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

Although many proteins are recognized to undergo folding via an intermediate, the microscopic nature of folding intermediates is less understood. In this study, F-19 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). F-19 NMR solvent isotope shifts, resulting from replacing H2O with D2O, and paramagnetic shifts arising from dissolved O-2 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 M the hydrophobic core, whereas the paramagnetic shifts show the increased hydrophobicity of this folding intermediate. N-15,H-1 and methyl C-13,H-1 HSQC NMR spectra demonstrate that this folding intermediate retains a near-native, tertiary structure whose hydrophobic interior is highly dynamic F-19 NMR CPMG relaxation dispersion measurements suggest the near-native state is transiently adopted well below the temperature associated with its onset.