Site-directed isotope labeling and FTIR spectroscopy: assignment of tyrosine bands in the bR → M difference spectrum of bacteriorhodopsin

Xiao Mei Liu, Sanjay Sonar, Chan Ping Lee, Matthew A Coleman, Uttam L. RajBhandary, Kenneth J. Rothschild

Research output: Contribution to journalArticle

23 Scopus citations

Abstract

Fourier transform infrared difference spectroscopy has been used extensively to probe structural changes in bacteriorhodopsin and other retinal proteins. However, the absence of a general method to assign bands to individual chemical groups in a protein has limited the application of this technique. While site-directed mutagenesis has been successful in special cases for such assignments, in general, this approach induces perturbations in the structure and function of the protein, thereby preventing unambiguous band assignments. A new approach has recently been reported (Sonar et al., Nature Struct. Biol., 1 (1994) 512-517) which involves cell-free expression of bacteriorhodopsin and site-directed isotope labeling (SDIL). We have now used this method to re-examine bands assigned in the bR → M difference spectrum to tyrosine residues. Our results show that out of 11 tyrosines in bR, only Tyr 185 is structurally active. This work further demonstrates the power of SDIL and FTIR to probe conformational changes at the level of individual amino acid residues in proteins.

Original languageEnglish (US)
Pages (from-to)63-70
Number of pages8
JournalBiophysical Chemistry
Volume56
Issue number1-2
DOIs
StatePublished - 1995
Externally publishedYes

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Keywords

  • Bacteriorhodopsin
  • Cell-free protein synthesis
  • FT-IR spectroscopy
  • Isotope labeling
  • M intermediate
  • Tyrosine

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Organic Chemistry
  • Physical and Theoretical Chemistry

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