Approaches for the measurement of solvent exposure in proteins by 19F NMR.

Department of Chemistry, University of Toronto, UTM, 3359 Mississauga Rd, North Mississauga, ON, L5L 1C6, Canada.
Journal of Biomolecular NMR (Impact Factor: 2.85). 09/2009; 45(3):255-64. DOI: 10.1007/s10858-009-9359-2
Source: PubMed

ABSTRACT Fluorine NMR is a useful tool to probe protein folding, conformation and local topology owing to the sensitivity of the chemical shift to the local electrostatic environment. As an example we make use of (19)F NMR and 3-fluorotyrosine to evaluate the conformation and topology of the tyrosine residues (Tyr-99 and Tyr-138) within the EF-hand motif of the C-terminal domain of calmodulin (CaM) in both the calcium-loaded and calcium-free states. We critically compare approaches to assess topology and solvent exposure via solvent isotope shifts, (19)F spin-lattice relaxation rates, (1)H-(19)F nuclear Overhauser effects, and paramagnetic shifts and relaxation rates from dissolved oxygen. Both the solvent isotope shifts and paramagnetic shifts from dissolved oxygen sensitively reflect solvent exposed surface areas.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Proper positioning of membrane proteins in the host membrane is often critical to successful protein function. While hydrophobic considerations play a dominant role in determining the topology of a protein in the membrane, amphiphilic residues, such as tryptophan, may 'anchor' the protein near the water-membrane interface. The SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) family of membrane proteins mediates intracellular membrane fusion. Correct positioning of the SNAREs is necessary if fusion is to occur. Synaptobrevins are integral vesicle membrane proteins that are well conserved across species. Interestingly, mammalian Synaptobrevins typically contain two adjacent tryptophans near the water-membrane interface whereas the Drosophila, neuronal-Synaptobrevin (n-Syb), contains a single tryptophan in this same region. To explore the role of these tryptophan residues in membrane positioning, we prepared a peptide containing residues 75-121 of D. melanogaster n-Syb in DPC micelles, biosynthetically labeled with 4-fluorophenylalanine and 5-fluorotryptophan for the examination by (19)F NMR spectroscopy. Mutations of this construct containing zero and two tryptophan residues near the water-membrane interface resulted in changes in the positioning of n-Syb in the micelle. Moreover, the addition of a second tryptophan appears to slow dynamic motions of n-Syb near the micelle-water interface. These data therefore indicate that juxtamembrane tryptophan residues are important determinants of the position of Synaptobrevin in the membrane.
    Biochimica et Biophysica Acta 07/2012; 1818(12):2994-9. · 4.66 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: In protein NMR experiments which employ nonnative labeling, incomplete enrichment is often associated with inhomogeneous line broadening due to the presence of multiple labeled species. We investigate the merits of fractional enrichment strategies using a monofluorinated phenylalanine species, where resolution is dramatically improved over that achieved by complete enrichment. In NMR studies of calmodulin, a 148 residue calcium binding protein, ¹⁹F and ¹H-¹⁵N HSQC spectra reveal a significant extent of line broadening and the appearance of minor conformers in the presence of complete (>95%) 3-fluorophenylalanine labeling. The effects of varying levels of enrichment of 3-fluorophenylalanine (i.e. between 3 and >95%) were further studied by ¹⁹F and ¹H-¹⁵N HSQC spectra, ¹⁵N T(1) and T(2) relaxation measurements, ¹⁹F T(2) relaxation, translational diffusion and heat denaturation experiments via circular dichroism. Our results show that while several properties, including translational diffusion and thermal stability show little variation between non-fluorinated and >95% ¹⁹F labeled samples, ¹⁹F and ¹H-¹⁵N HSQC spectra show significant improvements in line widths and resolution at or below 76% enrichment. Moreover, high levels of fluorination (>80%) appear to increase protein disorder as evidenced by backbone ¹⁵N dynamics. In this study, reasonable signal to noise can be achieved between 60-76% ¹⁹F enrichment, without any detectable perturbations from labeling.
    Journal of Biomolecular NMR 10/2010; 48(2):113-21. · 2.85 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Fluorine ((19) F) NMR is a valuable tool for studying dynamic biological processes. However, increasing the sensitivity of fluorinated reporter molecules is a key to reducing acquisition times and accessing transient biological interactions. Here, we evaluate the utility a novel amino acid, l-O-(perfluoro-t-butyl)-homoserine (pFtBSer), that can easily be synthesized and incorporated into peptides and provides greatly enhanced sensitivity over currently used (19) F biomolecular NMR probes. Incorporation of pFtBSer into the potent antimicrobial peptide MSI-78 results in a sharp (19) F NMR singlet that can be readily detected at concentrations of 5 µm and lower. We demonstrate that pFtBSer incorporation into MSI-78 provides a sensitive tool to study binding through (19) F NMR chemical shift and nuclear relaxation changes. These results establish future potential for pFtBSer to be incorporated into various proteins where NMR signal sensitivity is paramount, such as in-cell investigations. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.
    Journal of Peptide Science 03/2013; · 2.07 Impact Factor