Nanomolar pulse dipolar EPR spectroscopy in proteins : CuII-CuII and nitroxide-nitroxide cases
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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 solution, however the drive towards PDS at physiologically relevant sub-μM concentrations is limited by the currently achievable concentration sensitivity. Recently, PDS 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 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 - 3.5 nm), and are of particular relevance for applications where the achievable concentration is limiting.
Ackermann , K , Wort , J & Bode , B E 2021 , ' Nanomolar pulse dipolar EPR spectroscopy in proteins : Cu II -Cu II and nitroxide-nitroxide cases ' , Journal of Physical Chemistry B , vol. Articles ASAP . https://doi.org/10.1021/acs.jpcb.1c03666
Journal of Physical Chemistry B
Copyright © 2021 The Author(s). For the purpose of open access, the author has applied a Creative Commons Attribution (CC BY) public copyright licence to the author created accepted manuscript, following peer review. The research was funded in whole, or in part, by the Wellcome Trust. This version may differ slightly from the final published version, which is available at https://doi.org/10.1021/acs.jpcb.1c03666
DescriptionThis research was funded, in whole or in part, by the Wellcome Trust (099149/Z/12/Z and 204821/Z/16/Z). A CC BY or equivalent license is applied to the author-accepted manuscript arising from this submission, in accordance with the grant’s open-access conditions. B.E.B. and K.A. acknowledge support by the Leverhulme Trust (RPG-2018-397). J.L.W. was supported by the BBSRC DTP Eastbio. B.E.B. acknowledges equipment funding by BBSRC (BB/R013780/1).
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