Disulfide Bond Formation: Mechanisms for Isomerization and Novel Pathways

描述(申请人提供)：项目摘要：含有二硫键的蛋白质存在于所有生物体中。这些键对于将这些蛋白质折叠成其三级结构非常重要。生物体进化出蛋白质二硫键异构酶，这种异构酶确保在最终产物中找到正确的二硫键。在细菌大肠杆菌中，DsbC蛋白纠正错误形成的二硫键。DsbC被膜蛋白DsbD再生为一种活性酶。我们将从遗传学、生化和结构的角度研究DsbC和DsbD的作用机制。DsbC突变体的分离和鉴定、dsbC基因工程及其与错氧化底物相互作用的研究应提供有关其功能所需的氨基酸、对其正常工作至关重要的结构特征以及这种异构酶存在的二硫键形成的哪些方面的信息。DsbD通过二硫键还原级联将电子从硫氧还蛋白传递到细胞质周膜。DsbD基因突变体的分离和鉴定，结合对突变蛋白的生化研究和结构分析，可以解释DsbD膜包埋结构域的异常电子传递机制。我们将培育改变了细胞质氧化还原状态或影响二硫键形成的周质成分的菌株。获得的菌株提供了比正常细菌菌株产生更高产量的二硫键蛋白的方法。医学上重要的蛋白质--如胰岛素、人类生长激素和抗体--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有助于深入了解这些蛋白质在故障情况下的活动中断。了解二硫键形成的机制可以提高有效地生产其中一些用于医疗目的蛋白质的能力。相关性：许多对人类生长和生理很重要的蛋白质在两个半胱氨酸的硫化物之间含有化学键。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，可以为有效生产这些用于医疗目的的蛋白质提供信息。

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.