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Journal of Biochemistry Advance Access originally published online on July 23, 2007
Journal of Biochemistry 2007 142(3):325-333; doi:10.1093/jb/mvm135
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© 2007 The Japanese Biochemical Society.

Structural Change of Bacteriorhodopsin in the Purple Membrane above pH 10 Decreases Heterogeneity of the Irreversible Photobleaching Components

Yasunori Yokoyama1,*, Masashi Sonoyama1, Tatsuhiko Nakano2 and Shigeki Mitaku1

1Department of Applied Physics, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8603, Japan; and 2Scientific Instruments Division, Thermo Fisher Scientific K.K., C-2F, 3-9 Moriya-cho, Kanagawa-ku, Yokohama, Kanagawa 221-0022, Japan

*To whom correspondence should be addressed. Tel: +81-52-789-4465, Fax: +81-52-789-4465, E-mail: yokoyama{at}nuap.nagoya-u.ac.jp


  Abstract

Kinetic investigations of irreversible photobleaching of bacteriorhodopsin (bR) in purple membrane (PM) at high temperature have previously shown two kinds of bR species upon light illumination. The bR species consist of kinetically fast- and slow-denatured components, whose proportions were dependent upon structural changes in dark, as shown by CD. In order to elucidate electrostatic contribution on the heterogeneous stability and the bR structure in PM, photobleaching behaviour and structural changes over a wide pH range were investigated by kinetics as well as various spectroscopic techniques. Kinetics revealed that photobleaching below pH 9 obeyed double-exponential functions, whereas measurements above pH 10 were characterized by a single-decay component. FT-IR deconvoluted spectra showed a {alpha}II-to-{alpha}I transition in the transmembrane helices around pH 10. Near-IR Raman scattering spectra demonstrated the equilibrium shift of retinal isomers from all trans to 13-cis form. Near-UV CD spectra suggested configurational changes in the aromatic residues around the retinal pocket. An exciton-to-positive transition in visible CD spectrum was observed. This indicates disorganization in the 2D-crystalline lattice of PM, which occurred concomitantly with the changes above pH 10. A model for the changes in kinetic behaviour and molecular structure around pH 10 is discussed, focusing on changes in charge distribution upon alkalinization.

Key Words: denaturation kinetics, intermolecular interaction, irreversible photobleaching, retinal chromophore, transmembrane helix

Abbreviations: bR, bacteriorhodopsin; PM, purple membrane; PRG, proton release group; 2D, two-dimensional


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