THE METHANOL-INDUCED TRANSITION AND THE EXPANDED HELICAL CONFORMATIONIN HEN LYSOZYME

Methanol-induced conformational transitions of hen egg white lysozyme were investigated with a combined use of far- and near-UV CD and NMR s pectroscopies, ANS binding and small-angle X-ray scattering. Addition of methanol induced no global change in the native conformation itself , but induced a transition from the native state to the denatured stat e which was highly cooperative, as shown by the coincidence of transit ion curves monitored by the far- and near-UV CD spectroscopy, by isodi chroic points in the far- and near-UV CD spectra and by the concomitan t disappearance of individual H-1 NMR signals of the native state. The ANS binding experiments could detect no intermediate conformer simila r to the molten globule state in the process of the methanol denaturat ion. However, at high concentration of methanol, e.g., 60% (v/v) metha nol/water, a highly helical state (H) was realized. The H state had a helical content much higher than the native state, monitored by far-UV CD spectroscopy, and had no specific tertiary structure, monitored bo th by near-UV CD and NMR spectroscopy. The radius of gyration in the H state, 24.9 Angstrom, was significantly larger than that in the nativ e state (15.7 Angstrom). The Kratky plot for the H state did not show a clear peak and was quite similar to that for the urea-denatured stat e, indicating a complete lack of globularity. Thus we conclude that th e H state has a considerably expanded, flexible broken rod-like confor mation which is clearly distinguishable from the ''molten globule'' st ate. The stability of both N and H states depends on pH and methanol c oncentration. Thus a phase diagram involving N and H was constructed.