INTERMEDIATES IN PROTEIN FOLDING

在只给出初级序列信息的情况下，蛋白质的折叠机制仍然是生物化学中最困难和最具挑战性的问题之一。本提案的具体目的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。几种方法将被使用，包括停止流动核磁共振技术。位点定向诱变与氟标记氨基酸的结合以及我们建立的停止流动核磁共振装置将允许在折叠过程中检查蛋白质的特定区域。我们将继续用这种方法研究不同的氟标记氨基酸对大肠杆菌二氢叶酸还原酶的影响。正在研究的第二种蛋白质是脂肪酸结合蛋白。我们希望通过核磁共振技术制备稳定的中间体突变体来探索其结构。荧光相关光谱将用于检测折叠过程中野生型和稳定中间体的快速运动。我们还利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蛋白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。肠道脂肪酸结合蛋白是配体特异性（脂肪酸、类维生素a和胆盐）和组织特异性不同的蛋白质家族中的一种，被发现是肠道空间和时间分化的有用模型。PapD是致病菌中形成丝状菌毛蛋白的伴侣蛋白。

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.