Folding Mechanisms of Dihydrofolate Reductase

该项目的总体目标是了解蛋白质的氨基酸序列如何将其快速有效地折叠成天然构象的机制。生物物理和蛋白质工程技术将被用来研究一组同源蛋白，具有低序列相似性，但几乎相同的三维结构。来自大肠杆菌（ecDHFR）、干酪乳杆菌（lcDHFR）和人类（hDHFR）的二氢叶酸还原酶（DHFR）都属于α / β片蛋白类，并含有嵌入核苷结合结构域（NBD），该结构域在折叠中起关键作用。利用小角x射线散射和荧光共振能量转移（FRET）技术探测尿素变性ecDHFR的残留结构。突变分析将用于测试疏水簇对剩余结构的贡献，以及它们在形成平行折叠通道中的作用，这些通道先前在ecDHFR折叠反应中被发现。微秒级的折叠反应将通过将FRET技术连接到超快连续流混合系统来监测。突变分析将用于验证环路域和NBD之间的替代对接模式负责平行通道折叠机制的假设。系统的荧光和圆二色性研究将揭示lcDHFR的折叠机制。与ecDHFR和hDHFR的机制比较，将验证同源蛋白的折叠机制比氨基酸序列更保守的假设。由于NBD是在这三个超级王国中发现的十大基序之一，预计从这些对一组DHFRs折叠机制的研究中获得的见解将适用于一大类蛋白质的折叠。了解蛋白质序列决定其折叠途径和最终结构的机制，将对序列的三维结构预测、蛋白质的从头设计以及生物技术工业的蛋白质产品生产产生重大影响。这项研究将为本科生、研究生和博士后研究人员提供实验设计、现代生物物理和蛋白质化学技术以及基于计算机的数据分析方面的宝贵指导。

MatthewsMCB 0081076The overall goal of this project is to understand the mechanism by which the amino acid sequence of a protein directs its rapid and efficient folding to the native conformation. Biophysical and protein engineering techniques will be employed to study a set of homologous proteins that have low sequence similarity but nearly identical three-dimensional structures. Dihydrofolate reductases (DHFR) from Escherichia coli (ecDHFR), Lactobacillus casei (lcDHFR) and human (hDHFR) sources all belong to the alpha/beta sheet class of proteins and contain an embedded nucleoside-binding domain (NBD) that appears to play a critical role in folding. Small angle x-ray scattering and fluorescence resonance energy transfer (FRET) will be used to probe for residual structure in urea-denatured ecDHFR. Mutational analysis will be used to test for the contribution of hydrophobic clusters to the residual structure and to their role in the formation of parallel folding channels previously identified in the ecDHFR folding reaction. Microsecond folding reactions will be monitored by interfacing FRET technology to an ultrafast continuous-flow mixing system. Mutational analysis will be used to test the hypothesis that alternative docking modes between the loop domain and NBD are responsible for the parallel channel folding mechanism. Systematic fluorescence and circular dichroism studies will reveal the folding mechanism of lcDHFR. Comparison with the mechanisms for ecDHFR and hDHFR will test the hypothesis that folding mechanisms of homologous proteins are better conserved than the amino acid sequences. Because the NBD is one of the top ten motifs found in all three super-kingdoms, it is expected that the insights obtained from these studies of the folding mechanisms of a set of DHFRs will be applicable to the folding of a large class of proteins.An understanding of the mechanism by which the sequence of a protein dictates its folding pathway and, ultimately, structure would have a major impact on the prediction of the three-dimensional structure from sequence, on the de novo design of proteins, and on the production of protein products by the biotechnology industry. This research will provide valuable instruction in experimental design, modern biophysical and protein chemistry techniques, and computer-based data analysis to undergraduate students, graduate students and postdoctoral research associates.