Disulfide Bond Formation: Mechanisms for Isomerization and Novel Pathways

项目摘要：含有二硫键的蛋白质存在于所有生物体中。这些债券是 对这种蛋白质折叠成其三级结构很重要。生物进化出蛋白质二硫化物 确保在最终产物中找到正确的二硫键的异构酶。在细菌E。 在大肠杆菌中，DsbC蛋白纠正了错误形成的二硫键。DsbC被再生为一种活性酶 膜蛋白DsbD。我们将用Genetic，Genetic， 生物化学和结构方法。DsbC基因突变株的分离与鉴定 它的工程化及其与错氧化底物的相互作用的研究应该会产生关于 它的功能所需的氨基酸，对其正常工作重要的结构特征，以及哪些方面 二硫键的形成需要这种异构酶的存在。DsbD将电子转移到 细胞质膜由硫氧还蛋白通过二硫键还原级联进入周质DsbC。 DsbD基因突变体的分离鉴定及突变体的生化研究 蛋白质和结构分析应该能解释这种异常的电子转移机制 DsbD的膜包埋域。我们将培育出细胞质氧化还原能力发生改变的菌株 在影响二硫键形成的周质的状态或成分中。获得的菌株提供了 生产比正常细菌菌株更高产量的二硫键蛋白的方法。医学上的 重要的蛋白质--如胰岛素、人类生长激素和抗体--含有必需的二硫键 他们的活动。了解二硫键形成的特征可能有助于深入了解二硫键的破坏 这些蛋白质在故障情况下的活性。了解二硫键的作用机理 形成可以增强有效地生产其中一些用于医疗目的的蛋白质的能力。 相关性：许多对人类生长和生理很重要的蛋白质含有化学键 两种半胱氨酸的硫磺。这些二硫键结合的蛋白质包括大量的多肽激素(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 能够有效生产这些用于医疗目的的蛋白质的信息。

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.