Populating the equilibrium molten globule state of apomyoglobin under conditions suitable for structural characterization by NMR.

Overview

Conditions have been determined under which the equilibrium molten globule state of apomyoglobin is stable and remains monomeric for periods of time sufficient for the application of three-dimensional heteronuclear NMR experiments. The quality of initial two-dimensional NMR spectra suggests that sequence-specific assignments can be made for a majority of the protein resonances under these conditions. A pH titration of the protein followed using two-dimensional 1H-15N correlation experiments indicates that the equilibrium intermediate undergoes fast exchange on the chemical shift time scale with the unfolded state and intermediate time scale exchange with the native state, and suggests a strategy to assist with backbone resonance assignments. The conditions and techniques described may be applicable to the characterization of other equilibrium folding intermediates.