Biosynthetic Folding of Bacterial Luciferase

9513429鲍德温对蛋白质折叠过程的了解对于充分发挥现代生物技术的潜力至关重要。尽管在过去的大约30年里做出了巨大的努力，但仍然存在许多问题。今天最令人担忧的是伴侣在折叠过程中的作用。目前的学说认为，蛋白质的天然结构是最稳定的结构，但伴侣的存在增加了一种可能性，即伴侣可能会允许蛋白质在没有伴侣的情况下以足够快的速度形成具有生物学意义的构象。我们建议使用细菌荧光素酶来探索伴侣在决定天然结构和非天然结构形成速度中的作用。荧光素酶是这类研究的优秀模型系统，原因有很多，包括分析的简便性和敏感性(酶发出光)和酶的二聚体结构。由于该酶由两个不同的亚基组成，(和()，因此可以单独研究每个亚基的折叠，并在没有更复杂的折叠过程的情况下研究酶的组装。我们希望，通过这些研究，我们将能够确定伴侣与核糖体上生物合成过程中多肽链的折叠和相互作用的程度。本项目的目的是了解细菌荧光素酶亚单位的生物合成折叠途径以及分子伴侣在这一过程中所起的作用(S)。“生物合成折叠”是指在核糖体上合成多肽的过程中与折叠相关的过程。细胞外蛋白质合成策略是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，能够在没有辅助的情况下自发地从变性状态复性。研究表明，(亚基的折叠是共翻译的，有助于快速获得母语结构。核糖体结合的新生(亚基)的结构特征将通过有限的蛋白分解来探索。将分离出与紧接在释放之前的新生多肽相对应的(亚基)片段，即没有被核糖体保护的C-末端区域，并通过有限的蛋白分解、圆二色谱和荧光进行探测。这一大片段和代表()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动力学。这些实验将允许开发更完整的具有纯化成分的生物合成折叠/组装模型。从核糖体释放后能够与(亚基结合的(亚基物种)的性质将被分析并与在复性过程中形成的异二聚化能力(物种)进行比较，以了解(和(的重折叠过程中)生物合成过程中荧光素酶形成速度的明显差异。研究表明，亚基可以与分子伴侣相互作用，某些分子伴侣能够加速形成具有酶活性的杂二聚体。这一现象的机制及其与生物合成折叠的关系将被研究。在纯蛋白质的重折叠过程中，(亚基获得不能与(相互作用的替代构象。我们将研究分子伴侣对(亚基在这些不同构象之间分布的影响。我

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