PROTEIN FOLDING AND STABILITY OF SUBTILISIN

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)了解蛋白质折叠的动力学障碍 理解氨基酸序列与蛋白质的关系 结构和稳定性。成因、光谱和量热 将使用各种方法来研究异常折叠的能量学 丝氨酸蛋白酶枯草杆菌酶的反应。……的生物合成 枯草杆菌毒素依赖于一个77个氨基酸的N末端前体域， 它是自动加工的，以产生成熟形式的酶。 一旦加工，成熟的枯草杆菌菌素的天然构象是 很难从展开的状态中到达。折叠反应可以 然而，在体外，通过添加77个氨基酸来催化 前区域作为一个单独的多肽。这个机制是如何 前域催化枯草杆菌蛋白折叠将由以下因素决定 研究枯草杆菌菌素突变的动力学效应 前域。将通过研究探索折叠的动力学障碍 被困的、展开的形式的结构和稳定性 枯草杆菌毒素。最后是序性与稳定性的关系 将通过使用单价噬菌体展示来探索 在选择稳定突变的过程中的前域。 体外折叠效率低下是限制生产的一个因素 许多生物医学和生物技术的重组蛋白 利息。洞察折叠的能量障碍的本质 对折叠催化作用机理的准确认识 最终应该会导致新的蛋白质专一性的设计 折叠页。更好地了解如何稳定蛋白质 应该大大推进蛋白质工程领域，蛋白质 结构预测和从头蛋白质设计。

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.