Learning About Protein Unfolded States From Heterodimeric Fragment Complementation

Cuny City College, New York NY

Investigators

Abstract

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.