Approaches for the measurement of solvent exposure in proteins by 19F NMR.
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.
SourceAvailable from: Anna Maria Rydzik[Show abstract] [Hide abstract]
ABSTRACT: The New Delhi metallo-β-lactamase (NDM-1) is involved in the emerging antibiotic resistance problem. Development of metallo-β-lactamases (MBLs) inhibitors has proven challenging, due to their conformational flexibility. Here we report site-selective labeling of NDM-1 with 1,1,1-trifluoro-3-bromo acetone (BFA), and its use to study binding events and conformational changes upon ligand-metal binding using (19) F NMR spectroscopy. The results demonstrate different modes of binding of known NDM-1 inhibitors, including L- and D-captopril by monitoring the changing chemical environment of the active-site loop of NDM-1. The method described will be applicable to other MBLs and more generally to monitoring ligand-induced conformational changes.Angewandte Chemie International Edition 03/2014; DOI:10.1002/anie.201310866 · 11.34 Impact Factor
Clinical Biochemistry 09/2011; 44(13). DOI:10.1016/j.clinbiochem.2011.08.672 · 2.23 Impact Factor
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ABSTRACT: While many proteins are recognized to undergo folding via an intermediate, the microscopic nature of folding intermediates is less understood. In this study, 19F NMR and near UV circular dichroism (CD) are used to characterize a transition to a thermal folding intermediate of calmodulin, a water-soluble protein, which is biosynthetically enriched with 3-fluorophenylalanine (3F-Phe). 19F NMR solvent isotope shifts, resulting from replacing H2O with D2O, and paramagnetic shifts arising from dissolved O2 are used to monitor changes in water accessibility and hydrophobicity of the protein interior, as the protein progresses from a native state to an unfolded state, along a heat denaturation pathway. In comparison to the native state, solvent isotope shifts reveal the decreased presence of water in the hydrophobic core while paramagnetic shifts show increased hydrophobicity of this folding intermediate. 15N,1H and methyl 13C,1H HSQC NMR spectra identify that this folding intermediate retains a near-native tertiary structure, whose hydrophobic interior is highly dynamic. 19F NMR CPMG relaxation dispersion measurements suggest the near-native state is transiently adopted well below the temperature associated with its onset.Biochemistry 08/2013; 52(34). DOI:10.1021/bi4010057 · 3.38 Impact Factor