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Related Experiment Videos

Conformational changes in bacteriorhodopsin studied by infrared attenuated total reflection.

H Marrero1, K J Rothschild

  • 1Department of Physics, Boston University, Massachusetts 02215.

Biophysical Journal
|October 1, 1987
PubMed
Summary

Fourier transform infrared-difference spectroscopy using attenuated total reflection (ATR-FTIR) reveals new insights into bacteriorhodopsin

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Area of Science:

  • Biophysics
  • Spectroscopy
  • Membrane Protein Dynamics

Background:

  • Bacteriorhodopsin's photocycle involves significant conformational changes.
  • Previous infrared (IR) spectroscopic studies used transmittance methods.
  • Understanding these changes is crucial for deciphering protein function.

Purpose of the Study:

  • To introduce and validate a new attenuated total reflection Fourier transform infrared (ATR-FTIR)-difference spectroscopy method.
  • To investigate conformational changes in bacteriorhodopsin during its photocycle.
  • To compare ATR-FTIR results with traditional transmittance FTIR spectroscopy.

Main Methods:

  • Utilized attenuated total reflection (ATR) with Fourier transform infrared (FTIR)-difference spectroscopy.

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  • Immobilized purple membrane on a germanium crystal in a variable pH buffer.
  • Analyzed amide I and II absorbance using polarized IR light.

    • Main Results:

    • ATR-FTIR spectra of the light-to-dark transition differed from transmittance spectra, attributed to out-of-plane transition moments.
    • Altering pH (6.8 to 8.0) in La3+ substituted membranes slowed M-decay, enabling room temperature difference spectra.
    • Observed spectral changes at room temperature, not seen at -23°C, indicate localized protein backbone conformational changes in bacteriorhodopsin.

    Conclusions:

    • ATR-FTIR spectroscopy provides a valuable, complementary approach to transmittance FTIR for studying membrane protein dynamics.
    • The method reveals orientation-dependent spectral features and conformational changes.
    • Localized protein backbone conformational changes occur in bacteriorhodopsin during its photocycle, influenced by pH.