Site-specific 19 F NMR chemical shift and side chain relaxation analysis of a membrane protein labeled with an unnatural amino acid

National Laboratory for Physical Science at Microscale and School of Life Science, University of Science and Technology of China, Hefei, Anhui, People's Republic of China.
Protein Science (Impact Factor: 2.85). 01/2011; 20(1):224-8. DOI: 10.1002/pro.545
Source: PubMed


Site-specific ¹⁹F chemical shift and side chain relaxation analysis can be applied on large size proteins. Here, one-dimensional ¹⁹F spectra and T₁, T₂ relaxation data were acquired on a SH3 domain in aqueous buffer containing 60% glycerol, and a nine-transmembrane helices membrane protein diacyl-glycerol kinase (DAGK) in dodecyl phosphochoine (DPC) micelles. The high quality of the data indicates that this method can be applied to site-specifically analyze side chain internal mobility of membrane proteins or large size proteins.

Download full-text


Available from: Xi Zhaoyong
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Posttranslational modifications modulate the activities of most eukaryotic proteins and play a critical role in all aspects of cellular life. Understanding functional roles of these modifications requires homogeneously modified proteins that are usually difficult to purify from their natural sources. An emerging powerful tool for synthesis of proteins with defined posttranslational modifications is to genetically encode modified amino acids in living cells and incorporate them directly into proteins during the protein translation process. Using this approach, homogenous proteins with tyrosine sulfation, tyrosine phosphorylation mimics, tyrosine nitration, lysine acetylation, lysine methylation, and ubiquitination have been synthesized in large quantities. In this review, we provide a brief introduction to protein posttranslational modifications and the genetic noncanonical amino acid (NAA) incorporation technique, then discuss successful applications of the genetic NAA incorporation approach to produce proteins with defined modifications, and end with challenges and ongoing methodology developments for synthesis of proteins with other modifications.
    Full-text · Article · Nov 2010 · Molecular BioSystems
  • [Show abstract] [Hide abstract]
    ABSTRACT: Signals between a cell and its environment are often transmitted through membrane proteins; therefore, many membrane proteins, including G protein-coupled receptors (GPCRs) and ion channels, are important drug targets. Structural information about membrane proteins remains limited owing to challenges in protein expression, purification and the selection of membrane-mimicking systems that will retain protein structure and function. This review describes recent advances in solution NMR applied to the structural study of integral membrane proteins. The examples herein demonstrate that solution NMR spectroscopy will play a unique role not only in structural analysis, but also drug discovery of membrane proteins.
    No preview · Article · Jun 2011 · Current opinion in chemical biology
  • [Show abstract] [Hide abstract]
    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.
    No preview · Article · Sep 2011 · Biochemical and Biophysical Research Communications
Show more