A correlation between the loss of hydrophobic core packing interactions and protein stability

The hydrophobic core packing in four-α-helical bundles appears to be crucial for stabilizing the protein structure. To examine the structural basis of hydrophobic stabilization, the crystal structures of the Leu→Val (L41V) and Leu→Ala (L41A) substitutions of the core residue Leu41 of the ROP protein have been determined. Both substitutions are destabilizing and lead to formation of cavities. The main responses to mutations are the collapse of the central part of the α-helix containing the site of mutation, shifts of internal water molecules, and in L41A, the trapping of a water molecule in a cavity engineered by the mutation. For both mutants, these effects limit the increase in cavity size to less than 10 ų, while an increase of 37 ų and 100 ų is expected for L41V and L41A, respectively, in the absence of any cavity size reducing effects. The mobility of internal side-chains is increased and in L41A, it reaches values typical for exposed residues. A parameter (Δn(h)) is introduced as a measure of the number of van der Waals contacts lost. For ROP, barnase and T4 lysozyme mutants, there is a good correlation between Δn(h), and the free energy of unfolding ΔΔG relative to wild-type protein. The Δn(h) value turns out to be more suitable for analysing structural and energetic responses to mutation than other parameter, such as cavity volumes and packing densities. Possible evolutionary implications of the ΔΔG versus Δn(h) relationship are discussed.