PROTEIN FOLDING & UNFOLDING BY DQC CYTOCHROME C

蛋白质折叠

• 批准号: 8172051
• 负责人: Jack H Freed
• 金额: $ 0.29万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8172051
• 关键词: Biological Models; Computer Retrieval of Information on Scientific Projects Database; Cysteine; DNA Sequence Rearrangement; Development; Electrons; Freezing; Frequencies; Funding; Grant; Guanidinium Chloride; Heme Iron; Institution; Kinetics; Measurement; Methods; Phase; Preparation; Proteins; Relaxation; Reporting; Research; Research Personnel; Resources; Sampling; Signal Transduction; Source; Spin Labels; Structural Protein; Techniques; Technology; Testing; Time; United States National Institutes of Health; Work; base; cytochrome c; design; mutant; nanofabrication; operation; protein folding; tool

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.

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 细胞色素c是蛋白质折叠研究中的一个模型系统，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来报告蛋白质结构重排。它们包括DQC和双电子-电子共振(DER)。斯科尔斯博士已经成功地制备了异细胞色素c双半胱氨酸突变体，并用氮氧化物自旋标记了它们。他正在康奈尔纳米加工设施使用纳米加工技术开发一种合适的冷冻淬火设备。微型混合器/冷冻-淬灭组件被设计成在折叠开始后的50微秒内捕获初始折叠双自旋标记蛋白质的状态。距离及其分布将由DQC和17 GHz鹿测量得到，样品使用该冷冻淬火装置制备。最近我们对S47/K79双突变体的异细胞色素c折叠状态或通过添加不同浓度的氯化胍(GdmCL)展开的距离测量支持了这一技术的可行性。为了延长氮氧化物的相弛豫时间，在厌氧条件下还原血红素铁，完成样品的制备。这些测量是使用设置在17.4 GHz下运行的鹿进行的。这一工作频率使我们可以使用较小的样本并保持高灵敏度。使用的GdmCl浓度分别为0.7、1.5和2.8M。在折叠状态下，自旋标记之间的距离为15.8/0.5？当[GdmCl]为0.7M时，平均距离为30/3？，而1.5m和2.8m之间仅有微小差异，平均距离分别为53/5？和54/5？，在实验误差范围内相同。利用基于Tikhonov正则化的数值方法从偶极信号中提取距离分布的工作正在进行中。