Learning About Protein Unfolded States From Heterodimeric Fragment Complementation

这是对氧化大肠杆菌硫氧还蛋白(TRX)的一系列互补片段及其在不同界面附近的异源二聚体重组的研究。从本质上非结构蛋白质片段的DSC研究中估计的埋藏面的值将与残基水平上的构象偏好和骨架刚性的核磁共振研究相关联。该项目结合了三个专业领域(蛋白质片段互补、蛋白质量热法以及蛋白质结构和动力学的核磁共振分析)，并以两个主要假设为中心：(1)组成天然2和4链区域的分离的TRX片段之间具有实质性的非局部相互作用，但本质上仍然是非结构的。(2)不同异源二聚体重组的折叠/结合增量G的差异主要是由于分离片段的折叠程度和刚性不同所致。为了检验这些假设，将确定一系列互补片段的折叠/结合的增量G以及初始和最终状态的低分辨率结构表征。将对选定的分离片段和异二聚体重组进行高灵敏度的DSC研究，并将对选定的互补片段的初始态和终态的结构进行核磁共振研究，以研究这种选定对在两种状态下的动力学。这些研究的长期目标是通过实验和理论之间的协同作用来表征蛋白质未折叠状态的构象空间，并了解与结合相关的一系列亲和力和折叠程度的蛋白质-蛋白质相互作用。

This is a study of a family of complementary fragments from oxidized E. coli thioredoxin (Trx) and their heterodimeric reassemblies around different interfaces. The values of estimated buried surface from DSC studies of intrinsically unstructured protein fragments will be correlated with NMR studies of conformational preferences and rigidity of the backbone at the residue level. This project combines three areas of expertise (protein fragment complementation, calorimetry of proteins, and NMR analysis of structure and dynamics of proteins) and centers on two main hypotheses: (1) Isolated Trx fragments which comprise the regions of the native 2 and 4 strands have substantial nonlocal interactions between them, while remaining intrinsically unstructured. (2) The differences in delta G of folding/binding among the different heterodimeric reassemblies are mainly due to differences in the degree of folding and rigidity in the isolated fragments. In order to test these hypotheses, the delta G of folding/binding and low-resolution structural characterization of the initial and final states for a family of complementary fragments will be determined. High sensitivity DSC studies of selected isolated fragments and heterodimeric reassemblies will be undertaken, and NMR studies of the structures of the initial and final states of selected complementary fragments will be done to study the dynamics of such a selected pair in both states. The long-term objective of these studies is to characterize the conformational space of the unfolded state of proteins and understand protein-protein interactions covering a range of affinities and degree of folding associated with binding through a synergistic interaction between experimenation and theory.