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: 2181478
• 负责人: PHILIP N BRYAN
• 金额: $ 12.8万
• 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 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2181478
• 关键词: PROTEIN; FOLDING; STABILITY; SUBTILISIN

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.

枯草素被许多研究人员视为蛋白质无能为力 以成熟形式折叠。 本机的体内生产 枯作蛋白取决于77个氨基酸前肽，这是 最终从枯草菌素的N末端分裂以创建275 酶的氨基酸成熟形式。 该提议基于三个 我们最初项目的实验发现。 1）成熟的形式 枯草蛋白是一个不寻常的例子 激活能量折叠和展开。 2）高度纯化的77 氨基酸丙肽可以在体外催化枯草菌素折叠。 3） 枯草蛋白酶折叠的某些突变体迅速，定量，没有 参与丙肽。 光谱和 与X射线衍射方法结合的微浮力技术将 允许我们追求三个目标1）确定的性质 未催化的折叠反应中的动力学屏障。 学习如何 折叠异源表达的蛋白质是最烦人的问题之一 在生物技术中。 枯草菌素的折叠率的演示 可以通过突变大大加速，可以使其他希望 如果蛋白质折叠问题很难解决，如果 可以理解折叠这种蛋白质的动力学障碍。 2） 确定前肽对N的情况的作用可能是稳定的共同点 细胞外微生物蛋白酶的特征可能是因为它们 对热力学和动力学稳定性的贡献很大。 不幸的是，枯作素的主要工业用途是 包含高浓度金属螯合剂的环境 枯草菌素的带状钙并损害其稳定性。 这将是 创建高度稳定的枯草菌素具有很大的实际意义 这与钙无关。