Imprints of function on the folding landscape: functional role for an intermediate in a conserved eukaryotic binding protein

In the computational characterization of single domain protein folding, the effective free energies of numerous microstates are projected onto few collective degrees of freedom that in turn serve as well-defined reaction coordinates. In this regard, one-dimensional (1D) free energy profiles are widely used mainly for their simplicity. Since folding and functional landscapes are interlinked, how well can these reduced representations capture the structural and dynamic features of functional states while being simultaneously consistent with experimental observables? We investigate this issue by characterizing the folding of the four-helix bundle bovine acyl-CoA binding protein (bACBP), which exhibits complex equilibrium and kinetic behaviours, employing an Ising-like statistical mechanical model and molecular simulations. We show that the features of the 1D free energy profile are sufficient to quantitatively reproduce multiple experimental observations including millisecond chevron-like kinetics and temperature dependence, a microsecond fast phase, barrier heights, unfolded state movements, the intermediate structure and average f-values. Importantly, we find that the structural features of the native-like intermediate (partial disorder in helix 1) are intricately linked to a unique interplay between packing and electrostatics in this domain. By comparison with available experimental data, we propose that this intermediate determines the promiscuous functional behaviour of bACBP that exhibits broad substrate specificity. Our results present evidence to the possibility of employing the statistical mechanical model and the resulting 1D free energy profile to not just understand folding mechanisms but to even extract features of functionally relevant states and their energetic origins.