PROTEIN FOLDING AND STABILITY OF SUBTILISIN

枯草杆菌毒素被许多研究人员认为是一种不能 以成熟的形式折叠。土生土长的体内生产 枯草杆菌毒素依赖于77个氨基酸的前肽，它是 最终从枯草杆菌菌素的N-端裂解产生了275 氨基酸是酶的成熟形式。这项提议是基于三个方面 我们最初项目的实验结果。1)成熟的形式 枯草杆菌菌素是一种不寻常的单体蛋白质，具有高度的 折叠和展开的活化能。2)高纯度的77 氨基酸前肽在体外可催化枯草杆菌蛋白折叠。3) 枯草杆菌毒素的某些突变体可以快速、定量地折叠 前肽的参与。光谱分析和光谱分析技术的应用 微量热技术与x射线衍射法相结合将 允许我们追求三个目标1)确定 非催化折叠反应中的动力学势垒。学习如何 折叠异源表达的蛋白质是最令人烦恼的问题之一 在生物技术方面。证明枯草杆菌毒素的折叠率 突变可以极大地加速，这提供了其他 困难的蛋白质折叠问题可能也会得到类似的解决，如果 折叠这种蛋白质的动力学障碍是可以理解的。2) 确定前肽如何作用于n的情况可能是一个稳定的共同之处 胞外微生物蛋白水解酶的特性可能是由于其 对热力学和动力学稳定性都有很大贡献。 不幸的是，枯草杆菌毒素的主要工业用途是 含有高浓度金属螯合剂的环境，这 将钙从枯草杆菌菌素中剥离，并影响其稳定性。如果是这样的话 创造高度稳定的枯草杆菌具有重要的现实意义 它不依赖于钙。

Subtilisin had been regarded by many researchers as a protein incapable of folding in its mature form. The in vivo production of native subtilisin is dependent on a 77 amino acid propeptide, which is eventually cleaved from the N-terminus of subtilisin to create the 275 amino acid mature form of the enzyme. This proposal is based on three experimental findings from our initial project. 1) The mature form of subtilisin is an unusual example of a monomeric protein with a high activation energy to folding and unfolding. 2) The highly purified 77 amino acid propeptide can catalyze subtilisin folding in vitro. 3) Certain mutants of subtilisin fold rapidly and quantitatively without the participation of the propeptide. The application of spectroscopic and microcalorimetric techniques coupled with x-ray diffraction methods would allow us to pursue three aims 1) Determination of the nature of the kinetic barrier in the uncatalyzed folding reaction. Learning how to fold heterologously expressed proteins is one of the most vexing problems in biotechnology. The demonstration that the folding rate of subtilisin can be dramatically accelerated by mutation, offers hope that other difficult protein folding problems might be similarly addressed, if the kinetic barriers to folding such proteins can be understood. 2) Determination of how the propeptide acts to case of n may a stable common features of extracellular microbial proteases probably because of their large contribution to both thermodynamic and kinetic stability. Unfortunately, the major industrial uses of subtilisins are in environments containing high concentrations of metal chelators, which strip calcium from subtilisin and compromise its stability. It would be of great practical significance to create a highly stable subtilisin which is independent of calcium.