Dissecting C−H∙∙∙π and N−H∙∙∙π Interactions in Two Proteins Using a Combined Experimental and Computational Approach

C−H∙∙∙π and N−H∙∙∙π interactions can have an important contribution for protein stability. However, direct measurements of these interactions in proteins are rarely reported. In this work, we combined the mutant cycle experiments and molecular dynamics (MD) simulations to characterize C−H∙∙∙π and N−H∙∙∙π interactions and their cooperativity in two model proteins. It is shown that the average C−H∙∙∙π interaction per residue pair is ~ −0.5 kcal/mol while the N−H∙∙∙π interaction is slightly stronger. The triple mutant box measurement indicates that N−H∙∙∙π∙∙∙C−H∙∙∙π and C−H∙∙∙π∙∙∙C−H∙∙∙π can have a positive or negative cooperativity. MD simulations suggest that the cooperativity, depending on the local environment of the interactions, mainly arises from the geometric rearrangement when the nearby interaction is perturbed.