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 19F-containing unnatural amino acid (trimethylfluoro-phenylalanine, tfmF) was applied to accomplish site-specific 19F 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 19F spins, a standard curve for 19F-tfmF chemical shifts was drawn for varying solvent H2O/D2O ratios. Further site-specific 19F 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.
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