螺旋形成事件以及可能的N末端和C末端螺旋相互作用。还在平衡条件下研究了半胱氨酸标记的蛋白质，以表征探针的流动性以及探针对蛋白质热力学的影响。折叠和变性蛋白质之间自旋探针流动性的差异很明显，并且在折叠蛋白质中，探针的运动受到各向异性限制。折叠蛋白质中连接的氮氧化物的运动似乎受到将其连接到半胱氨酸的碳和硫键的限制。半胱氨酸硫连接的原始点在C末端螺旋内靠近其与N末端螺旋界面处距离血红素铁约11埃，但低温EPR自旋探针线宽表明探针距离血红素铁更远（> 15埃）。从所有物理证据来看，在半胱氨酸102处标记的蛋白质发生了折叠，但在这个原型系统中的自旋探针扰乱了堆积，从而降低了热解链温度、折叠自由能、去折叠转变中点处的胍浓度、变性剂的m参数以及螺旋圆二色性特征。这项研究为利用EPR光谱研究放置在特定半胱氨酸突变位点的自旋标记的蛋白质折叠/去折叠动力学铺平了道路。
