PROTEIN FOLDING & UNFOLDING BY DQC CYTOCHROME C

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 细胞色素c是蛋白质折叠研究的模型系统，它是新方法的试验场。由于2D-FT的动力学版本，这将是我们在这项研究中的主要工具，正在ACERT开发中，我们正在应用其他方法，可以报告蛋白质结构重排。它们包括DQC和双电子-电子共振（DEER）。Scholes博士成功地制备了异细胞色素c双半胱氨酸突变体，并用氮氧自由基对其进行了自旋标记。他正在康奈尔纳米纤维工厂开发一种使用纳米纤维技术的合适的冷冻淬火装置。微混合器/冷冻淬灭组件被设计成在折叠开始的50微秒内捕获初始折叠双自旋标记蛋白质的状态。距离和它们的分布将从DQC和17 GHz的DEER测量使用该冷冻淬火装置制备的样品上获得。这种技术的可行性是支持最近的距离测量，我们已经完成了S47/K79双突变体的异细胞色素c在其折叠状态或展开加入氯化胍（GdmCL）在几个浓度。为了延长氮氧相弛豫时间，血红素铁被还原，样品制备在厌氧条件下完成。 使用设置为在17.4 GHz下操作的DEER进行测量。 这种工作频率使我们能够使用更小的样品并保持高灵敏度。使用的GdmCl浓度为0.7、1.5和2.8M。 在折叠状态下，自旋标签之间的距离为15.8/0.5英寸。 在[GdmCl]为0.7 M时，平均距离为30/3，而1.5 M和2.8 M的信号之间仅存在轻微差异，两种情况的平均距离为53/5和54/5，在实验误差范围内相同。 工作正在进行中，以提取距离分布的偶极信号的数值方法的基础上吉洪诺夫正则化。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Cytochrome c is a model system in protein folding studies and it serves as a testing ground for new methods. Since the kinetic version of 2D-FT, which will be our major tool in this study, is under development at ACERT, we are applying other methods that can report on protein structural rearrangement. They include DQC and double electron-electron resonance (DEER). Dr. Scholes has succeeded in the preparation of iso-Cytochrome c double cysteine mutants and their spin-labeling with nitroxides. He is developing a suitable freeze-quench apparatus using nanofabrication technology at the Cornell Nanofabrication Facility. The micromixer/freeze-quench assembly was designed to capture the state of incipiently folding doubly spin-labeled protein within 50 microseconds of the start of folding. The distances and their distributions will be obtained from DQC and 17 GHz DEER measurements on the samples prepared using this freeze-quench apparatus. The feasibility of this technique is supported by recent distance measurements that we have completed for S47/K79 double mutants of iso-Cytochrome c in its folded state or unfolded by adding guanidium chloride (GdmCL) in several concentrations. In order to lengthen the nitroxide phase relaxation time Heme iron was reduced and the sample preparations were accomplished under anaerobic conditions. The measurements were performed using DEER set up for operation at 17.4 GHz. This working frequency let us use smaller samples and maintain high sensitivity. The GdmCl concentrations used were 0.7, 1.5, and 2.8M. In the folded state, the distance between spin labels was 15.8/0.5 ¿. At [GdmCl] of 0.7 M the average distance was 30/3 ¿, whereas there was only a slight difference between the signals for 1.5M and 2.8M with average distances of 53/5 ¿ and 54/5 ¿ for the two cases, the same to within experimental error. Work is in progress to extract distance distributions from the dipolar signals by numerical methods based on Tikhonov regularization.