Disulfide Bond Formation: Mechanisms for Isomerization and Novel Pathways

项目摘要：在所有生物体中均发现含有二硫键的蛋白质。这些债券 这对于将这些蛋白质折叠成其三级结构很重要。生物进化出了蛋白质二硫化物 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作为活性酶再生 由膜蛋白DsbD。我们将研究DsbC和DsbD的作用机制， 生物化学和结构方法。dsbC基因突变体的分离和表征 它的工程和研究其相互作用与misoxidized基板应产生的信息， 氨基酸所需的功能，结构特征重要，它的工作和哪些方面 二硫键的形成需要这种异构酶的存在。DsbD将电子转移到 通过二硫键还原级联反应将细胞质膜从硫氧还蛋白转移到周质DsbC。 dsbD基因突变体的分离、鉴定及其生化特性的研究 蛋白质和结构分析应该阐明不寻常的电子转移机制， DsbD的膜包埋结构域。我们将开发出细胞质氧化还原 状态或在影响二硫键形成的周质成分中。获得的菌株提供 这意味着产生比正常细菌菌株更高产量的二硫键结合蛋白。医学上 重要的蛋白质-例如胰岛素、人生长激素和抗体-含有二硫键， 他们的活动。了解二硫键形成的特征可能会导致对破坏 这些蛋白质的活动在故障的情况下。理解二硫键的机制 形成可以增强有效地产生用于医学目的的这些蛋白质中的一些的能力。 相关性：许多对人类生长和生理重要的蛋白质在其分子之间含有化学键。 两个半胱氨酸的硫。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）。了解二硫键如何正确形成， 这些信息允许有效生产这些蛋白质用于医疗目的。

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.