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.

Solvent-accessibility of discrete residue positions in the polypeptide hormone glucagon by 19F-NMR observation of 4-fluorophenylalanine

“…1a). This is consistent with previous reports on other systems (Hansen et al 1985; Kitevski-LeBlanc et al 2009; Shi et al 2011). …”

“…The solvent isotope effect on the 19 F chemical shift at variable D 2 O concentrations (Hansen et al 1985; Kitevski-LeBlanc et al 2009; Shi et al 2011) was used to investigate site-specific solvent accessibility of glucagon (Fig. 1).…”

Protein 19F NMR in Escherichia coli

“…The broad protein resonances observed in the cell slurry narrow on lysis, but the width at half height for the broadest resonance is still >0.5 ppm. The observation of CAM crosspeaks43,44 in the HSQC spectrum of the lysate (Figure 6F) proves that detectable amounts of the protein are present. The HSQC spectrum of the lysate also shows that CAM is not fully Ca 2+ loaded 43…”

“…The sharp resonance is from free 3FY. The other two, one on either side of the 3FY resonance, are from the protein 43. The HSQC spectrum from the slurry (Figure 6B) shows only metabolite signals 38,39 Figure 6C and 6D.…”

Paramagnetic relaxation enhancement for protein-observed 19F NMR as an enabling approach for efficient fragment screening

“…6–11 The application of paramagnetic additives to 19 F NMR has also been described in the context of NMR with molecular oxygen. 12 However, the specialized NMR tubes required would not be ideal for routine small molecule screening. These studies did not explore if the beneficial effects of paramagnetic metal additives are retained in the presence of bound ligands of varying affinity, a question we examine herein.…”