The compact and expanded denatured conformations of apomyoglobin in the methanol-water solvent

We have performed a detailed study of methanol-induced conformational trans itions of horse heart apomyoglobin (apoMb) to investigate the existence of the compact and expanded denatured states. A combination of far- and near u ltraviolet circular dichroism, NMR spectroscopy, and small-angle X-ray scat tering (SAXS) was used, allowing a phase diagram to be constructed as a fun ction of pH and the methanol concentration. The phase diagram contains four conformational states, the native (N), acid-denatured (U-A), compact denat ured (I-M), and expanded helical denatured (H) states, and indicates that t he compact denatured state (I-M) is stable under relatively mild denaturing conditions, whereas the expanded denatured states (U-A and H) are realized under extreme conditions of pH (strong electric repulsion) or alcohol conc entration (weak hydrophobic interaction). The results of this study, togeth er with many previous studies in the literature, indicate the general exist ence of the compact denatured states not only in the salt-pH plane but also in the alcohol-pH plane. Furthermore, to determine the general feature of the H conformation we used several proteins including ubiquitin, ribonuclea se A, alpha-lactalbumin, beta-lactoglobulin, and Streptomyces subtilisin in hibitor (SSI) in addition to apoMb. SAXS studies of these proteins in 60% m ethanol showed that the H states of these all proteins have expanded and no nglobular conformations. The qualitative agreement of the experimental data with computer-simulated Kratky profiles also supports this structural feat ure of the H state.