Thermodynamics of protein unfolding: questions pertinent to testing the validity of the two-state model.

We discuss a number of questions pertaining to the analysis of data to extract thermodynamic parameters for the reversible unfolding of proteins. Simulations are presented to illustrate problems in trying to test the validity of the two-state model, vis-a-vis a more complicated unfolding model. A conceptual and practical problem is how to consider the unfolded state and how to relate the observed signal to this state. We discuss the idea that the unfolded state can be described as a single macrostate, comprising a distribution of microstates having different degrees of solvent-accessible surface area. We also discuss the possibilities and thermodynamic consequences of having more than one unfolded state and of having a denaturant which both stabilizes and destabilizes the protein's native state.