Get access
Advertisement

Characterizing the Structure and Photocycle of PR 2D Crystals with CD and FTIR Spectroscopy

Authors

  • Gabriela Schäfer,

    1. Institute of Biophysics, Johann Wolfgang Goethe-University Frankfurt, Frankfurt, Germany
    Search for more papers by this author
  • Sarika Shastri,

    1. Institute of Biophysical Chemistry and Center of Biomolecular Magnetic Resonance, Johann Wolfgang Goethe-University Frankfurt, Frankfurt, Germany
    Search for more papers by this author
  • Mirka-Kristin Verhoefen,

    1. Institute of Physical and Theoretical Chemistry, Johann Wolfgang Goethe-University Frankfurt, Frankfurt, Germany
    Search for more papers by this author
  • Vitali Vogel,

    1. Institute of Biophysics, Johann Wolfgang Goethe-University Frankfurt, Frankfurt, Germany
    Search for more papers by this author
  • Clemens Glaubitz,

    1. Institute of Biophysical Chemistry and Center of Biomolecular Magnetic Resonance, Johann Wolfgang Goethe-University Frankfurt, Frankfurt, Germany
    Search for more papers by this author
  • Josef Wachtveitl,

    1. Institute of Biophysics, Johann Wolfgang Goethe-University Frankfurt, Frankfurt, Germany
    2. Institute of Physical and Theoretical Chemistry, Johann Wolfgang Goethe-University Frankfurt, Frankfurt, Germany
    Search for more papers by this author
  • Werner Mäntele

    Corresponding author
    1. Institute of Biophysics, Johann Wolfgang Goethe-University Frankfurt, Frankfurt, Germany
      *Corresponding author email: maentele@biophysik.uni-frankfurt.de (Werner Mäntele)
    Search for more papers by this author

  • This paper is part of the Proceedings of the 13th International Conference on Retinal Proteins, Barcelona, Spain, 15–19 June 2008.

*Corresponding author email: maentele@biophysik.uni-frankfurt.de (Werner Mäntele)

Abstract

We present here a study on proteorhodopsin (PR) 2D crystals with analytical ultracentrifugation, circular dichroism and Fourier transform infrared (FTIR) spectroscopy. The aim of our experiments was to test the activity of 2D crystal sample preparations and to gain further insight in PR structure, stability and function with these techniques. Our results demonstrate higher stability compared to detergent-solubilized or reconstituted samples. For different pH values, low pH 2D crystals tend to form bigger aggregates and are less stable than at basic pH. The pH 9 sample shows a sharp phase transition during heat denaturation and there is also evidence for protein–protein interaction due to the close proximity of the proteins in the 2D crystals. In the FTIR measurements at cryogenic temperatures (77 K), we characterized the first step in the PR photocycle. At pH 9, the K intermediate could be observed and the samples showed no orientation effects. At pH 5, we could trap the K/L intermediate, characterized by its negative IR signal at 1741 cm−1. In rapid-scan FTIR experiments, we could also identify the M intermediate of the photocycle at basic pH. We conclude that the PR 2D crystals exhibit a fully functional photocycle and are therefore well suited for further studies on the proton transport mechanism of PR.

Get access to the full text of this article

Ancillary