Sanjay M. Sonar

TIME‐RESOLVED FOURIER TRANSFORM INFRARED SPECTROSCOPY OF THE BACTERIORHODOPSIN MUTANT TYR‐185→E: ASP‐96 REPROTONATES DURING O FORMATION; ASP‐85 AND ASP‐212 DEPROTONATE DURING O DECAY

Abstract— The protonation state of key aspartic acid residues in the O intermediate of bacteriorhodopsin (bR) has been investigated by time‐resolved Fourier transform infrared (FTIR) difference spectroscopy and site‐directed mutagenesis. In an earlier study (Bouschéet al., J. Biol Chem. 266, 11063–11067, 1991) we found that Asp‐96 undergoes a deprotonation during the M→N transition, confirming its role as a proton donor in the reprotonation pathway leading from the cytoplasm to the Schiff base. In addition, both Asp‐85 and Asp‐212, which protonate upon formation of the M intermediate, remain protonated in the N intermediate. In this study, we have utilized the mutant Tyr‐185→Phe (Y185F), which at high pH and salt concentrations exhibits a photocycle similar to wild type bR but has a much slower decay of the O intermediate. Y185F was expressed in native Halobacterium halobium and isolated as intact purple membrane fragments. Time‐resolved FTIR difference spectra and visible difference spectra of this mutant were measured from hydrated multilayer films. A normal N intermediate in the photocycle of Y185F was identified on the basis of characteristic chromophore and protein vibrational bands. As N decays, bands characteristic of the all‐trans O chromophore appear in the time‐resolved FTIR difference spectra in the same time range as the appearance of a red‐shifted photocycle intermediate absorbing near 640 nm. Based on our previous assignment of the carboxyl stretch bands to the four membrane embedded Asp groups: Asp‐85, Asp‐96, Asp‐115 and Asp‐212, we conclude that during O formation: (i) Asp‐96 undergoes reprotonation. (ii) Asp‐85 may undergo a small change in environment but remains protonated. (iii) Asp‐212 remains partially protonated. In addition, reisomerization of the chromophore during the N→O transition is accompanied by a major reversal of protein conformational changes which occurred during the earlier steps in the photocycle. These results are discussed in terms of a proposed mechanism for proton transport.

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.

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.