INTERMEDIATES IN PROTEIN FOLDING

蛋白质折叠的机制，只给出一级序列中的信息，仍然是生物化学中最困难和最具挑战性的问题之一。这个建议的具体目标是通过确定折叠途径上早期中间体的性质和结构特性来解决这个问题。将使用几种方法，包括停流NMR技术。定点诱变加上掺入氟标记的氨基酸和一个停流核磁共振装置，我们已经建立将允许检查特定区域的蛋白质折叠过程中。我们将继续研究E.使用不同的氟标记氨基酸通过该方法研究大肠杆菌二氢叶酸还原酶。第二种被检测的蛋白质是脂肪酸结合蛋白。我们希望通过核磁共振技术，使突变体是稳定的中间体探索结构。荧光相关光谱将被用来检查在折叠过程中的野生型和稳定的中间体的快速运动。我们还使用二氢叶酸还原酶来研究细菌伴侣GroEL的作用机制以及金属，核苷酸和GroES的影响。最后，我们计划用细菌伴侣PapD进行折叠实验。二氢叶酸还原酶是许多化疗、抗菌和抗寄生虫药物的靶点。肠脂肪酸结合蛋白是在配体特异性（脂肪酸、类维生素A和胆汁盐）和组织特异性方面不同的蛋白质家族中的一种，并且被发现是用于肠道中的空间和时间分化的有用模型。PapD是在病原菌中形成丝状皮利的蛋白质的伴侣。

The mechanism by which a protein folds, given only the information in the primary sequence, remains one of the most difficult and challenging problems in biochemistry. The specific aims of this proposal are to approach this problem by determining the nature and structural properties of early intermediates on the folding pathway. Several methods are to be used including stopped 0flow NMR techniques. Site directed mutagenesis coupled with incorporation of fluorine labeled amino acids and a stopped flow NMR device we have built will allow examination of specific regions of a protein during folding. We will continue to study the E. coli dihydrofolate reductase by this method using different fluorine labeled amino acids. A second protein being examined is the fatty acid binding protein. We wish to make mutants that are stable intermediates to explore structure by NMR techniques. Fluorescence correlation spectroscopy will be used to examine rapid motions in wild-type and stable intermediates in the folding process. We are also using dihydrofolate reductase to examine the mechanism of action of the bacterial chaperone GroEL and effects of metals, nucleotides and GroES. Finally, we plan folding experiments with the bacterial chaperone PapD. Dihydrofolate reductase is the target for numerous chemotherapeutic, antibacterial and antiparasitic drugs. The intestinal fatty acid binding protein is one of a family of proteins that differ in ligand specificity (fatty acids, retinoids and bile salts) and tissue specificity and are found to be useful models for spatial and temporal differentiation in the gut. PapD is a chaperone for proteins that form filamentous pili in pathogenic bacteria.