Nanomolar Pulse Dipolar EPR Spectroscopy in Proteins: CuII-CuII 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 spectroscopy (PDS) is a powerful tool that provides highly precise geometric constraints in frozen solutions; however, the drive toward PDS at physiologically relevant sub-mu M concentrations is limited by the currently achievable concentration sensitivity. Recently, PDS using a combination of nitroxide- and Cu-II-based spin labels allowed measuring a 500 nM concentration of a model protein. Using commercial instrumentation and spin labels, we demonstrate Cu-II-Cu-II 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-mu M PDS measurements at short to intermediate distances (similar to 1.5 to 3.5 nm), and are of particular relevance for applications where the achievable concentration is limiting.

Automated summary

The study utilizes biophysical methods such as pulse dipolar electron paramagnetic resonance spectroscopy to investigate complex biomolecular assemblies implicated in human health and disease, demonstrating the feasibility of PDS measurements at sub-micromolar concentrations and highlighting the significance for applications with concentration limitations.