Pulse dipolar electron paramagnetic resonance spectroscopy distance measurements at low nanomolar concentrations : the CuII-trityl case
Abstract
Recent sensitivity enhancements in pulse dipolar EPR spectroscopy (PDS) have afforded distance measurements at submicromolar spin concentrations. This development opens the path for new science, as more biomolecular systems can be investigated at their respective physiological concentrations. Here, we demonstrate that the combination of orthogonal spin labelling using CuII ions and trityl yields a more than 3-fold sensitivity increase compared to the established CuII-nitroxide labelling strategy. Application of the recently developed variable-time RIDME method yields a further approximately 2.5-fold increase compared to the commonly used constant-time RIDME. This overall increase in sensitivity of almost an order of magnitude makes distance measurements in the range of 3 nm with protein concentrations as low as 10 nM feasible, more than two times lower than previously reported. We expect that experiments at single digit nanomolar concentrations are imminent, which has the potential to transform biological PDS applications.
Citation
Ackermann , K , Heubach , C , Schiemann , O & Bode , B E 2024 , ' Pulse dipolar electron paramagnetic resonance spectroscopy distance measurements at low nanomolar concentrations : the Cu II -trityl case ' , The Journal of Physical Chemistry Letters , vol. 15 , pp. 1455-1461 . https://doi.org/10.1021/acs.jpclett.3c03311
Publication
The Journal of Physical Chemistry Letters
Status
Peer reviewed
ISSN
1948-7185Type
Journal article
Description
Funding: To meet institutional and research funder open access requirements, any accepted manuscript arising shall be open access under a Creative Commons Attribution (CC BY) reuse licence with zero embargo. The authors acknowledge support by a University of St Andrews-University of Bonn Collaborative Research Grant, by the Wellcome Trust (204821/Z/16/Z), and by the EPSRC (EP/X016455/1). B.E.B. acknowledges equipment funding by BBSRC (BB/R013780/1 and BB/T017740/1). O.S. thanks the DFG for funding (420322655). C.A.H. thanks the DAAD for a travel and research scholarship. The authors thank the StAnD (St Andrews and Dundee) EPR grouping for long-standing support and the St Andrews mass spectrometry and proteomics facility for equipment access.Collections
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