Clusters of isoleucine, leucine, and valine side chains define cores of stability in high-energy states of globular proteins: Sequence determinants of structure and stability.

Measurements of protection against exchange of main chain amide hydrogens (NH) with solvent hydrogens in globular proteins have provided remarkable insights into the structures of rare high-energy states that populate their folding free-energy surfaces. Lacking, however, has been a unifying theory that rationalizes these high-energy states in terms of the structures and sequences of their resident proteins. The Branched Aliphatic Side Chain (BASiC) hypothesis has been developed to explain the observed patterns of protection in a pair of TIM barrel proteins. This hypothesis supposes that the side chains of isoleucine, leucine, and valine (ILV) residues often form large hydrophobic clusters that very effectively impede the penetration of water to their underlying hydrogen bond networks and, thereby, enhance the protection against solvent exchange. The linkage between the secondary and tertiary structures enables these ILV clusters to serve as cores of stability in high-energy partially folded states. Statistically significant correlations between the locations of large ILV clusters in native conformations and strong protection against exchange for a variety of motifs reported in the literature support the generality of the BASiC hypothesis. The results also illustrate the necessity to elaborate this simple hypothesis to account for the roles of adjacent hydrocarbon moieties in defining stability cores of partially folded states along folding reaction coordinates.