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: 3301207
• 负责人: PHILIP N BRYAN
• 金额: $ 16.7万
• 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 1993-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3301207
• 关键词: Bacillus subtilis; aminoacid; chemical stability; chemical substitution; computer data analysis; fluorescence spectrometry; intermolecular interaction; microcalorimetry; molecular cloning; molecular dynamics; mutant; protein denaturation; protein engineering; protein folding; protein purification; protein structure function; site directed mutagenesis; subtilisins; thermodynamics

The goal of this project is to study protein folding and stability of subtilisin BPN' and using in vitro mutagenesis, thermodynamic analysis, x- ray crystallography and theoretical methods. The subtilisin system was chosen for its convenience in genetic manipulation, expression, purification and crystallographic characterization of mutant proteins. Mutants which lack proteolytic activity unfold and refold in a highly reversible manner and will be used as vehicles for studying stability mutations by thermodynamic methods. A collection of over 100 mutant subtilisins has been accumulated over the past four years, of which about 20 are significantly more stable that the wild type subtilisin BPN'. Differential scanning calorimetry and denaturation in urea and guanidine- HCI will be used to study the unfolding of mutant subtilisins. The goal is to understand the consequences of specific amino acid changes on the folded, unfolded and transition states in the unfolding process. X-ray crystallography will be used to correlate structural changes in the folded protein with the free energy changes measured by thermodynamic experiments. Various strategies for protein stabilization including attempts to change hydrophobic and electrostatic interactions and decrease chain entropy of the unfolded enzyme will be examined. By studying mutants differing only slightly from the wild type protein, measuring the free energy changes resulting from the modification and correlating specific structural elements to the observed changes in free energy, the theoretical basis for predicting the energetic consequences of a mutation should be improved.

该项目的目标是研究蛋白质折叠和稳定性， 枯草杆菌蛋白酶BPN'和使用体外诱变，热力学分析，x- 射线晶体学和理论方法。 枯草杆菌蛋白酶系统是 选择它是因为它在遗传操作，表达， 突变蛋白的纯化和晶体学表征。 缺乏蛋白水解活性的突变体以高度折叠的方式展开和重折叠。 可逆的方式，并将用作研究稳定性的车辆 突变的热力学方法。 100多个变种人 在过去的四年中，枯草杆菌蛋白酶已经积累，其中大约 20的枯草杆菌蛋白酶BPN '的稳定性显著高于野生型枯草杆菌蛋白酶BPN'。 尿素和胍中的差示扫描量热法和变性- HCI将用于研究突变型枯草杆菌蛋白酶的解折叠。 目标是 为了了解特定氨基酸变化的后果， 展开过程中的折叠、展开和过渡状态。 X射线晶体学将用于关联晶体的结构变化 通过热力学方法测量折叠蛋白质的自由能变化 实验 用于蛋白质稳定化的各种策略，包括 试图改变疏水和静电相互作用， 将检查未折叠酶的链熵。 通过研究突变体 与野生型蛋白质仅略有不同，测量游离的 能量变化导致的修改和相关的具体 结构元素的观察到的自由能的变化，理论 预测突变的能量后果的基础应该是 提高