The SIR provided L-[4- 13 ClAspartic Acid; 2g L-[1,5_13C 2]Glutamic Acid; 0.5g FTIR difference spectroscopy can be used to probe conformational changes at the level of individual amino acid residues in a membrane protein. However, a key problem limiting progress is the absence of a general method for the incorporation of isotope labels at specific positions in a protein. Until recently this was not possible except through chemical synthesis, which is generally limited to polypeptides of 50 residues. We have recently introduced a new method which circumvents this limitation. Termed site-directed isotope labeling (SDIL), it is based on the use of suppressor tRNAs and in vitro synthesis. In the case of bacteriorhodopsin, the light- driven proton pump, we have used this method to label several tyrosine and peptide carbonyl positions. Combined with FrIR-difference spectroscopy, this has allowed us to pinpoint structural activity in Tyr residue 185 which may serve as a hinge for conformational changes in that protein. Our goal is now to extend these methods so that they can be used to label routinely any residue in bacteriorhodopsin; can be applied to other proteins and can be used in conjunction with biophysical techniques such as solid state NMR, neutron diffraction and FTIR. For this purpose new methods will be developed for both enzymatic and chemical arninoacylation of suppressor tRNAs to optimize in vitro expression, isolation and refolding and to characterize SDIL proteins spectroscopically. An extensive set of SDIL bacteriorhodopsin analogs will be produced including labels placed in Asp, Glu, Pro, Thr, Ser and Trp positions. These studies will directly lead to a detailed picture of how this protein functions. Similar research will be conducted on sensory rhodopsin I, the phototactic receptor in Halobacterium. salinarium.
| Martinez, Rodolfo A; Glass, David R; Ortiz, Erick G et al. (2014) Synthesis of isotopically labeled 1,3-dithiane. J Labelled Comp Radiopharm 57:338-41 |
| Martinez, Rodolfo A; Glass, David R; Ortiz, Erick G et al. (2013) Large-scale preparation of (13) C-labeled 2-(phenylthio)acetic acid and the corresponding labeled sulfoxides and sulfones. J Labelled Comp Radiopharm 56:31-5 |
| Creager, Melinda S; Jenkins, Janelle E; Thagard-Yeaman, Leigh A et al. (2010) Solid-state NMR comparison of various spiders' dragline silk fiber. Biomacromolecules 11:2039-43 |
| Jenkins, Janelle E; Creager, Melinda S; Lewis, Randolph V et al. (2010) Quantitative Correlation between the protein primary sequences and secondary structures in spider dragline silks. Biomacromolecules 11:192-200 |
| Kim, Sun Hee; Aznar, Constantino; Brynda, Marcin et al. (2004) An EPR, ESEEM, structural NMR, and DFT study of a synthetic model for the covalently ring-linked tyrosine-histidine structure in the heme-copper oxidases. J Am Chem Soc 126:2328-38 |
| Ollivault-Shiflett, Morgane; Kimball, David B; Silks, L A Pete (2004) Synthesis of chiral 13C,77Se-labeled selones. J Org Chem 69:5150-2 |
| Schmidt, Bryan; Hillier, Warwick; McCracken, John et al. (2004) The use of stable isotopes and spectroscopy to investigate the energy transducing function of cytochrome c oxidase. Biochim Biophys Acta 1655:248-55 |
| Van Dien, Stephen J; Strovas, Tim; Lidstrom, Mary E (2003) Quantification of central metabolic fluxes in the facultative methylotroph methylobacterium extorquens AM1 using 13C-label tracing and mass spectrometry. Biotechnol Bioeng 84:45-55 |
| Shiflett, Patrick R; Taylor-McCabe, Kirsten J; Michalczyk, Ryszard et al. (2003) Structural studies on the hairpins at the 3' untranslated region of an anthrax toxin gene. Biochemistry 42:6078-89 |
| Nilsson, Bradley L; Hondal, Robert J; Soellner, Matthew B et al. (2003) Protein assembly by orthogonal chemical ligation methods. J Am Chem Soc 125:5268-9 |
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