Pulse dipolar EPR for determining nanomolar binding affinities

Protein interaction studies often require very low concentrations and highly sensitive biophysical methods. Here, we demonstrate that pulse dipolar electron paramagnetic resonance spectroscopy allows measuring dissociation constants in the nanomolar range. This approach is appealing for concentration-limited biomolecular systems and medium-to-high-affinity binding studies, demonstrated here at 50 nanomolar protein concentration. Cu II -nitroxide RIDME measurements at 100 nM protein concentration allow reliable extraction of dissociation constants and distances, while measurements at 50 nM protein concentration allow reliable extraction of dissociation constants only.