Structures and relative free energies of partially folded states of proteins

The ability of proteins to fold to well defined compact structures is one of the most remarkable examples of the effect of natural selection on biological molecules. To understand their properties, including the stability, the mechanism of folding, and the possibilities of misfolding and association, it is necessary to know the protein free energy landscape. We use NMR data as restraints in a Monte Carlo sampling procedure to determine the ensemble of structures populated by human α-lactalbumin in the presence of increasing concentrations of urea. The ensembles of structures that represent the partially folded states of the protein show that two structural cores, corresponding to portions of the α and β domains of the native protein, are preserved even when the native-like interactions that define their existence are substantially weakened. Analysis of the network of residual contacts reveals the presence of a complex interface region between the two structural cores and indicates that the development of specific interactions within this interface is the key step in achieving the native structure. The relative probabilities of the conformations determined from the NMR data are used to construct a coarse-grained free energy landscape for α-lactalbumin in the absence of urea. The form of the landscape, together with the existence of distinct cores, supports the concept that robustness and modularity are the properties that make possible the folding of complex proteins.