Disulfide Bond Formation: Mechanisms for Isomerization and Novel Pathways

描述（由申请方提供）：项目总结：在所有生物体中均发现含有二硫键的蛋白质。这些键对于将这些蛋白质折叠成其三级结构是重要的。生物体已经进化出蛋白质二硫键异构酶，其确保在最终产物中发现正确的二硫键。在细菌E.在大肠杆菌中，DsbC蛋白纠正不正确形成的二硫键。DsbC通过膜蛋白DsbD再生为活性酶。我们将使用遗传、生化和结构方法研究DsbC和DsbD的作用机制。dsbC的突变体的分离和表征，它的基因工程和研究其相互作用与misoxidized基板应产生的信息，其功能所需的氨基酸，重要的结构特征，它的正常工作和什么方面的二硫键形成需要存在这样的异构酶。DsbD通过二硫键还原级联反应将电子从硫氧还蛋白跨细胞质膜转移到周质DsbC。dsbD基因的突变体的分离和表征结合突变蛋白和结构分析的生化研究应阐明不寻常的电子传递机制，涉及膜嵌入域的DsbD。我们将开发改变细胞质氧化还原状态或影响二硫键形成的周质组分的菌株。所获得的菌株提供了比正常细菌菌株产生更高产率的二硫键结合蛋白的手段。医学上重要的蛋白质-例如胰岛素、人生长激素和抗体-含有对其活性至关重要的二硫键。了解二硫键形成的特征可能会导致对这些蛋白质在故障情况下的活性破坏的深入了解。了解二硫键形成的机制可以提高有效生产这些蛋白质用于医疗目的的能力。相关性：许多对人类生长和生理重要的蛋白质在两个半胱氨酸的硫之间含有化学键。这些二硫键结合的蛋白质包括大量的肽激素（例如胰岛素）和免疫球蛋白（抗体）。了解二硫键是如何正确形成的，可以提供信息，使这些蛋白质的有效生产用于医疗目的。

DESCRIPTION (provided by applicant): Project Summary: Proteins containing disulfide bonds are found in all organisms. These bonds are important for the folding of such proteins into its tertiary structure. Organisms have evolved protein disulfide isomerases which insure that the correct disulfide bonds are found in the final product. In the bacterium E. coli, the DsbC protein corrects incorrectly formed disulfide bonds. DsbC is regenerated as an active enzyme by the membrane protein DsbD. We will study the mechanisms of action of DsbC and DsbD using genetic, biochemical and structural approaches. Isolation and characterization of mutants of dsbC, genetic engineering of it and studies on its interaction with misoxidized substrates should yield information on the amino acids required for its functioning, structural features important for it to work properly and what aspects of disulfide bond formation necessitate the existence of such isomerases. DsbD transfers electrons across the cytoplasmic membrane from thioredoxin to the periplasmic DsbC by a disulfide bond reduction cascade. Isolation and characterization of mutants of the dsbD gene combined with biochemical studies on mutant proteins and structural analysis should illuminate the unusual electron transfer mechanism involving the membrane-embedded domain of DsbD. We will develop strains altered either in their cytoplasmic redox state or in components of the periplasm that influence disulfide bond formation. Strains obtained provide means of producing higher yields of disulfide-bonded proteins than normal bacterial strains. Medically important proteins- e.g. insulin, human growth hormone and antibodies- contain disulfide bonds essential for their activity. Understanding features of disulfide bond formation may lead to insights into the disruption of these proteins' activities in cases of malfunction. Understanding mechanisms involved in disulfide bond formation can enhance the ability to efficiently produce some of these proteins for medical purposes. Relevance: Many proteins important to human growth and physiology contain chemical bonds between the sulfurs of two cysteines. These disulfide-bonded proteins include a large number of peptide hormones (e.g. insulin) and immunoglobulins (antibodies). Understanding how disulfide bonds are formed correctly gives information allowing efficient production of these proteins for medical purposes.