19F NMR studies of a desolvated near-native protein folding intermediate

摘要

Although many proteins are recognized to undergo folding via an intermediate, the microscopic nature of folding intermediates is less understood. In this study, ~(19)F 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). ~(19)F NMR solvent isotope shifts, resulting from replacing H_2O with D_2O, 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 in the hydrophobic core, whereas the paramagnetic shifts show the increased hydrophobicity of this folding intermediate. ~(15)N,~1H and methyl ~(13)C, ~1H HSQC NMR spectra demonstrate that this folding intermediate retains a near-native tertiary structure whose hydrophobic interior is highly dynamic. ~(19)F NMR CPMG relaxation dispersion measurements suggest the near-native state is transiently adopted well below the temperature associated with its onset.