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Nanomolar Pulse Dipolar EPR Spectroscopy in Proteins Using Commercial Labels and Hardware

preprint
submitted on 13.12.2020, 23:17 and posted on 15.12.2020, 11:44 by Katrin Ackermann, Joshua Wort, Bela Bode
The study of complex biomolecular assemblies implicated in human health and disease is increasingly performed under native conditions. Pulse Dipolar Electron paramagnetic resonance (PDEPR) spectroscopy is a powerful tool that provides highly precise geometric constraints in frozen solution, however the drive towards in cellulo EPR is limited by the currently achievable concentration sensitivity in the low μM regime. Achieving PDEPR at physiologically relevant sub-μM concentrations is currently very challenging. Recently, relaxation induced dipolar modulation enhancement (RIDME) measurements using a combination of nitroxide and double-histidine CuII based spin labels allowed measuring 500 nM concentration of a model protein. Herein, we demonstrate CuII-CuII RIDME and nitroxide-nitroxide PELDOR measurements down to 500 and 100 nM protein concentration, respectively. This is possible using commercial instrumentation and spin labels. These results herald a transition towards routine sub-μM PDEPR measurements at short to intermediate distances (~1.5-3.5 nm), without the necessity of specialized instrumentation or spin-labelling protocols, particularly relevant for applications in near physiological conditions.

Funding

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

Wellcome Trust

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Cryogen-Free Arbitrary Waveform EPR for Structural Biology and Biophysics

Biotechnology and Biological Sciences Research Council

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Leverhulme RPG-2018–397

BBSRC DTP Eastbio

ISSF support to the University of St Andrews from The Well-come Trust

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