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

Citations Over TimeTop 19% of 2013 papers

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.

Related Papers

• → Unification of the Folding Mechanisms of Non-two-state and Two-state Proteins(2004)88 cited
• → Shape of the Free Energy Barriers for Protein Folding Probed by Multiple Perturbation Analysis(2006)13 cited
• → Folding kinetics of Che Y mutants with enhanced native α-helix propensities 1 1Edited by A. R. Fersht(1997)62 cited
• → Unification of the Folding Mechanisms of Non-two-state and Two-state Proteins(2004)4 cited
• → Statistical Analysis of the Relationship between the Folding Rate and Structure-Based Parameters of Globular Proteins(2006)