posters 5th Asia-Pacific NMR Symposium 2013

Site-Specific Incorporation of 19F Labled Unnatural Amino Acids for in site Membrane Protein NMR Analysis and Phosphorylation Quantification (#168)

PAN SHI 1 , DONG LI 1 , SANLING LIU 2 , Changlin Tian 1
  1. University of Science and Technology of China, Hefei, ANHUI, China
  2. High Magnetic Field Laboratory, Chinese Academy of Sciences, Hefei, Anhui, China

19F labeled Unnatural amino acids provided a mechanism for protein specific and site-specific isotope labeling in bacterial cells. Here, 19F-trifluoromethyl- phenylalanine was incorporated to different sites of diacylglycerol kinase for further in situ NMR analysis, without protein purification. Differences of 19F chemical shift and T1 relaxation values of 19F-tfmF-DAGK in native E. coli membrane versus in detergent micelles were observed, indicating strong influences of hydrophobic environment to membrane proteins. 19F-Tyrosine was incorporated to E. coli tyrosine kinase and acquired 19F NMR signals illustrate small portion of phosphorylation of the kinase, in the presence of ATP and Mg2+ ions.