Probing the non-covalent forces key to the thermodynamics of β-hairpin unfolding.

Although it is well understood that the graph of the free energy of unfolding (Δ G ) of a globular protein with temperature approximates to a negative parabola, there is as yet no link between this global (G) Δ G G ( T ) function and the individual structural elements-residue type and the non-covalent forces between groups-contributing to it. As such, there is little understanding of how each structural element contributes to the globally assessed changes of enthalpy (Δ H G ), entropy (Δ S G ), and heat capacity (Δ C p(G) ) of unfolding calculated from the Δ G G ( T ) function. To address this situation, we consider here an alternative approach to examining fold stability. Specifically, we examine the local (L) reporting of the thermodynamics of unfolding provided by each residue. By using 1 H NMR spectroscopy to monitor the response of the individual mainchain amide N-H groups of β-hairpin peptides with temperature, we generate local Δ G L ( T ) functions, using these to calculate the local enthalpy (Δ H L ), entropy (Δ S L ), and heat capacity (Δ C p(L) ) of unfolding. Mapping the thermodynamic changes in this way, for specific point-mutations, provides new information about how specific residues, non-covalent forces, and secondary structure type, contribute to folding. This type of information provides new details of the factors contributing to the typically measured global Δ G G ( T ) function.