Nanomolar Pulse Dipolar EPR Spectroscopy in Proteins; the Copper(II)-Copper(II) and Nitroxide-Nitroxide Cases

The study of ever more complex biomolecular assemblies implicated in human health and disease is facilitated by a suite of complementary biophysical methods. Pulse Dipolar Electron Paramagnetic Resonance (PDEPR) spectroscopy is a powerful tool that provides highly precise geometric constraints in frozen solution, however the drive towards PDEPR at physiologically relevant sub-μM concentrations is limited by the currently achievable concentration sensitivity. Recently, PDEPR using a combination of nitroxide and Cu^II based spin labels allowed measuring 500 nM concentration of a model protein. Using commercial instrumentation and spin labels we demonstrate Cu^II-Cu^II and nitroxide-nitroxide PDEPR measurements at protein concentrations more than an order of magnitude below previous examples reaching 500 and 100 nM, respectively. These results demonstrate the general feasibility of sub-μM PDEPR measurements at short to intermediate distances (~1.5 - 3.5 nm), and are of particular relevance for applications where the achievable concentration is limiting.