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Nanomolar Pulse Dipolar EPR Spectroscopy in Proteins: Cu^II-Cu^II and Nitroxide-Nitroxide Cases.

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 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-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.

SUBMITTER: Ackermann K

PROVIDER: S-EPMC7611071 | biostudies-literature |

REPOSITORIES: biostudies-literature

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