Nanomolar Pulse Dipolar EPR Spectroscopy in Proteins: Cu <sup>II</sup> –Cu <sup>II</sup> and Nitroxide–Nitroxide Cases
Katrin Ackermann, Joshua L. Wort, Bela E. Bode
Abstract
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 spectroscopy (PDS) is a powerful tool that provides highly precise geometric constraints in frozen solutions; however, the drive toward PDS at physiologically relevant sub-μM concentrations is limited by the currently achievable concentration sensitivity. Recently, PDS using a combination of nitroxide- and CuII-based spin labels allowed measuring a 500 nM concentration of a model protein. Using commercial instrumentation and spin labels, we demonstrate CuII–CuII and nitroxide–nitroxide PDS measurements at protein concentrations below previous examples reaching 500 and 100 nM, respectively. These results demonstrate the general feasibility of sub-μM PDS measurements at short to intermediate distances (∼1.5 to 3.5 nm), and are of particular relevance for applications where the achievable concentration is limiting.