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，长期目标是阐明规则 控制蛋白质折叠。 牛胰腺胰蛋白酶抑制剂（BPTI）为 选择为模型系统，因为它是一个简单的球状多肽和 大量的结构，生物物理和理论数据已经存在 可用于野生型蛋白质。 BPTI也是最好的理解 Kunitz类蛋白酶抑制剂的成员，包括 与人类生理学和疾病有关的几个成员，包括 脂蛋白相关凝血抑制剂和替代抑制剂 阿尔茨海默氏症的淀粉样β蛋白前体的域。 该研究的总体基本目的是使 具有干扰折叠特性的遗传修饰的BPTI形式， 在数量上表达这些蛋白质，并以生物物理为特征。 该赠款的具体研究重点是： 1）通过制造BPTI解决“设计”二硫键的问题 突变体缺少两个或多个本地二硫化物； 2）对所有稳定相互作用进行定量调查 通过系统地突变到丙氨酸的每个非丙氨酸残基和 表征所得突变体的稳定自由能； 3）测量蛋白质折叠的“纸板箱”模型 静电对5-55个二硫键形成的贡献和 全球多肽稳定性； 4）促进针对合作者实验室的正在进行的工作 研究选定的BPTI突变体的结构和动力学。 据推测，这种结合的遗传和生物物理方法 非常简单模型蛋白的折叠机理的解剖会将 阐明蛋白质折叠的过程仍然很差 一般的。