25-30 August 2019
Henry Ford Building
Europe/Berlin timezone

Quantitative sub-micromolar pulse dipolar EPR spectroscopy evidences high copper(II) labeling efficiency for double-histidine motifs

28 Aug 2019, 11:30
25m
Lecture Hall D (Henry Ford Building)

Lecture Hall D

Henry Ford Building

Talk EPR development and applications EPR

Speaker

Dr Bela Bode (University of St Andrews)

Description

Electron paramagnetic resonance (EPR) distance measurements provide highly accurate and precise geometric constraints. These have made valuable contributions to studies of the structures and conformations of biomolecules. Recently, application of double-histidine (dHis) motifs, coupled with CuII spin-labels has shown promise in even higher precision distance measurements.1 However, the non-covalent CuII-coordination approach is vulnerable to low binding-affinity. Earlier estimations of dissociation constants (KD) revealed micromolar to low millimolar KDs under EPR distance measurmeent conditions.1 As many challenging biomolecular targets are only stable at or below low micromolar concentration higher KDs are likely to limit the usefulness of this approach.

We have investigated the binding affinity directly from primary pulse dipolar (PD) EPR data.2 By combining spectroscopically orthogonal CuII and nitroxide spin-labels and performing RIDME (relaxation-induced dipolar modulation enhancement) distance measurements the uncertainty from speciation of the CuII spin-label is largely mitigated. By exploiting the superb sensitivity of this experiment and label combination we have demonstrated significant loading of dHis sites at submicromolar concentrations. This study demonstrates that the affinity for CuII-chelators is not limiting for PDEPR studies in the low micromolar range and that the combination of RIDME and the orthogonal spin-labels CuII and nitroxide makes submicromolar PDEPR experiments feasible.3

  1. (a) T.F. Cunningham, M.R. Putterman, A. Desai, W.S. Horne, S. Saxena, ACIE 2015, 54, 6330; (b) S. Ghosh, M.J. Lawless, G.S. Rule, S. Saxena, JMR 2018, 286, 163.
  2. (a) K. Ackermann, A. Giannoulis, D.B. Cordes, A.M.Z. Slawin, B.E. Bode, ChemComm 2015, 51, 5257; (b) A. Giannoulis, M. Oranges, B.E. Bode, ChemPhysChem 2017, 18, 2318; (c) A. Giannoulis, K. Ackermann, P. Spindler, C. Higgins, D.B. Cordes, A.M.Z. Slawin, T.F. Prisner, B.E. Bode, PCCP, 2018, 20, 11196.
  3. J.L. Wort, K. Ackermann, A. Giannoulis, A.J. Stewart, D.G. Norman, B.E. Bode, under review.

Primary authors

Mr Joshua Wort (University of St Andrews) Dr Katrin Ackermann (University of St Andrews) Dr Angeliki Giannoulis (University of St Andrews) Dr Alan Stewart (University of St Andrews) Dr David Norman (University of Dundee) Dr Bela Bode (University of St Andrews)

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