RhNGF slow unfolding is not due to proline isomerization: Possibility of a cystine knot loop‐threading mechanism

The unfolding of recombinant human β‐NGF (NGF) in guanidine hydrochloride (GdnHCl) was found to be time dependent with the denaturation midpoint moving to lower GdnHCl concentration over time. Dissociation and extensive unfolding of the NGF dimer occurred rapidly in 5 M GdnHCl, but further unfolding of the molecule occurred over many days at 25 °C. Fluorescence spectroscopy, size‐exclusion and reversed‐phase HPLC, ultra‐centrifugation, and proton NMR spectroscopy were used to ascertain that the slow unfolding step was between two denatured monomeric states of NGF (M1 and M2). Proton NMR showed the monomer formed at early times in GdnHCl (M1) had little β‐sheet structure, but retained residual structure in the tryptophan indole and high‐field methyl regions of the spectrum. This residual structure was lost after prolonged incubation in GdnHCl giving a more fully unfolded monomer, M2. From kinetic unfolding experiments in 5 M GdnHCl it was determined that the conversion of M1 to M2 had an activation energy of 26.5 kcal/mol, a half‐life of 23 h at 25 °C, and the rate of formation of M2 was dependent on the GdnHCl concentration between 5 and 7.1 M GdnHCl. These properties of the slow unfolding step are inconsistent with a proline isomerization mechanism. The rate of formation of the slow folding monomer M2 increases with truncation of five and nine amino acids from the NGF N‐terminus. A model for the slow unfolding reaction is proposed where the N‐terminus threads through the cystine knot to form M2, a loop‐threading reaction, increasing the conformational freedom of the denatured state.