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: 2181477
• 负责人: PHILIP N BRYAN
• 金额: $ 12.42万
• 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/2181477
• 关键词: X ray crystallography; calorimetry; catalyst; chemical binding; chemical kinetics; chemical stability; conformation; crosslink; disulfide bond; enzyme complex; intermolecular interaction; molecular chaperones; mutant; nuclear magnetic resonance spectroscopy; protein denaturation; protein engineering; protein folding; protein structure function; site directed mutagenesis; stop flow technique; subtilisins; thermodynamics; zymogens

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.

枯草杆菌蛋白酶被许多研究者认为是一种 在成熟的形态下折叠。 体内产生天然的 枯草杆菌蛋白酶依赖于77个氨基酸的前肽， 最终从枯草杆菌蛋白酶的N-末端切割，产生275个 酶的氨基酸成熟形式。 这一建议基于三个 我们最初项目的实验结果。1)的成熟形式 枯草杆菌蛋白酶是一种不寻常的单体蛋白质， 折叠和展开的活化能。2)高纯度77 氨基酸前肽在体外可催化枯草杆菌蛋白酶折叠。第三章 枯草杆菌蛋白酶的某些突变体快速定量地折叠， 前肽的参与。 本文介绍了分光光度法和 与X射线衍射方法结合的微量热技术将 让我们追求三个目标1）确定的性质， 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棘手的问题之一 在生物技术领域。 枯草杆菌蛋白酶的折叠速度 可以通过突变显著加速，为其他人提供了希望， 困难的蛋白质折叠问题可能会得到类似的解决，如果 可以理解折叠这些蛋白质的动力学障碍。（二） 确定前肽如何作用于n的情况可能是一个稳定的共同点。 细胞外微生物蛋白酶的特征可能是因为它们的 对热力学和动力学稳定性都有很大贡献。 不幸的是，枯草杆菌蛋白酶的主要工业用途是在 含有高浓度金属螯合剂的环境， 从枯草杆菌蛋白酶中除去钙并损害其稳定性。 这将是 具有重要的现实意义，创造一个高度稳定的枯草杆菌蛋白酶 它不依赖于钙。