Disulfide Bond Formation: Isomerization and Pathways

二硫键的形成：异构化和途径

• 批准号: 7037217
• 负责人: JONATHAN BECKWITH
• 金额: $ 51.55万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-02-01 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7037217
• 关键词: Escherichia coli; NAD(P)H oxidoreductase; bacterial genetics; bacterial proteins; biotechnology; directed evolution; disulfide bond; electron transport; functional /structural genomics; gene mutation; isomer; isomerase; molecular cloning; molecular genetics; oxidation reduction reaction; protein disulfide reductase (glutathione); protein engineering; protein folding; recombinant proteins; thiols; thioredoxin

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.

描述（由申请人提供）： 项目摘要：在所有生物体中都发现含有二硫键的蛋白质。这些键对于将此类蛋白质折叠成其三级结构非常重要。生物体已经进化出蛋白质二硫键异构酶，确保在最终产品中找到正确的二硫键。在大肠杆菌中，DsbC 蛋白可纠正错误形成的二硫键。 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 突变体的分离和表征、其基因工程以及对其与错氧化底物相互作用的研究，应能提供有关其功能所需的氨基酸、对其正常工作重要的结构特征以及二硫键形成的哪些方面需要存在的信息。此类异构酶。 DsbD 通过二硫键还原级联将电子穿过细胞质膜从硫氧还蛋白转移到周质 DsbC。 dsbD 基因突变体的分离和表征结合突变蛋白的生化研究和结构分析应该阐明涉及 DsbD 膜嵌入结构域的不寻常的电子转移机制。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质的方法。医学上重要的蛋白质 - 例如胰岛素、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了解这些蛋白质在发生故障时活性的破坏。了解二硫键形成所涉及的机制可以增强有效生产某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学重要的蛋白质在两个半胱氨酸的硫之间含有化学键。这些二硫键蛋白质包括大量肽激素（例如胰岛素）和免疫球蛋白（抗体）。了解二硫键如何正确形成可以提供有效生产这些用于医疗目的的蛋白质的信息。