PROTEIN FOLDING AND STABILITY OF SUBTILISIN

本项目的目标是研究蛋白质折叠和稳定性。 枯草杆菌毒素BPN‘和使用体外诱变，热力学分析，x- 射线结晶学和理论方法。枯草杆菌菌素系统是 选择它是因为它在基因操作、表达、 突变蛋白的纯化及结晶学性质研究。 缺乏蛋白分解活性的突变体在高度的 可逆方式，并将用作研究稳定性的工具 用热力学方法进行突变。100多个变种人的集合 枯草杆菌毒素在过去四年中积累，其中约 20的稳定性明显高于野生型枯草杆菌毒素BPn‘。 差示扫描量热法研究尿素和胍的变性 HCI将被用于研究突变的枯草杆菌毒素的展开。我们的目标是 为了了解特定氨基酸变化对人体健康的影响 展开过程中的折叠、展开和过渡态。 X射线结晶学将被用来关联结构变化 用热力学方法测定折叠蛋白质的自由能变化 实验。蛋白质稳定的各种策略包括 尝试改变疏水和静电相互作用并减少 将检测未折叠的酶的链熵。通过研究突变体 与野生型蛋白仅略有不同，测量游离 由修饰引起的能量变化以及与特定的 结构元素对观察到的自由能的变化，理论上 预测突变的能量后果的基础应该是 改进了。

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.