Yawen Bai

Future directions in folding: The multi‐state nature of protein structure

All possible protein folding intermediates exist in equilibrium with the native protein at native as well as non‐native conditions, with occupation determined by their free energy level. The study of these forms can illuminate the fundamental principles of protein structure and folding. Hydrogen exchange methods can be used to detect and characterize these partially unfolded forms at native conditions and as a function of mild denaturant and temperature. This information illuminates the requirements that govern the ability of kinetic and equilibrium methods to study folding intermediates.

[15] Thermodynamic parameters from hydrogen exchange measurements

Just as exchangeable hydrogens that are controlled by global unfolding can be used to measure thermodynamic parameters at a global level, hydrogens that are exposed to exchange by local unfolding reactions may be used to obtain locally resolved energy parameters. Results with the hemoglobin system demonstrate the ability of HX methods to locate functionally important changes in a protein and to measure the energetic contribution of each. These results offer the promise that HX measurements may be used to delineate, in terms of definable bonds and their energies and interactions, the network of interactions that Hb and other proteins use to produce their various functions.

The Cooperative Substructure of Protein Molecules

A new hydrogen exchange method makes it possible to study the unfolded state of a protein and its partially unfolded forms under native conditions. The hydrogen bonded groups that are exposed in each state, identified by NMR-detected hydrogen exchange measurements, define the structure of each intermediate. The free energy level of each state can be obtained from the measured hydrogen exchange rates. Initial results with cytochrome c depict four structural units which together account for the entire protein structure. The intermediate forms reveal the cooperative substructure of the protein and appear to represent the major kinetic intermediates in the protein folding pathway.