Matthew Coleman

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.

DETECTION OF THREONINE STRUCTURAL CHANGES UPON FORMATION OF THE M-INTERMEDIATE OF BACTERIORHODOPSIN : EVIDENCE FOR ASSIGNMENT TO THR-89

The behavior of threonine residues in the bacteriorhodopsin (bR) photocycle has been investigated by Fourier transform infrared difference spectroscopy. L-Threonine labeled at the hydroxyl group with 18O (L-[3-(18)O]threonine) was incorporated into bR and the bR-->M FTIR difference spectra measured. Bands are assigned to threonine vibrational modes on the basis of 18O induced isotope frequency shifts and normal mode calculations. In the 3500 cm-1 region, a negative band is assigned to the OH stretch of threonine. In the 1125 cm-1 region, a negative band is assigned to a mixed CH3 rock/CO stretch mode. The frequency of both these bands indicates the presence of at least one hydrogen bonded threonine hydroxyl group in light adapted bR which undergoes a change in structure by formation of the M intermediate. Spectral changes induced by the substitution Thr-89-->Asn but not Thr-46-->Asn or Asp-96-->Asn are consistent with the assignment of these bands to Thr-89. These results along with another related study on the mutant Thr-89-->Asn indicate that the active site of bR includes Thr-89 and that its interaction with the retinylidene Schiff base and Asp-85 may play an important role in regulating the color of bacteriorhodopsin and the transfer of a proton to the Schiff base.

Site-directed isotope labeling of membrane proteins: A new tool for spectroscopists

Publisher Summary This chapter introduces a method for assigning bands in Fourier transform infrared (FTIR)-difference spectra based on a technique termed as site-directed isotope labeling (SDIL). The key element in SDIL is the use of a suppressor tRNA aminoacylated with an isotopically labeled amino acid. This tRNA is targeted to insert the isotopic amino acid at the proper position in the nascent protein by using an amber codon at the corresponding position in the gene. Cell-free synthesis (in vitro translation) and exogenous addition of the aminoacylated suppressor tRNA prevent aminoacylation of non-suppressor tRNAs with the isotopic amino acid, similar to the approach used for site-directed non-native amino acid replacement. The integral membrane protein bacteriorhodopsin (bR) was chosen as a model system for demonstrating the application of SDIL-FTIR. Studies on bacteriorhodopsin demonstrate that the FTIR-SDIL approach can probe the local environment and structural changes of specific residues and backbone carbonyl groups in a protein.