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

Abstract

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.

Anatomy of protein structures: Visualizing how a one-dimensional protein chain folds into a three-dimensional shape

“…Consistently, there is a local minimum fragment 48-105 with a score of 2.94 in the complete list of 20 local minima of the fragment map (not shown). Fifth, the 111-120 peptide populates a nonnative structured conformation, in rapid exchange with a random coil state in aqueous solution (26). This further supports fragment 106-123 (with a score of Ϫ9.10 as compared with the score of fragment 3-38, Ϫ0.53) as being stabilized in its native conformation by associating with fragment 3-38.…”

Local and Global Cooperativity in the Human α-Lactalbumin Molten Globule

“…36 -38 Peptides encompassing residues 101-111 and 101-120 of the a-LA sequence do show some degree of helicity in aqueous solution but the latter appears to be largely non-native in character. 39,40 A peptide consisting of residues 1 -38 and 95 -120 cross-linked by the C28 -C111 disulfide bond, however, shows not only significant helical structure but also characteristics of a molten globule including the enhancement of ANS fluorescence. 41 Deletion of residues 95 -100 has little effect on the structure in this peptide, indicating that the C-helix is not important for the stability of its molten globule structure.…”

A protein dissection study demonstrates that two specific hydrophobic clusters play a key role in stabilizing the core structure of the molten globule state of human ?-lactalbumin

“…We have used a protein dissection approach to examine the role‐specific regions of the protein chain play in stabilizing structure within the α‐domain of human α‐lactalbumin 11–14. The α‐domain contains 4 α‐helices denoted A through D as well as a 3 10 helix.…”

“…It is of interest that 1 H NMR studies of two peptides consisting of residues 101–111 and 111–120 of αLA have shown that these fragments have significant propensity to form remarkably well‐defined, locally stabilized, non‐native structure. The structure is localized within residues 101–105 and residues 115–12011, 14, 15 and is stabilized by non‐native clusters of hydrophobic residues. Most of the contacts between the C‐terminal portion of the protein and the B‐helix are formed by residues contained within these two clusters.…”