9419059 Rothschild The central goal of this project is the elucidation of the molecular mechanism of light-driven proton transport in bacteriorhodopsin (bR). In earlier work, it was demonstrated that Fourier transform infrared (FTIR) difference spectroscopy can provide detailed information about the protonation state, local environment and orientation of specific amino acid residues which are structurally active during the bR photocycle. However, a key problem limiting future progress is the assignment of FTIR bands to individual groups in a protein. While site-directed mutagenesis (SDM) has been successfully applied in special cases for this purpose, its use is limited due to perturbations induced in the structure and function of the protein as illustrated by recent studies on the mutant Y185F. In addition, SDM does not permit the assignment of bands to the vibrations of specific peptide groups, an essential next step for investigating protein backbone conformational changes such as have been detected between the M and N intermediates of bR. A general method of band assignment has now been developed based on site-directed isotope labeling (SDIL). SDIL analogs of bR are produced by cell-free synthesis using specially aminoacylated suppressor tRNAs. These SDIL-bR analogs have identical properties as native bR and are suitable for FTIR-difference spectroscopy. In the first application of this approach, bands have been assigned to the vibrations of specific tyrosine side-chains as well as to individual groups in the bR backbone structure. In this research, SDIL-FTIR will be used to investigate systematically the role of specific residues in bR proton pumping . Our experiments will be guided by earlier studies which have provided information about specific residues which undergo protonation changes, may participate in a hypothesized proton wire, act to couple the retinal chromophore to the protein and are involved in secondary structural changes of the protei n. Recently developed methods for time-resolved FTIR-difference spectroscopy, polarized FTIR and attenuated total reflection FTIR will also be used in this research. In collaboration with the J. Spudich laboratory, the SDIL-FTIR approach will be extended to sensory-rhodopsin I (sRI), a phototaxis light-receptor in Halobacteria salinarium. %%% The primary goal of this project is understanding how bacteriorhodopsin functions as a light-driven proton pump. Bacteriorhodopsin serves as a prototype for other membrane proteins which carry out essential process in cells including ion transport and energy transduction. A promising new approach for studying how membrane proteins function is FTIR-difference spectroscopy. When combined with biochemical and genetic methods it has the power to provide information about the protonation state, local environment and even orientation of specific groups in a protein. Unlike solid-state NMR, infrared spectroscopy can also probe rapid dynamic conformational changes in proteins. In this project, FTIR- difference spectroscopy will be combined with three methods: i) uniform isotope labeling of specific amino acids; ii) site-directed mutagenesis (SDM) and site-directed isotope labeling (SDIL) to investigate bacteriorhodopsin. If successful, these studies will provide a detailed picture of how bacteriorhodopsin functions and provide clues to how other membrane proteins work. ***
