BIOCHEMISTRY AND BIOPHYSICS OF BPTI FOLDING MUTANTS

这项工作的广泛和长期目标是阐明规则， 控制蛋白质折叠。 牛胰蛋白酶抑制剂（BPTI）， 选择它作为模型系统，因为它是一种简单的球状多肽， 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蛋白酶抑制剂家族的成员，该家族包括 与人类生理学和疾病相关的几个成员，包括 脂蛋白相关凝血抑制剂和选择性剪接 阿尔茨海默氏症淀粉样β蛋白前体的结构域。 这项研究的总体基本目标是， 具有扰动折叠特性的BPTI的遗传修饰形式， 大量表达这些蛋白质，并对它们进行生物学表征。 该补助金的具体研究优先事项是： 1)通过制造BPTI解决“设计出”二硫键的问题 缺失两个或更多天然二硫键的突变体; 2)进行定量调查的所有稳定的相互作用， 通过系统地将每个非丙氨酸残基突变为丙氨酸来修饰分子， 表征所得突变体的稳定化自由能; 3)测试蛋白质折叠的“纸板箱”模型， 静电对5-55二硫键形成的贡献以及对 全局多肽稳定性; 4)促进合作者实验室正在进行的工作， 研究所选BPTI突变体的结构和动力学。 据推测，这种结合了遗传学和生物物理学的方法， 对一个非常简单的蛋白质模型的折叠机制的剖析， 阐明了蛋白质折叠的过程仍然知之甚少， 将军

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.