FOLDING OF PHAGE P22 STRUCTURAL PROTEINS

在细胞内，新合成的多肽在复杂的环境中折叠 DNA、RNA和极高浓度的蛋白质。在这么高的地方 蛋白质浓度多肽在体外的折叠失败， 导致聚集而不是有效折叠。然而，蛋白质是 能够在体内折叠。这个悖论最近得到了解决， 发现了一类被称为分子伴侣的蛋白质。分子 分子伴侣通过促进蛋白质的一个子集的折叠而起作用， 多肽通过与它们的折叠中间体相互作用， 聚集。显然，分子伴侣可以区分 折叠中间体，因为只有一个子集需要帮助， 生产性折叠。然而，是什么区分了公认的折叠 从一个未被识别的中间是一个谜。的长期目标 本项目旨在了解折叠中间体的特性， 被分子伴侣识别，以及分子伴侣如何促进 体内底物多肽。沙门氏菌的外壳蛋白 噬菌体P22提供了一个独特的模型系统，用于研究 体内折叠中间体的特征， 分子伴侣，GroEL和GroES;外壳蛋白突变体，其折叠 在高温下有缺陷，需要GroEL和GroES进行生产 折叠和组装。因此，这些单个氨基酸取代 干扰突变体外壳多肽的折叠， 它们成为GroEL和GroES的底物。这是一种额外的力量 模型系统是这种能力，可以很容易地选择突变体影响， 折页. 在这项建议中，生物物理学、生物化学和遗传学将用于 探测GroEL和GroES与外壳多肽底物的相互作用 在体内和体外通过关联从体内折叠获得的结果 与那些从体外动力学研究中获得的实验。效果 氨基酸取代对外壳蛋白动力学折叠的影响 将使用内源性和外源性荧光进行体外分析， 圆二色性GroEL的结合口袋将通过 荧光猝灭和底物多肽对 蛋白酶拯救突变体外壳正确折叠的条件 通过GroEL和GroES在体外测定蛋白质。的能力 GroEL和GroES在体内的正确折叠将在正常情况下被确定， GroEL和GroES水平升高或降低。GroEL和GroES， 功能已被改变的突变将被测试的能力， 在体内正确折叠底物多肽。 此外，基因 方法将被用来调查期间发生的相互作用 折叠，在折叠中间和折叠之间 中间体和GroEL，通过分离基因内第二位点抑制子 在产生野生型或缺陷型的细胞中， GroEL。所提出的各种方法将使人们对以下问题有独特的见解： 蛋白质在体内折叠的机制。这一观点很重要，因为 许多严重的疾病，如阿尔茨海默氏病，是由 蛋白质的错误折叠

Within cells, newly synthesized polypeptides fold within a complex milieu of DNA, RNA and extremely high concentrations of proteins. At these high protein concentrations the folding of polypeptides in vitro fails, resulting in aggregation rather than productive folding. Yet, proteins are able to fold in vivo. This paradox has recently been resolved by the discovery of a class of proteins known as molecular chaperones. Molecular chaperones function by facilitating the folding of a subset of polypeptides by interacting with their folding intermediates to keep them from aggregating. Clearly, molecular chaperones differentiate among folding intermediates since only a subset require assistance for productive folding. However, what distinguishes a recognized folding intermediate from a non-recognized one is an enigma. The long term goal of this project is to understand the features of folding intermediates that are recognized by chaperones and how chaperones facilitate the folding of substrate polypeptides in vivo. Coat protein from the Salmonella bacteriophage P22 provides a unique model system with which to study the features of in vivo folding intermediates that are recognized by the molecular chaperones, GroEL and GroES; coat protein mutants, whose folding is defective at high temperature, require GroEL and GroES for productive folding and assembly. Consequently, these single amino acid substitutions perturb the folding of the mutant coat polypeptides in such a way that they become substrates of GroEL and GroES. An additional strength of this model system is this ability to easily select for mutants affected in folding. In this proposal biophysics, biochemistry, and genetics will be used to probe the interactions of GroEL and GroES with coat polypeptide substrates in vivo and in vitro by correlating results obtained from in vivo folding experiments with those obtained from kinetic studies in vitro. The effect of the amino acid substitutions on the kinetics folding of coat protein will be analyzed in vitro using intrinsic and extrinsic fluorescence, and circular dichroism. The binding pocket of GroEL will be probed by fluorescence quenching and accessibility of substrate polypeptides to protease. Conditions for the rescue of proper folding of the mutant coat proteins by GroEL and GroES in vitro will be determined. The ability of GroEL and GroES to correct folding in vivo will be ascertained at normal, increased, or decreased levels of GroEL and GroES. GroEL and GroES whose function has been altered by mutation will be tested for the ability to properly fold substrate polypeptides in vivo. Moreover, a genetic approach will be used to investigate the interactions that occur during folding, both within a folding intermediate and between folding intermediates and GroEL, by isolating intragenic second site suppressors of the defective coat proteins in cells producing wild-type or defective GroEL. The variety of approaches proposed will allow singular insight in the mechanism of protein folding in vivo. This insight is important since many serious diseases, such as Alzheimer's disease, are caused by the misfolding of proteins.