Native-like beta-hairpin retained in the cold-denatured state of bovine beta-lactoglobulin

Bovine P-lactoglobulin is denatured by increased temperature (heat denatura tion) and by decreased temperature (cold-denaturation) in the presence of 4 M urea at pH 2.5. We characterized the structure of the cold-denatured sta te of beta -lactoglobulin using circular dichroism (CD), small-angle X-ray scattering (SAXS) and heteronuclear nuclear magnetic resonance (NMR). CD an d SAXS indicated that the cold-denatured state, in comparison with the high ly denatured state induced by urea, is rather compact, retaining some secon dary structure, but no tertiary structure. The location of the residual str uctures in the cold-denatured state and their stability were characterized by H-1/H-2 exchange combined with heteronuclear NMR. The results indicated that the residues adjacent to the disulfide bond (C106-C119) connecting bet a -strands G and H had markedly high protection factors, suggesting the pre sence of a native-like beta -hairpin stabilized by the disulfide bond. Sinc e this beta -hairpin is conserved between different conformational states, including the kinetic refolding intermediate, it should be of paramount imp ortance for the folding and stability of beta -lactoglobulin. On the other hand, the non-native a-helix suggested for the folding intermediate was not detected in the cold-denatured state. The H-1/H-2 exchange experiments sho wed that the protection factors of a mixture of the native and cold-denatur ed states is strongly biased by that of the labile cold-denatured state, co nsistent with a two-process model of the exchange. (C) 2001 Academic Press.