FTIR difference spectroscopy can provide detailed information about changes in the hydrogen bonding, protonation state and orientation of specific chemical groups in a membrane protein with sub- microsecond time resolution. However, this technique is limited by the absence of a general method for placing isotope labels at specific positions in a protein. During the initial phase of this project, a new approach for labeling membrane proteins, termed site-directed isotope labeling (SDIL), was demonstrated. This approach is based on expressing a protein in an in vitro reaction mixture containing a suppressor tRNA, aminoacylated with isotopically labeled amino acid. In the first application, SDIL analogs of bacteriorhodopsin (bR) were produced containing 2H and 13C labels selectively incorporated into both specific tyrosine residues and backbone peptide carbonyl groups. FTIR analysis of these SDIL analogs led to the identification of structurally active groups which may be involved in proton transport and the coupling of chromophobe isomerization to protein conformational changes. Recently, scaled-up procedures for the low-cost production of SDIL analogs has been demonstrated, opening the door for solid-state NMR studies. The Investigator proposes to continue the development of the SDIL approach so that it can be routinely used along with both FTIR and NMR spectroscopy to investigate membrane proteins. New methods will be developed for: i) enzymatic and chemical charging of suppressor tRNAs; ii) in vitro expression, isolation, refolding and reconstitution of a variety of nascent proteins and iii) the routine production of SDIL analogs for solid-state NMR analysis. These methods will first be applied to bacteriorhodosin, in order to assign FTIR difference bands arising from the structural changes of individual residues during the bR photocycle. The SDIL approach will be also applied to sensor ry rhodopsin I (SRI), a receptor protein involved in phototaxis. In addition to an increased understanding of how these membrane proteins function, the proposed research will have an important impact in other areas of biophysics and biology, including enzyme catalysis and protein folding.
