The Stabilization Mechanism of the Intermediate Structure of Equine Beta-Lactoglobulin

Abstract

A single-disulfide mutant of equine β-lactoglobulin, C66A/C160A, forms an expanded and helical conformation at an acidic pH and a low anion concentration (C state). The C state is a model of an early folding intermediate of βlactoglobulin. The secondary structures in the C state are in the regions corresponding to F, G, H strands and the major α-helix, and they assume the native-like or nonnative helices. Peptides encompassing those helices did not form stable helices. A longer fragment, CHIBL, which encompasses the structured region in the C states, has a helical structure similar to the corresponding region of the full-length protein in the C state. This result indicates that non-local interactions responsible for the helix formation in the C state reside in the sequence of CHIBL. The disulfide bond, Cys106-Cys119, links two nonnative helices in the C state. This is one of the possible long-range interactions stabilizing the helical structures. The CD spectrum of disulfide-reduced C66A/C160A shows decreased helical content. This indicates that the helices are stabilized by this disulfide bond. The detailed structural analysis of the reduced C66A/C160A by using proline mutants suggests that the nonnative helix in the G strand region can form without the disulfide bond despite Cys106 is contained in that region. It also suggests that the disulfide bond is crucial to the formation of the nonnative helix in the H strand region. We synthesized a peptide encompassing two helices formed in the C state. The GssH peptide is disulfide-linked G-peptide and H-peptide. This peptide did not form stable helical structures. Therefore G and H strands and major helix regions must interact with each other simultaneously to form stable helices in the C state.