Biosynthetic Folding of Bacterial Luciferase

细菌荧光素酶的生物合成折叠

• 批准号: 9513429
• 负责人: Thomas Baldwin
• 金额: $ 27万
• 依托单位: Texas A&M AgriLife Research
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-01 至 2000-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9513429&HistoricalAwards=false
• 关键词: Biosynthetic; Folding; Bacterial; Luciferase

9513429 Baldwin An understanding of the processes of protein folding is crucial to full realization of the potential of modern biotechnology. In spite of intense efforts over the past ca. 30 years, many questions remain. Of central concern today is the role of chaperones in the folding process. Current dogma holds that the native structure of a protein is the most stable structure possible, but the existence of chaperones raises the possibility that chaperones might allow proteins to assume conformations that do not form fast enough in their absence to be of biological significance. We propose to use bacterial luciferase to probe the roles of chaperones in determining the rates of formation of native and nonnative structures. Luciferase is an excellent model system for such studies for numerous reasons, including the ease and sensitivity of the assay (the enzyme emits light) and the dimeric structure of the enzyme. Since the enzyme is composed of two nonidentical subunits, ( and (, it is possible to investigate the folding of each subunit in isolation, and to investigate the assembly of the enzyme in the absence of more complicating folding processes. We expect that, through these studies, we will be able to ascertain the extent to which the chaperones interact with and guide the folding of thepolypeptide chain during biosynthesis on the ribosome. %%% The objective of this project is to understand the biosynthetic folding pathway of the ( subunit of bacterial luciferase and the role(s) played by molecular chaperones in this process. "Biosynthetic folding" refers to the processes associated with folding during synthesis of the polypeptide on a ribosome. The cell free protein synthesis strategies serve as a model for protein synthesis and folding within the living cell. Bacterial luciferase is a cytoplasmic heterodimer capable of unassisted spontaneous refolding from the denatured state. It has been shown that the ( subunits folds cotranslationally, contributing to the fast acqui sition of native structure. Structural features of the ribosome-bound nascent ( subunit will be probed by limited proteolysis. A fragment of the ( subunit corresponding to the nascent polypeptide immediately prior to release, i.e., without the C-terminal region which is sheltered by the ribosome, will be isolated and probed by limited proteolysis, circular dichroism and fluorescence. This large fragment and a fragment representing the C-terminal portion of ( will be used to analyze the kinetics of folding and assembly of bacterial luciferase. These experiments will permit development of a more complete model of biosynthetic folding/assembly with purified components. The nature of the ( subunit species capable of association with the ( subunit following release from the ribosome will be analyzed and compared with the heterodimerization-competent ( species formed during refolding, to understand apparent differences in the rates of formation of luciferase during biosynthesis of ( and refolding of (. It has been shown that ( subunit can interact with chaperones, and that certain chaperones are able to accelerate formation of enzymatically active heterodimer. The mechanism of this phenomenon and its relation to biosynthetic folding will be studied. During refolding of the pure protein, the ( subunit acquires alternative conformations which cannot interact with (. The effect of chaperones on the distribution of ( subunit between these alternative conformations will be examined. I

9513429 Baldwin了解蛋白质折叠的过程对于充分发挥现代生物技术的潜力至关重要。 尽管在过去的一年里，我们付出了巨大的努力。 30年过去了，很多问题依然存在。 目前关注的焦点是分子伴侣在折叠过程中的作用。 目前的教条认为，蛋白质的天然结构是可能的最稳定结构，但伴侣蛋白的存在提出了这样一种可能性，即伴侣蛋白可能允许蛋白质呈现在它们不存在时形成得不够快而具有生物学意义的构象。 我们建议使用细菌荧光素酶来探测分子伴侣在确定天然和非天然结构的形成速率中的作用。 荧光素酶是用于此类研究的极好模型系统，原因有很多，包括测定的容易性和灵敏度（酶发光）以及酶的二聚体结构。 由于该酶由两个不同的亚基组成，（和（，它是可能的研究每个亚基在隔离的折叠，并研究在更复杂的折叠过程的情况下，酶的组装。 我们希望通过这些研究，我们将能够确定在核糖体上生物合成过程中，分子伴侣与多肽链相互作用并指导多肽链折叠的程度。 本项目的目的是了解细菌荧光素酶亚基的生物合成折叠途径以及分子伴侣在此过程中所起的作用。 “生物合成折叠”是指在核糖体上多肽合成期间与折叠相关的过程。 无细胞蛋白质合成策略充当活细胞内蛋白质合成和折叠的模型。 细菌荧光素酶是一种胞质异源二聚体，能够从变性状态自发重折叠。 已经表明，亚基互补折叠，有助于快速获得天然结构。 核糖体结合的新生亚基的结构特征将通过有限的蛋白水解来探测。 对应于释放前新生多肽的α亚基片段，即，没有被核糖体保护的C-末端区域，将被分离并通过有限的蛋白水解、圆二色性和荧光探测。 该大片段和代表的C-末端部分的片段将用于分析细菌荧光素酶的折叠和组装的动力学。 这些实验将允许开发具有纯化组分的生物合成折叠/组装的更完整模型。 将分析从核糖体释放后能够与β亚基结合的β亚基种类的性质，并与具有异源二聚化能力的β亚基种类进行比较。 在重折叠过程中形成的物种，以了解在生物合成和重折叠过程中荧光素酶形成速率的明显差异。 已经显示α亚基可以与分子伴侣相互作用，并且某些分子伴侣能够加速酶活性异二聚体的形成。 将研究这种现象的机制及其与生物合成折叠的关系。 在纯蛋白质的重折叠过程中，（亚基获得了不能与（相互作用的替代构象。 将检查分子伴侣对这些替代构象之间的亚基分布的影响。 我