MECHANISMS OF CHAPERONIN-ASSISTED PROTEIN FOLDING

许多体内蛋白质折叠和组装反应已经被证明是有效的。 发现需要一组必需的辅助蛋白质， 伴侣蛋白 其作用机制尚不清楚。 我们将使用 十二聚体大肠杆菌谷氨酰胺合成酶（GS），线粒体 rhodanese和酵母α-葡糖苷酶作为E.杆菌 伴侣蛋白系统，groEL和groES。 罗丹语要求所有的 伴侣蛋白组分和ATP用于折叠，而GS和α-葡萄糖苷酶仅用于折叠 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 关于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伴侣蛋白增加了正确折叠的 寡聚体和单体蛋白质。 要检验的主要假设是 1）GroEL调节其对部分折叠的 通过结合核苷酸（ATP、ADP、ATP类似物）和 生长以快速释放先前结合的底物; 2） 伴侣蛋白系统不同需求的分子起源 a）性质和强度 和B）是否释放 中间体仍然具有错误折叠或聚集的趋势。 我们的实验方法是：1）确定结合力， 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测定中间体; 2)确定是否仍然需要伴侣系统后， PFI已从固定化的（但功能性的）groEL释放;和3） 测定动力学（监测时间依赖性活性、荧光 伴侣蛋白辅助折叠（和组装） 反应作为增加浓度的稳定 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.