¹⁹F NMR Studies of a Desolvated Near-Native Protein Folding Intermediate

Abstract

Although 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 the 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, the solvent isotope shifts reveal the decreased presence of water in the hydrophobic core, whereas the paramagnetic shifts show the increased hydrophobicity of this folding intermediate. ¹⁵N, ¹H and methyl ¹³C,¹H HSQC NMR spectra demonstrate 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.

19F NMR Reveals Multiple Conformations at the Dimer Interface of the Nonstructural Protein 1 Effector Domain from Influenza A Virus

“…In the context of the broader 19 F protein NMR literature, these dynamical results on the NS1A ED dimer interface constitute a novel application of 19 F NMR relaxation to directly probe slow motion dynamics within a protein:protein interface The NMR properties of 19 F have, have been exploited recently to illuminate slow motion conformational dynamic processes in other types of interactions including protein-ligand interactions (Ahmed et al, 2007), peptide-bicelleinteractions (Buer et al, 2010), changes in dynamics accompanying protein complex formation (Acchione et al, 2012), and the thermodynamic and kinetic aspects of domain swapping (Liu et al, 2012). In addition, 19 F CPMG relaxation approaches were recently employed to characterize the conformational exchange behavior of fluorinated Phe residues in the thermal folding intermediate of calmodulin (Kitevski-Leblanc et al, 2013). …”

Glycosaminoglycan Binding and Non-Endocytic Membrane Translocation of Cell-Permeable Octaarginine Monitored by Real-Time In-Cell NMR Spectroscopy

“…Labeling of R8 with 4CF 3 -Phe is found to be an effective method to detect peptide uptake to cells with minimal perturbation [23]. In addition, 19 F NMR spectroscopy enables us to make a quantitative (concentration) analysis relevant to the molecular dynamics of biological interest without perturbing the system [33,34,35,36]. Here we observe the direct membrane translocation of 19 F-R8 at 4 °C, the temperature low enough to assure no endocytic pathway of the cellular uptake [37].…”

A suite of 19F based relaxation dispersion experiments to assess biomolecular motions

“…Recently, fluorine ( 19 F) NMR has regained attraction (Hellmich et al 2009;Liu et al 2012;Kim et al 2013;Kitevski-Leblanc et al 2013;Aramini et al 2014;Hoang and Prosser 2014;Manglik et al 2015;Matei and Gronenborn 2016;Lu et al 2019;Huang et al 2020). This spin is absent from virtually all biomolecules, however, 19 F probes can be artificially introduced into proteins by incorporation of fluorinated amino acids (Crowley et al 2012) or through post-translational modification with fluorine-containing tags (Brauer and Sykes 1986).…”