MECHANISMS OF CHAPERONIN-ASSISTED PROTEIN FOLDING

许多体内的蛋白质折叠和组装反应已经被 发现需要一组必要的辅助蛋白，称为 陪伴者。它们的作用机制尚不清楚。我们将使用 十二聚体大肠杆菌谷氨酰胺合成酶(GS)，线粒体 罗丹尼斯和酵母α-葡萄糖苷酶作为产酶底物的研究 伴侣系统、GroEL和Groes。罗丹尼斯人要求所有的 伴侣成分和用于折叠的ATP，而只有GS和a-葡萄糖苷酶 需要GroEL和ATP才能启动折叠。确定分子 这些观察到的差异的起源将提供重要的机制 有关伴侣蛋白辅助折叠和组装的信息。 我们研究的长期目标是确定 Groe分子伴侣增加了正确折叠的产品产量 寡聚和单体蛋白质。需要检验的主要假设是 即：1)GroEL调节其与部分折叠的结合亲和力 通过结合核苷酸(ATP、ADP、ATP类似物)和 快速释放先前结合的底物；2) 伴侣系统的不同需求的分子起源 对不同底物的观察取决于a)性质和强度 初始折叠中间体的结合以及b)是否释放 中间产品仍然有错误折叠或聚合的趋势。 我们的实验方法是：1)测定结合热和 GroEL与核苷酸、GroE之间的自由能和稳定折叠 差示停流滴定微量热法测定中间体； 2)确定之后是否仍需要监护人系统 PFI已从固定化的(仍有功能的)GroEL中释放出来；以及3) 确定动力学(监测与时间相关的活性、荧光 和焓变化)伴侣蛋白辅助折叠(和组装) 作为稳定剂浓度增加的函数的反应 伴侣蛋白-PFI复合体。

A number of in vivo protein folding and assembly reactions have been found to require an essential set of accessory proteins called chaperonins. Their mechanism of action is unknown. We will use dodecameric Escherichia coli glutamine synthetase (GS), mitochondria rhodanese, and yeast alpha-glucosidase as substrates for the E. coli chaperonin system, groEL and groES. Rhodanese requires all the chaperonin components and ATP for folding while GS and a-glucosidase only require groEL and ATP to initiate folding. Determining the molecular origins for these observed differences will provide important mechanistic information about chaperonin-assisted folding and assembly. The long range goal of our research is to define the mechanism by which the groE chaperonins increase the product yields of correctly folded oligomeric and monomeric proteins. The main hypotheses to be tested are that; 1) GroEL modulates its binding affinity for partially folded intermediates (PFI) by binding nucleotide (ATP, ADP, ATP analogs) and groES to rapidly release the previously bound substrate and; 2)the molecular origins of the different requirements of the chaperonin system observed with various substrates are dictated by a) nature and strength of binding of the initial folding intermediate and b) whether the release intermediate still has a tendency to misfold or aggregate. Our experimental approach is to; 1) determine the binding enthalpies and free energies between groEL and nucleotides, groES, and stable folding intermediates by differential stopped-flow titration microcalorimetry; 2) determine whether the chaperonin system is still required after the PFI has been released from immobilized (yet functional) groEL; and 3) determine the kinetics (monitoring time-dependent activity, fluorescence and enthalpic changes) of chaperonin-assisted folding (and assembly) reactions as a function of increasing concentrations of stable chaperonin-PFI complexes.