Protein Folding and Stability of Subtilisin

枯草杆菌素BPN是一种由土壤细菌解淀粉芽孢杆菌分泌的275个氨基酸的丝氨酸蛋白酶。这是一个不寻常的，但不是唯一的例子，一个蛋白质的天然状态很难从未折叠状态获得。枯草杆菌素的生物合成依赖于77个氨基酸，N端原域，它被自动加工以产生成熟形式的酶。一旦加工，成熟枯草菌素的天然构象很难从未折叠状态到达。然而，折叠反应可以在体外通过添加77个氨基酸原氨酸作为单独的多肽来催化。本课题的长期目标是：1)了解枯草菌素折叠的动力学屏障。2)了解prodomain在降低屏障中的作用。3)阐明热力学稳定性、动力学稳定性与易折叠之间的一般关系。为了实现这一目标，我们建议使用快速动力学方法结合二维核磁共振实验，从热力学稳定性（由折叠平衡常数定义）和动力学稳定性（由展开速率定义）的角度来表征枯草菌素核心结构中每个残基的折叠。在残基特定能量参数定义后，我们将使用这些信息设计体外进化实验，以选择易折叠和动力学稳定的突变体。通过确定具有高动力学稳定性的蛋白质的折叠能量和原结构域在降低动力学屏障中的作用，我们希望揭示以下问题：1)中间体的稳定性是否与折叠速率相关？2)是否以牺牲中间体的稳定性为代价来获得高的动力学稳定性？3)稳定折叠中间体是否倾向于以牺牲整体稳定性为代价？加速折叠的突变是否模拟原蛋白结合的效果？5)在折叠催化剂存在下进化的蛋白质是否倾向于变得更稳定，但更不稳定？低效率的体外折叠是许多生物医学和生物技术感兴趣的重组蛋白生产的限制因素。深入了解折叠的能量障碍的本质和对折叠催化机制的精确理解将最终导致新的蛋白质特异性折叠酶的设计。了解易折叠与稳定性之间的关系将推动蛋白质工程、蛋白质结构预测和从头蛋白质设计领域的发展。

Subtilisin BPN is a 275 amino acid, serine protease secreted from the soil bacterium Bacillus amyloliquefaciens. It is an unusual, but not unique, example of a protein for which the native state is difficult to access from the unfolded state. 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 prodomain as a separate polypeptide. The long term goals of this proposal are: 1) To understand the kinetic barrier to subtilisin folding. 2) To understand the role of the prodomain in reducing the barrier. 3) To elucidate general relationships between thermodynamic stability, kinetic stability and facile folding. To accomplish this we propose to use rapid kinetic methods coupled with 2D NMR experiments to characterize the folding of each residue in the core structure of subtilisin in terms of thermodynamic stability (as defined by an equilibrium constant for folding) and kinetic stability (as defined by an unfolding rate). After residue specific energetic parameters are defined we will use this information to design in vitro evolution experiments to select for facile folding vs. kinetically stable mutants. By determining the folding energetics of a protein with high kinetic stability and the role of the prodomain in reducing the kinetic barrier we hope to shed light on the following questions: 1) Does the stability of intermediates correlate with folding rate? 2) Is high kinetic stability gained at the expense of the stability of intermediates? 3) Does stabilizing folding intermediates tend to occur at the expense of the stability of the whole? 4) Do mutations which accelerate folding mimic the effects of prodomain binding? 5) Do proteins which evolve in the presence of a folding catalyst tend to become more kinetically stable but less thermodynamically stable? 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. Understanding the relationships between facile folding and stability should advance the fields of protein engineering, protein structure prediction and de novo protein design.