FOLDING OF PHAGE P22 STRUCTURAL PROTEINS

在细胞内，新合成的多肽折叠成一个复合体。 大分子浓度高的环境。因此，体内的蛋白质 能够在会导致体外折叠失败的条件下折叠。在……里面 为了防止聚集，分子伴侣促进了折叠 某些蛋白质通过与未折叠的中间体相互作用或部分 折叠的表格。分子伴侣似乎能够区分 折叠中间体，以确定生产所需的子集 折叠。区分伴侣底物和伴侣底物的因素 未被识别的多肽仍然未知。P22噬菌体外壳蛋白为 一个适当的系统来调查这个问题。这其中的一个力量 系统是选择需要伴侣折叠的突变体的能力。 野生型蛋白不需要伴侣GroEL或GroES折叠 但是已经确定了点突变的特征，这些突变需要这些伴侣来 折叠。拯救这些突变体的全球抑制突变体也被 被发现了。此外，外壳蛋白的折叠可以与它的 程序集，从而允许对外部控件进行单独调查 由这些过程中的主要氨基酸序列决定。在这项提案中， 生物化学、生物物理和遗传方法的结合将用于 猪瘟病毒外壳蛋白的体内和体外折叠组装研究 噬菌体P22。定量体外实验与实验结果的比较 体内测量的结果将有助于阐明伯氨基是如何 酸序列指导折叠和组装。这些实验还将提供 深入了解折叠所需的与GroEL和GroE的交互。 这项研究的目标是：1)确定关键氨基酸的作用 在外壳蛋白的折叠和组装中；2)研究相互作用 含有GroES和GroEL的底物多肽的体内外和3) 检验亚单位界面突变对折叠和 集合。方法的范围将允许深入了解以下因素 控制蛋白质在体内的折叠和组装。这种洞察力很重要，因为 错误折叠和/或蛋白质错误组装已被认为与严重疾病有关。

Within cells, newly synthesized polypeptides fold in a complex environment of high concentrations of macromolecules. Thus, in vivo proteins are able to fold under conditions that would cause in vitro folding to fail. In order to prevent aggregation, molecular chaperones facilitate the folding of certain proteins by interacting with unfolding intermediates or partially folded forms. Molecular chaperones appear to be able to differentiate among folding intermediates in order to identify the subset required for productive folding. The factors that distinguish a substrate for a chaperone from a non-recognized polypeptide are still unknown. Coat protein of the P22 phage is an appropriate system for investigating this question. A strength of this system is the ability to select for mutants that require chaperones to fold. The wild type protein does not require the chaperones GroEL or GroEs to fold but point mutants have been characterized which require these chaperones for folding. Global suppressor mutants that rescue these mutants have also been discovered. In addition, the folding of coat protein can be decoupled from its assembly, thereby allowing the separate investigation of the control exterted by the primary amino acid sequence on these processes. In this proposal, a combination of biochemical, biophysical and genetic approaches will be used to study the in vivo and the in vitro folding and assembly of the coat protein of phage P22. The comparison of quantitative in vitro experiments with the results of in vivo measurements will aid in the elucidation of how the primary amino acid sequence directs folding and assembly. These experiments will also provide insight into interactions with GroEL and GroES that are required for folding. The goals of the research are: 1) to determine the role of crucial amino acids in the folding and assembly of coat protein; 2) investigate the interaction of substrate polypeptides with GroES and GroEL in vitro and in vivo and; 3) examine the effects of mutations at the subunit interface on folding and assembly. The range of approaches will allow insight into the factors which control protein folding and assembly in vivo. This insight is important since misfolding and/or protein misassembly has been implicated in serious diseases.