Solution structure of a peptide model of a region important for the folding of alpha-lactalbumin provides evidence for the formation of nonnative structure in the denatured state.

Elucidating the properties of the denatured state of proteins under conditions relevant for their folding is a key factor in understanding the folding process. We show that a peptide corresponding to residues 111-120 of human alpha-lactalbumin has a pronounced propensity to adopt nonnative structure in aqueous solution. Two-dimensional NMR provides evidence for a structured, nonnative conformation in fast exchange with a random coil ensemble. A total of 78 Rotating Frame Overhauser Effects (ROEs) were used to calculate the conformation of the structured population. A nonnative cluster of hydrophobic residues involving the side chains of K114, W118, Ll119, and A120 is observed, which helps to stabilize a turn-like conformation in the vicinity of residues 115-118. The structure in 30% (vol/vol) TFE was also calculated. Interestingly, the addition of TFE did not simply amplify the population of the structured conformer observed in H2O, but instead induced a new conformation. The implications for the folding of the intact protein are discussed. We also discuss the implications of this study for the relevance of the use of mixed TFE/H2O solvent systems to study isolated peptides.