FOLDING OF PHAGE P22 STRUCTURAL PROTEINS

在细胞内，新合成的多肽折叠成复合物， 高浓度的大分子环境。因此，体内蛋白质 能够在导致体外折叠失败的条件下折叠。在 为了防止聚集，分子伴侣促进 某些蛋白质通过与解折叠中间体或部分 折叠形式。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 折页.区分伴侣蛋白底物与非伴侣蛋白底物的因素是： 非识别多肽仍然是未知的。P22噬菌体的外壳蛋白是 一个合适的系统来调查这个问题。这种力量 系统是选择需要伴侣蛋白折叠的突变体的能力。 野生型蛋白质不需要伴侣GroEL或GroE来折叠 但是点突变体的特征在于需要这些分子伴侣， 折页.拯救这些突变体的全球抑制突变体也被 发现了此外，外壳蛋白的折叠可以与其 组装，从而允许单独调查控制外部 由这些过程中的一级氨基酸序列决定。在这一建议中，A 生物化学、生物物理学和遗传学方法的组合将用于 研究了壳蛋白在体内和体外的折叠和组装 噬菌体P22。体外定量实验与结果的比较 的体内测量将有助于阐明如何伯氨基 酸性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GroEL和GroES的相互作用。 本研究的目标是：1）确定关键氨基酸的作用 在外壳蛋白的折叠和组装中的作用; 2）研究 在体外和体内用GroES和GroEL底物多肽; 3） 检查亚基界面突变对折叠的影响， 组装件.各种方法将使我们能够深入了解 控制蛋白质在体内的折叠和组装。这一观点很重要，因为 错误折叠和/或蛋白质错误组装与严重疾病有关。

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.