Transient protein encounters characterized by paramagnetic NMRElectronic supplementary information (ESI) available: Deconvoluted mass spectra of the EDTA(Mn2+)-conjugated proteins, PRE profiles for the control experiments, the distribution of the intermolecular ET distances in the control ensemble refinement run, discussion of the practical aspects of the PRE measurements, and the results of the error analysis of the ensemble calculations from the measured PREs. See DOI: 10.1039/c4sc01232a

• Main Authors: Van de Water, K, van Nuland, N. A. J, Volkov, A. N
• Format: Journal Article
• Language: English
• Published: 29.09.2014
• ISSN: 2041-6520; 2041-6539
• DOI: 10.1039/c4sc01232a

Invisible to most biophysical techniques, transient intermediates formed during 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. The 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 a 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 protein polypeptides rather than heme cofactors. Multiple encounters, sampled by interacting proteins in search of the dominant, functionally active orientation, are visualized by paramagnetic NMR.