BIOCHEMISTRY AND BIOPHYSICS OF BPTI FOLDING MUTANTS

BPTI 折叠突变体的生物化学和生物物理学

• 批准号: 3122364
• 负责人: STEPHEN P ANDERSON
• 金额: $ 13.45万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-01 至 1994-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3122364
• 关键词: Alzheimer's disease; X ray crystallography; amyloid proteins; bioenergetics; cystine; disulfide bond; globular protein; ionic bond; mutant; nuclear magnetic resonance spectroscopy; pancreatic polypeptide; protease inhibitor; protein folding; protein structure; thiols; trypsin inhibitors

The broad, long-term objective of this work is to elucidate the rules that govern protein folding. Bovine pancreatic trypsin inhibitor (BPTI) was chosen as a model system because it is a simple globular polypeptide and a large amount of structural, biophysical, and theoretical data is already available for the wild type protein. BPTI is also the best understood member of the Kunitz class of protease inhibitors, a family that includes several members relevant to human physiology and disease including the lipoprotein-associated coagulation inhibitor and the alternatively-spliced domain of the Alzheimer's amyloid beta protein precursor. The overall basic thrust of the research has been to make genetically-modified forms of BPTI that have perturbed folding properties, express these proteins in quantity, and characterize them biophysically. The specific research priorities for this grant are: 1) Address the problem of "designing out" disulfide bonds by making BPTI mutants missing two or more of the native disulfides; 2) Conduct a quantitative survey of all stabilizing interactions in the molecule by systematically mutating each non-alanine residue to alanine and characterizing the stabilization free energies of the resulting mutants; 3) Test the "cardboard box" model of protein folding by measuring the contribution of electrostatics to 5-55 disulfide bond formation and to global polypeptide stability; 4) Facilitate ongoing work in collaborators' laboratories aimed at investigating the structure and dynamics of selected BPTI mutants. Presumably, this combined genetic and biophysical approach to the dissection of the folding mechanism of a very simple model protein will illuminate the still poorly-understood process of protein folding in general.

这项工作的广泛、长期目标是阐明以下规则： 控制蛋白质折叠。 牛胰蛋白酶抑制剂（BPTI） 选择作为模型系统是因为它是一种简单的球状多肽和 大量的结构、生物物理和理论数据已经 可用于野生型蛋白质。 BPTI也是最好理解的 Kunitz 类蛋白酶抑制剂的成员，该家族包括 一些与人类生理学和疾病相关的成员，包括 脂蛋白相关凝血抑制剂及其可变剪接 阿尔茨海默病淀粉样β蛋白前体的结构域。 该研究的总体基本主旨是 具有扰动折叠特性的 BPTI 基因修饰形式， 大量表达这些蛋白质，并对其进行生物物理表征。 该资助的具体研究重点是： 1）通过制作BPTI解决“设计出”二硫键的问题 缺失两个或多个天然二硫化物的突变体； 2) 对所有稳定相互作用进行定量调查 通过系统地将每个非丙氨酸残基突变为丙氨酸来形成分子 表征所得突变体的稳定自由能； 3) 通过测量蛋白质折叠的“纸板箱”模型 静电对 5-55 二硫键形成的贡献 整体多肽稳定性； 4) 促进合作者实验室正在进行的工作，旨在 研究所选 BPTI 突变体的结构和动力学。 据推测，这种结合遗传和生物物理的方法 剖析一个非常简单的模型蛋白质的折叠机制将 阐明目前仍知之甚少的蛋白质折叠过程 一般的。