Denaturation of proteins. III. N.M.R. studies on lysozyme and α-lactalbumin

Abstract

The denaturation process is considered with particular reference to the application of N.M.R. spectroscopy for its study. All known examples of exchange between native and unfolded states are shown to be cases of slow exchange on the N.M.R. time scale. Furthermore, providing the denaturation is two-state, the fraction of unfolded molecules α can be obtained from measurements of the heights of various N.M.R. resonances, including those which are composites of several closely related types of protons. The experimentally measured parameter F does not equal α if intermediates are present or in the very unlikely event of the denaturation process occurring rapidly on the n.m.r, time scale. Multiple-state denaturations can be observed by the non-coincidence of curves of F (determined from different resonances) against denaturant parameter or from the occurrence of proton resonances which are only observed in the transition region.     Lysozyme is not denatured in 8M urea at pH > 3.0. At pH 2.8 the NH resonances of arginine sharpen before those of many other proton resonances in the molecule which sharpen simultaneously. It is concluded that three clusters of arginine side-chains, which involve seven arginine residues, unfold before the bulk of the molecule. Denaturation of α-lactalbumin in urea at pH 2.5 and pH 6.0 in D₂O occurs over a broad range of concentrations and is clearly multiple state at pH 6.0.     The N.M.R. study shows the presence of an exposed methionine residue in α-lactalbumin, no evidence of high field perturbed methyl resonances, and a considerably reduced stability to denaturation as compared with lysozyme. The dearth of aliphatic-aromatic interactions in α-lactalbumin may account for its more open structure compared with lysozyme as well as its reduced stability towards denaturation.