Site-directed isotope labeling and FTIR spectroscopy: assignment of tyrosine bands in the bR-->M difference spectrum of bacteriorhodopsin.
Fourier transform infrared difference spectroscopy has been used extensively to probe structural changes in bacteriorthodopsin 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.
Liu
X M
XM
Physics Department and Molecular Biophysics Laboratory, Boston University, MA 02215, USA.
Sonar
S
S
Lee
C P
CP
Coleman
M
M
RajBhandary
U L
UL
Rothschild
K J
KJ
D000595:Amino Acid Sequence; D001436:Bacteriorhodopsins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D003001:Cloning, Molecular; D003903:Deuterium; D004926:Escherichia coli; D015870:Gene Expression; D007553:Isotope Labeling / Q000379:methods; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D014443:Tyrosine
D011994:Recombinant Proteins; D014443:Tyrosine; D001436:Bacteriorhodopsins; D003903:Deuterium