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

Abstract

Although it is well understood that the graph of the free energy of unfolding (ΔG) of a globular protein with temperature approximates to a negative parabola, there is as yet no link between this global (G) ΔG_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 (ΔH_G), entropy (ΔS_G), and heat capacity (ΔC_p(G)) of unfolding calculated from the ΔG_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 ¹H NMR spectroscopy to monitor the response of the individual mainchain amide N–H groups of β-hairpin peptides with temperature, we generate local ΔG_L(T) functions, using these to calculate the local enthalpy (ΔH_L), entropy (ΔS_L), and heat capacity (ΔC_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 ΔG_G(T) function.