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

Published online by Cambridge University Press:  01 April 1999

YUJI O. KAMATARI ,
SHOKO OHJI ,
TAKASHI KONNO ,
YASUTAKA SEKI ,
KUNITSUGU SODA ,
MIKIO KATAOKA  and
KAZUYUKI AKASAKA
YUJI O. KAMATARI
Affiliation:
The Graduate School of Science and Technology, Kobe University, Kobe 657-8501, Japan Oxford Centre for Molecular Sciences, University of Oxford, Oxford OX1 3QT, United Kingdom
SHOKO OHJI
Affiliation:
The Graduate School of Science and Technology, Kobe University, Kobe 657-8501, Japan
TAKASHI KONNO
Affiliation:
National Institute for Physiological Science, Myodaiji, Okazaki 444-8585, Japan
YASUTAKA SEKI
Affiliation:
Department of Bioengineering, Faculty of Engineering, Nagaoka University of Technology, Nagaoka 940-2188, Japan
KUNITSUGU SODA
Affiliation:
Department of Bioengineering, Faculty of Engineering, Nagaoka University of Technology, Nagaoka 940-2188, Japan
MIKIO KATAOKA
Affiliation:
Graduate School of Materials Science, Nara Institute of Science and Technology, Ikoma 630-0101, Japan
KAZUYUKI AKASAKA
Affiliation:
The Graduate School of Science and Technology, Kobe University, Kobe 657-8501, Japan Department of Chemistry, Faculty of Science, Kobe University, Kobe 657-8501, Japan
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Abstract

We have performed a detailed study of methanol-induced conformational transitions of horse heart apomyoglobin (apoMb) to investigate the existence of the compact and expanded denatured states. A combination of far- and near-ultraviolet circular dichroism, NMR spectroscopy, and small-angle X-ray scattering (SAXS) was used, allowing a phase diagram to be constructed as a function of pH and the methanol concentration. The phase diagram contains four conformational states, the native (N), acid-denatured (UA), compact denatured (IM), and expanded helical denatured (H) states, and indicates that the compact denatured state (IM) is stable under relatively mild denaturing conditions, whereas the expanded denatured states (UA and H) are realized under extreme conditions of pH (strong electric repulsion) or alcohol concentration (weak hydrophobic interaction). The results of this study, together with many previous studies in the literature, indicate the general existence 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, ribonuclease A, α-lactalbumin, β-lactoglobulin, and Streptomyces subtilisin inhibitor (SSI) in addition to apoMb. SAXS studies of these proteins in 60% methanol showed that the H states of these all proteins have expanded and nonglobular conformations. The qualitative agreement of the experimental data with computer-simulated Kratky profiles also supports this structural feature of the H state.

Keywords

apomyoglobinexpanded helical denatured statemethanol-induced conformational transitionmolten globule statesmall-angle X-ray scattering
Type
Research Article
Information
Protein Science , Volume 8 , Issue 4 , April 1999 , pp. 873 - 882
Copyright
© 1999 The Protein Society

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