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Backbone dynamics of bacteriorhodopsin as studied by 13C solid-state NMR spectroscopy

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Abstract

The surface dynamics of bacteriorhodopsin was examined by measurements of site-specific 13C–1H dipolar couplings in [3-13C]Ala-labeled bacteriorhodopsin. Motions of slow or intermediate frequency (correlation time <50 µs) scale down 13C–1H dipolar couplings according to the motional amplitude. The two-dimensional dipolar and chemical shift (DIPSHIFT) correlation technique was utilized to obtain the dipolar coupling strength for each resolved peak in the 13C MAS solid-state NMR spectrum, providing the molecular order parameter of the respective site. In addition to the rotation of the Ala methyl group, which scales the dipolar coupling to 1/3 of the rigid limit value, fluctuations of the Cα–Cβ vector result in additional motional averaging. Typical order parameters measured for mobile sites in bacteriorhodopsin are between 0.25 and 0.29. These can be assigned to Ala103 of the C–D loop and Ala235 at the C-terminal α-helix protruded from the membrane surface, and Ala196 of the F–G loop, as well as to Ala228 and Ala233 of the C-terminal α-helix and Ala51 from the transmembrane α-helix. Such order parameters departing significantly from the value of 0.33 for rotating methyl groups are obviously direct evidence for the presence of fluctuation motions of the Ala Cα–Cβ vectors of intact preparations of fully hydrated, wild-type bacteriorhodopsin at ambient temperature. The order parameter for Ala160 from the expectantly more flexible E–F loop, however, is unavailable under highest-field NMR conditions, probably because increased chemical shift anisotropy together with intrinsic fluctuation motions result in an unresolved 13C NMR signal.

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Acknowledgements

The junior research group is funded by the Saxon State Ministry of Higher Education, Research and Culture. The work was supported by the Deutsche Forschungsgemeinschaft (Ar-195/8-1). This work was also supported in part by a grant-in-aid for Scientific Research from KAKENHI from MEXT of Japan.

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Authors and Affiliations

  1. Junior Research Group "Solid-state NMR Studies of the Structure of Membrane-associated Proteins", Biotechnological-Biomedical Center, University of Leipzig, Liebigstrasse 27, 04103 , Leipzig, Germany

    Daniel Huster

  2. Institute of Medical Physics and Biophysics, University of Leipzig, Liebigstrasse 27, 04103 , Leipzig, Germany

    Patrick Barré

  3. Department of Life Science, Himeji Institute of Technology, Harima Science Garden City, Kamigori, 678-1297, Hyogo , Japan

    Satoru Yamaguchi & Hazime Saitô

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  1. Patrick Barré
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  2. Satoru Yamaguchi
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  3. Hazime Saitô
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  4. Daniel Huster
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Correspondence to Hazime Saitô or Daniel Huster.

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Barré, P., Yamaguchi, S., Saitô, H. et al. Backbone dynamics of bacteriorhodopsin as studied by 13C solid-state NMR spectroscopy. Eur Biophys J 32, 578–584 (2003). https://doi.org/10.1007/s00249-003-0305-z

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  • DOI: https://doi.org/10.1007/s00249-003-0305-z

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