Conformational studies of peptides spanning the helical sequence in the molten globule α-lactalbumin

Abstract

Elucidation of the mechanism of globular protein folding is a major issue in structural biology, and recent progress in the experimental studies on protein folding has improved understanding of transient structural intermediates along the folding pathway. Recent excellent techniques have shown the presence of the intermediate at an early stage of folding, which has the native-like secondary structure in the same regions as those in the native molecule and the destroyed specific tertiary structure. Also, these intermediates have been shown to be identical with the molten globule (MG) state observed for some proteins such as -lactalbumin ( -LA) as a compact equilibrium unfolding intermediate. Therefore studies on the MG state of -LA are important for understanding protein folding [I]. The native αLA is composed of fourα-helices (A,B,C,and D) and aβ-sheet. Recently it has been shown that in the MG state a native-like backbone topology is partially retained (at least the native Band C -helices), and a nonnative hydrophobic core is formed in a region located between the Band Chelices in the native state. Therefore, to get further information on stabilization of the MG state, the conformational properties of the peptides that encompass the helices and the hydrophobic core region of -LA should be investigated in detail. We synthesized four peptides, B 14(22-33, C 17(84100), C24(84-107), and H7( 101-107), which correspond to the Band C-helices, the C-helix combined with the hydrophobic core, and only the core region, respectively, in bovine LA [2]. Also, to study the folding mechanism of -LA in vivo, interactions of these peptides with a molecular chaperone, GroEL, were studied. The chaperone accelerates in vivo protein folding and inhibits misfolding. It binds to the MG proteins, but the binding mechanism is not yet clear.