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折叠失败， 导致聚合，而不是富有成效的折叠。然而，蛋白质是 能够在体内折叠。这一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 伴侣通过促进折叠的子集 多肽通过与其折叠中间体相互作用来保持它们 通过聚合。显然，分子伴侣区分了 折叠中间体，因为只有一个子集需要辅助 高效折叠。然而，是什么区别于公认的折叠 未被识别的生物的中间产物是一个谜。的长期目标是 这个项目是为了了解折叠中间体的特征 被伴侣识别，以及伴侣如何促进折叠 体内底物多肽。沙门氏菌外壳蛋白 噬菌体P22提供了一个独特的模型系统来研究 可识别的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才能生产 折叠和组装。因此，这些单一的氨基酸替换 以这样一种方式干扰突变外壳多肽的折叠 它们成为GroEL和Groes的底物。这方面的一个额外的力量 模型系统是这种轻松选择受影响的突变体的能力 折叠。 在这项提案中，生物物理学、生物化学和遗传学将被用于 探索GroEL和GroES与包被多肽底物的相互作用 通过关联体内折叠获得的结果进行体内和体外实验 用体外动力学研究得到的结果进行实验。其效果 氨基酸取代对外壳蛋白动力学折叠的影响 将在体外使用本征和外源荧光进行分析，以及 圆二向色性。GroEL的绑定口袋将被探测到 底物多肽的荧光猝灭与可及性 蛋白酶。突变体毛被正确折叠的挽救条件 将测定GroEL和GroES体外培养的蛋白质。的能力 体内正确折叠的GroEL和GroES将在正常情况下确定， 增加或降低GroEL和GroES水平。GroEL和Groes的 功能已经被改变的突变将被测试的能力 在体内适当折叠底物多肽。此外，一种基因 将使用一种方法来调查在 折叠，无论是在折叠中间还是在折叠之间 中间体和GroEL，通过分离基因内第二位点抑制物 产生野生型或缺陷型的细胞中的缺陷外壳蛋白 格罗尔。建议的各种方法将允许对 蛋白质在体内折叠的机制。这种洞察力很重要，因为 许多严重的疾病，如阿尔茨海默氏症，都是由 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.