Transient Protein Encounters Characterized by Paramagnetic NMR

Invisible to most biophysical techniques, lowly-populated transient intermediates formed on the path of biomolecular association orchestrate protein recognition and binding. Here we study such minor species mediating the interaction between physiological partners cytochrome c and cytochrome c peroxidase by paramagnetic relaxation enhancement NMR spectroscopy.
Visualization of multiple protein-protein orientations constituting the transient encounter state reveals a broad spatial distribution, which is in striking agreement with that obtained in earlier theoretical simulations. Being inactive in the intermolecular electron transfer, the encounter complex pre-orients the interacting molecules, enabling the reduced dimensionality search of the dominant, functionally active bound form. The encounter complex is insensitive to the redox and spin states of the interacting molecules, suggesting that its properties are determined by the protein polypeptides rather than their heme cofactors.