Thermodynamic hierarchy and local energetics of folded proteins.

The cooperativity of local fluctuations within the folded state of a protein is demonstrated based on applying the "multiple-probe principle" to the hydrogen exchange (HX) rates of a set of amide protons of the protein. A hierarchical thermodynamic analysis is presented for the protein energetics, which includes both global two-state folding-unfolding transition and local cooperative fluctuations within the folded state. The analysis dissects the local fluctuations from the global unfolding; both lead to the HX but from the folded and unfolded states, respectively. It provides the newly observed HX behavior of proteins in mild denaturant, namely solvent and temperature dependent HX rates from the folded state, with a quantitative interpretation in terms of a set of local energetic parameters. These local energetic parameters contain much information concerning the structures and stabilities of native proteins.