FAST KINETICS OF DESIGNED SERPINS

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。 这是一个旨在通过实验和计算技术了解蛋白质如何折叠的项目。后者可以指导快速文件夹的搜索，也可以指导结构上一致的突变体。我们将采用的计算技术使用统计热力学来识别蛋白质序列中的受挫位置，以及结构上一致和不那么令人沮丧的突变。此方法以前曾用于指导搜索FAST文件夹。在这种情况下，将其应用于20个残基的微小蛋白Trp-CAGE，以进一步缩短其4微秒的折叠时间。我们将使用的实验技术包括激光诱导温度跳跃动力学实验(红外或紫外光探测)以及使用荧光、红外光谱和圆二色谱的静态实验。 我们进一步建议使用计算机辅助的多肽设计，结合荧光相关光谱(FCS)和基于荧光共振能量转移(FRET)的单分子技术来设计和表征短肽，这些短肽也可以阻碍和逆转蛇类的聚合。其具体目的是从理论和实验的角度设计和研究这些多肽，并研究解聚的潜在机制细节，从而为潜在的治疗分子的设计提供信息。

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. This is a project aimed to understand how proteins fold using both experimental and computational techniques. The latter can guide the search for fast folders and also tructurally consistent mutants. The computational technique we will employ uses statiscal thermodynamics to identify frustrated positions in the protein sequence as well as structurally consistent and less frustrating mutations. This method has been used previously to guide the search for fast folders. In this case, it has been applied to the 20 residue miniprotein, Trp-cage, in an effort to decrease its 4 microsecond folding time further. The experimental techniques we will use include laser induced temperature-jump kinetics experiments (with IR or UV detection) as well as static experiments using fluorescence, infrared spectroscopy, and circular dichroism. We further propose to use computationally assisted peptide design in association with fluorescence correlation spectroscopy (FCS) and fluorescence resonance energy transfer (FRET) based single molecule techniques to design and characterize short peptides that also impede and reverse the polymerization of serpins. The specific aims are to design and study these peptides both from the theoretical and experimental points of view and to investigate the underlying mechanistic details of depolymerization, thus informing the design of potential therapeutic molecules.