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

preprint
revised on 30.03.2021, 16:30 and posted on 31.03.2021, 05:28 by Katrin Ackermann, Joshua Wort, Bela Bode
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 CuII based spin labels allowed measuring 500 nM concentration of a model protein. Using commercial instrumentation and spin labels we demonstrate CuII-CuII 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.

Funding

State of the art pulse EPR instrumentation for long range distance measurements in biomacromolecules.

Wellcome Trust

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Institutional Strategic Support Fund

Wellcome Trust

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Leverhulme Trust RPG-2018–397

Cryogen-Free Arbitrary Waveform EPR for Structural Biology and Biophysics

Biotechnology and Biological Sciences Research Council

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East of Scotland Bioscience Doctoral Training Partnership

Biotechnology and Biological Sciences Research Council

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History

Email Address of Submitting Author

beb2@st-andrews.ac.uk

Institution

University of St Andrews

Country

United Kingdom

ORCID For Submitting Author

0000-0002-3384-271X

Declaration of Conflict of Interest

none to declare

Exports