These are preliminary reports that have not been peer-reviewed. They should not be regarded as conclusive, guide clinical practice/health-related behavior, or be reported in news media as established information. For more information, please see our FAQs.
Nanomolar Pulse Dipolar EPR Spectroscopy in Proteins Using Commercial Labels and Hardware
preprintsubmitted 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.
State of the art pulse EPR instrumentation for long range distance measurements in biomacromolecules.
Wellcome TrustFind out more...
Cryogen-Free Arbitrary Waveform EPR for Structural Biology and Biophysics
Biotechnology and Biological Sciences Research CouncilFind out more...