PROTEIN FOLDING AND STABILITY OF SUBTILISIN

枯草杆菌蛋白酶的蛋白质折叠和稳定性

• 批准号: 6018778
• 负责人: PHILIP N BRYAN
• 金额: $ 15.1万
• 依托单位: UNIVERSITY OF MD BIOTECHNOLOGY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6018778
• 关键词: X ray crystallography; calorimetry; catalyst; chemical binding; chemical kinetics; chemical stability; circular dichroism; conformation; intermolecular interaction; molecular chaperones; nuclear magnetic resonance spectroscopy; protein engineering; protein folding; protein sequence; protein structure function; recombinant proteins; subtilisins; thermodynamics

The long term objectives of this project are: 1) To understand the mechanism of how one protein can catalyze the folding of another; 2) To understand kinetic barriers to protein folding; 3) To understand relationships between amino acid sequence and protein structure and stability. Genetic, spectroscopic and calorimetric methods will be used to study the energetics of the unusual folding reaction of the serine protease subtilisin. The biosynthesis of subtilisin is dependent on a 77 amino acid, N-terminal prodomain, which is auto-processed to create the mature form of the enzyme. Once processed, the native conformation of mature subtilisin is difficult to reach from the unfolded state. The folding reaction can be catalyzed in vitro, however, by the addition of the 77 amino acid proregion as a separate polypeptide. The mechanism of how the prodomain catalyzes subtilisin folding will be determined by studying the kinetic effects of mutations in subtilisin and the prodomain. Kinetic barriers to folding will be explored by studying the structure and stability of the trapped, unfolded forms of subtilisin. Finally the relationships between sequence and stability will be explored by using monovalent phage display of the prodomain in a procedure which selects for stabilizing mutations. Inefficient in vitro folding is a limiting factor in the production of many recombinant proteins of biomedical and biotechnological interest. Insight into the nature of the energetic barriers to folding and precise understanding of the mechanism of folding catalysis should lead eventually to the design of novel protein-specific foldases. Better knowledge concerning how to stabilize proteins should greatly advance the fields of protein engineering, protein structure prediction and de novo protein design.

该项目的长期目标是：1）了解 一种蛋白质如何催化另一种蛋白质折叠的机制; 2)了解蛋白质折叠的动力学障碍; 3） 了解氨基酸序列和蛋白质之间的关系 结构和稳定性。遗传、光谱和量热 方法将被用来研究不寻常的折叠的能量学 丝氨酸蛋白酶枯草杆菌蛋白酶的反应。的生物合成 枯草杆菌蛋白酶依赖于77个氨基酸的N末端前结构域， 其被自动加工以产生酶的成熟形式。 一旦加工，成熟枯草杆菌蛋白酶的天然构象是 很难从未折叠状态到达。 折叠反应可以 然而，通过添加77个氨基酸， 前区作为单独的多肽。 的机制如何 前结构域催化枯草杆菌蛋白酶折叠将由 研究枯草杆菌蛋白酶突变的动力学效应， 前结构域折叠的动力学障碍将通过研究 被困的，展开的形式的结构和稳定性 枯草杆菌蛋白酶。最后讨论了序列与稳定性的关系 将探索通过使用单价噬菌体展示的 在选择稳定突变的程序中使用前域。 体外折叠效率低是生产重组蛋白的限制因素。 许多生物医学和生物技术的重组蛋白 兴趣洞察折叠的能量障碍的本质 以及对折叠催化机制的精确理解 最终将导致设计新的蛋白质特异性 折叠酶更好地了解如何稳定蛋白质 将极大地推进蛋白质工程、蛋白质 结构预测和从头蛋白质设计。