Non-Native α-Helices in the Initial Folding Intermediate Facilitate the Ordered Assembly of the ß-Barrel in ß-Lactoglobulin.

ABSTRACT

The roles of non-native α-helices frequently observed in the initial folding stage of ß-sheet proteins have been examined for many years. We herein investigated the residue-level structures of several mutants of bovine ß-lactoglobulin (ßLG) in quenched-flow pH-pulse labeling experiments. ßLG assumes a collapsed intermediate with a non-native α-helical structure (I0) in the early stage of folding, although its native form is predominantly composed of ß-structures. The protection profile in I0 of pseudo-wild type (WT*) ßLG was found to deviate from the pattern of the "average area buried upon folding" (AABUF). In particular, the level of protection at the region of strand A, at which non-native α-helices form in the I0 state, was significantly low compared to AABUF. G17E, the mutant with an increased helical propensity, showed a similar protection pattern. In contrast, the protection pattern for I0 of E44L, the mutant with an increased ß-sheet propensity, was distinct from that of WT* and resembled the AABUF pattern. Transverse relaxation measurements demonstrated that the positions of the residual structures in the unfolded states of these mutants were consistent with those of the protected residues in the respective I0 states. On the basis of the slower conversion of I0 to the native state for E44L to that for WT*, non-native α-helices facilitate the ordered assembly of the ß-barrel by preventing interactions that trap folding.