MECHANISMS OF CHAPERONIN-ASSISTED PROTEIN FOLDING

许多体内蛋白质折叠和组装反应已被证实 发现需要一组必需的辅助蛋白，称为 伴侣蛋白。 它们的作用机制尚不清楚。 我们将使用 十二聚大肠杆菌谷氨酰胺合成酶 (GS)、线粒体 硫氰酸酶和酵母 α-葡萄糖苷酶作为大肠杆菌的底物 伴侣蛋白系统、groEL 和 groES。 罗丹尼斯要求所有 用于折叠的伴侣蛋白成分和 ATP，而仅 GS 和 a-葡萄糖苷酶 需要 groEL 和 ATP 来启动折叠。 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机制 groE 伴侣蛋白可提高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 那个； 1) GroEL 调节其对部分折叠的结合亲和力 通过结合核苷酸（ATP、ADP、ATP 类似物）产生中间体 (PFI) 和 groES 快速释放先前结合的底物； 2） 伴侣蛋白系统不同要求的分子起源 用各种基材观察到的结果取决于 a) 性质和强度 初始折叠中间体的结合力和 b) 是否释放 中间体仍然有错误折叠或聚集的倾向。 我们的实验方法是： 1) 确定结合焓和 groEL 和核苷酸之间的自由能、groES 和稳定折叠 通过差示停流滴定微量热法制备中间体； 2) 判断是否还需要伴侣系统 PFI 已从固定化（但具有功能性）groEL 中释放出来；和 3) 确定动力学（监测时间依赖性活性、荧光 伴侣蛋白辅助折叠（和组装）的热函变化） 反应作为稳定浓度增加的函数 伴侣蛋白-PFI 复合物。

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.