Genetic Studies on Mechanisms of Disulfide Bond Formation

9406275与许多从细胞输出的蛋白质和许多膜蛋白一样，它们的序列中含有半胱氨酸之间的二硫键。这些二硫键通常对蛋白质的折叠和功能都很重要。1991年，人们发现细菌表达一种蛋白质DsbA，这种蛋白质是蛋白质中有效形成二硫键所必需的。从那时起，人们发现酿酒酵母也表达了类似的活性。本项目的一个主要目的是研究DsbA蛋白催化蛋白质中的半胱氨酸对氧化的机制。为此，正在分离出DsbA活性有缺陷的突变株，对突变基因进行测序，并进行体内研究，以确定这一过程中的缺陷步骤。此外，与生物化学家和X射线结晶学家的互动和合作将有助于阐明缺陷的基础。这些研究应该会对DsbA的结构-功能关系有一个详细的描述。为了进一步了解DsbA的作用机制，在体内进行的实验旨在表明DsbA蛋白在蛋白质合成和折叠的哪个阶段作用于输出蛋白以催化二硫键的形成。体内和体外实验将测试DsbA是否可以作用于已经折叠的蛋白质。体内实验将分析膜转位过程中输出的蛋白质，以确定在蛋白质完全转位之前是否可以形成二硫键。已经设计了一种基因选择，可以分离出口蛋白质的突变，这些突变不能被促进二硫键形成的系统作用，即使它们保留了适当的半胱氨酸。β-内酰胺酶将被用作模型系统，因为它只有一个二硫键，可以输出到周质，在没有二硫键的情况下是有效的，并且其三维结构已被确定为1.8个焦虑罗姆。这些突变体可能改变蛋白质的折叠途径或其二级或三级结构。对突变蛋白的序列和结构的分析应该揭示允许二硫键形成的重要特征。实验还旨在检测细菌周质中催化二硫键形成的其他系统。在没有DsbA体系的情况下，发现了背景二硫键形成活性。将寻找同时增加和降低这一背景活性的突变体。此外，还将启动遗传学研究，以确定在厌氧生长条件下负责二硫键形成的系统。这些研究应该有助于确定与这些其他活动有关的基因和基因产物。这些研究对基础科学和应用科学具有广泛的影响。这些结果与蛋白质折叠问题有关，蛋白质折叠是生物学中主要的新发展领域之一。此外，细菌系统中表达的外源蛋白中二硫键的形成也带来了许多问题。操纵二硫键形成系统的能力应该可以更容易地解决这些表达问题。含有二硫键的一系列重要蛋白质包括肽激素、膜受体、细菌毒素和免疫球蛋白。***

9406275 Beckwith Many proteins exported from cells and many membrane proteins contain disulfide linkages between cysteines within their sequences. These disulfide bonds are often important both for the folding and the functioning of the protein. In 1991, it was found that bacteria express a protein, DsbA, that is required for efficient formation of disulfide bonds in proteins. Since then, it has been found that the yeast Saccharomyces cerevisiae expresses a similar activity. A major purpose of this project is to study the mechanism by which the DsbA protein catalyzes oxidation of pairs of cysteines in proteins. To this end, mutants are being isolated that are defective in DsbA activity, the mutant genes sequenced and in vivo studies carried out to define the defective step in the process. In addition, interaction and collaborations with biochemists and an X-ray crystallographer will help elucidate the basis of the defect. These studies should lead to a detailed picture of the structurefunction relationships for DsbA. To further understand the mechanism of DsbA action, experiments done in vivo are designed to indicate at what stage of protein synthesis and folding the DsbA protein acts on exported proteins to catalyze disulfide bond formation. In vivo and in vitro experiments will test whether DsbA can act on an already folded protein. In vivo experiments will analyze exported proteins during the process of membrane translocation to determine whether disulfide bonds can form before the proteins are fully translocated. A genetic selection has been devised which may allow the isolation of mutants of exported proteins that are unable to be acted on by systems that promote disulfide bond formation even though they retain the appropriate cysteines. beta-lactamase will be used as a model system since it has a single disulfide bond, is exported to the periplasm, is functional in the absence of its disulfide bond and has had its 3-dimensional structure determined to 1.8 Angst roms. These mutants may alter the folding, pathway for the protein or its secondary or tertiary structure. Analysis of the sequence and structure of the mutant proteins should reveal the important features that allow disulfide bond formation to take place. Experiments are also designed to detect other systems in the bacterial periplasm that catalyze disulfide bond formation. In the absence of the DsbA system, a background disulfide bond-forming activity is found. Mutants that both increase and decrease the activity of this background activity will be sought. In addition, genetic studies will be initiated to identify the system responsible for disulfide bond formation under conditions of anaerobic growth. These studies should lead to the identification of genes and gene products responsible for these other activities. %%% These studies have wide implications for basic and applied science. The results are relevant to questions of protein folding, one of the major new developing areas in biology. In addition, the formation of disulfide bonds in expressed foreign proteins in bacterial systems has presented many problems. The ability to manipulate the disulfide bond-forming systems should allow more ready solution to these expression problems. The range of important proteins that contain disulfide bonds includes peptide hormones, membrane receptors, bacterial toxins and immunoglobulins. ***