title: Site-specific solvent exposure analysis of a membrane protein using unnatural amino acids and 19F nuclear magnetic resonance.
abstract: Membrane proteins play an essential role in cellular metabolism, transportation and signal transduction across cell membranes. The scarcity of membrane protein structures has thus far prevented a full understanding of their molecular mechanisms. Preliminary topology studies and residue solvent exposure analysis have the potential to provide valuable information on membrane proteins of unknown structure. Here, a (19)F-containing unnatural amino acid (trimethylfluoro-phenylalanine, tfmF) was applied to accomplish site-specific (19)F spin incorporation at different sites in diacylglycerol kinase (DAGK, an Escherichia coli membrane protein) for site-specific solvent exposure analysis. Due to isotope effect on (19)F spins, a standard curve for (19)F-tfmF chemical shifts was drawn for varying solvent H(2)O/D(2)O ratios. Further site-specific (19)F solvent isotope shift analysis was conducted for DAGK to distinguish residues in water-soluble loops, interfacial areas or hydrophobic membrane regions. This site-specific solvent exposure analysis method could be applied for further topological analysis of other membrane proteins.
authors.affiliation: Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, PR China.
authors.forename: Pan
authors.initials: P
authors.lastname: Shi
authors.forename: Dong
authors.initials: D
authors.lastname: Li
authors.forename: Hongwei
authors.initials: H
authors.lastname: Chen
authors.forename: Ying
authors.initials: Y
authors.lastname: Xiong
authors.forename: Changlin
authors.initials: C
authors.lastname: Tian
mesh_terms: D000596:Amino Acids / Q000737:chemistry*; D017666:Deuterium Oxide / Q000737:chemistry; D019852:Diacylglycerol Kinase / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology; D005462:Fluorine Radioisotopes / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000737:chemistry; D011487:Protein Conformation; D012997:Solvents / Q000737:chemistry
chemical_list: D000596:Amino Acids; D005462:Fluorine Radioisotopes; D008565:Membrane Proteins; D012997:Solvents; D010649:Phenylalanine; D019852:Diacylglycerol Kinase; D017666:Deuterium Oxide