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 Citations (Scopus)

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

Fingerprint

Isotope Labeling
Bacteriorhodopsins
tyrosine
Fourier Transform Infrared Spectroscopy
Isotopes
Labeling
marking
Tyrosine
Spectrum Analysis
isotopes
Spectroscopy
proteins
spectroscopy
Proteins
mutagenesis
Mutagenesis
probes
Site-Directed Mutagenesis
amino acids
Fourier transforms

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

Cite this

Site-directed isotope labeling and FTIR spectroscopy : assignment of tyrosine bands in the bR → M difference spectrum of bacteriorhodopsin. / Liu, Xiao Mei; Sonar, Sanjay; Lee, Chan Ping; Coleman, Matthew A; RajBhandary, Uttam L.; Rothschild, Kenneth J.

In: Biophysical Chemistry, Vol. 56, No. 1-2, 1995, p. 63-70.

Research output: Contribution to journalArticle

Liu, Xiao Mei ; Sonar, Sanjay ; Lee, Chan Ping ; Coleman, Matthew A ; RajBhandary, Uttam L. ; Rothschild, Kenneth J. / Site-directed isotope labeling and FTIR spectroscopy : assignment of tyrosine bands in the bR → M difference spectrum of bacteriorhodopsin. In: Biophysical Chemistry. 1995 ; Vol. 56, No. 1-2. pp. 63-70.
@article{82103988a6d44eceb76a9e5c1ef714ad,
title = "Site-directed isotope labeling and FTIR spectroscopy: assignment of tyrosine bands in the bR → M difference spectrum of bacteriorhodopsin",
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.",
keywords = "Bacteriorhodopsin, Cell-free protein synthesis, FT-IR spectroscopy, Isotope labeling, M intermediate, Tyrosine",
author = "Liu, {Xiao Mei} and Sanjay Sonar and Lee, {Chan Ping} and Coleman, {Matthew A} and RajBhandary, {Uttam L.} and Rothschild, {Kenneth J.}",
year = "1995",
doi = "10.1016/0301-4622(95)00016-Q",
language = "English (US)",
volume = "56",
pages = "63--70",
journal = "Biophysical Chemistry",
issn = "0301-4622",
publisher = "Elsevier",
number = "1-2",

}

TY - JOUR

T1 - Site-directed isotope labeling and FTIR spectroscopy

T2 - assignment of tyrosine bands in the bR → M difference spectrum of bacteriorhodopsin

AU - Liu, Xiao Mei

AU - Sonar, Sanjay

AU - Lee, Chan Ping

AU - Coleman, Matthew A

AU - RajBhandary, Uttam L.

AU - Rothschild, Kenneth J.

PY - 1995

Y1 - 1995

N2 - 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.

AB - 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.

KW - Bacteriorhodopsin

KW - Cell-free protein synthesis

KW - FT-IR spectroscopy

KW - Isotope labeling

KW - M intermediate

KW - Tyrosine

UR - http://www.scopus.com/inward/record.url?scp=0029089393&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0029089393&partnerID=8YFLogxK

U2 - 10.1016/0301-4622(95)00016-Q

DO - 10.1016/0301-4622(95)00016-Q

M3 - Article

VL - 56

SP - 63

EP - 70

JO - Biophysical Chemistry

JF - Biophysical Chemistry

SN - 0301-4622

IS - 1-2

ER -