DEER sensitivity between iron centers and nitroxides in heme-containing proteins improves dramatically using broadband, high-field EPR
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This work demonstrates the feasibility of making sensitive nanometer distance measurements between Fe(III) heme centers and nitroxide spin labels in proteins using the double electron-electron resonance (DEER) pulsed EPR technique at 94 GHz. Techniques to measure accurately long distances in many classes of heme proteins using DEER are currently strongly limited by sensitivity. In this paper we demonstrate sensitivity gains of more than 30 times compared to previous lower frequency (X-band) DEER measurements on both human neuroglobin and sperm whale myoglobin. This is achieved by: taking advantage of recent instrumental advances; employing wideband excitation techniques based on composite pulses and exploiting more favorable relaxation properties of low-spin Fe(III) in high magnetic fields. This gain in sensitivity potentially allows the DEER technique to be routinely used as a sensitive probe of structure and conformation in the large number of heme and many other metalloproteins.
Motion , C L , Lovett , J E , Bell , S , Cassidy , S L , Cruickshank , P A S , Bolton , D R , Hunter , R I , El Mkami , H , Van Doorslaer , S & Smith , G M 2016 , ' DEER sensitivity between iron centers and nitroxides in heme-containing proteins improves dramatically using broadband, high-field EPR ' Journal of Physical Chemistry Letters , vol. 7 , no. 8 , pp. 1411-1415 . DOI: 10.1021/acs.jpclett.6b00456
Journal of Physical Chemistry Letters
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DescriptionClaire Motion would like to acknowledge funding from EPSRC as part of the iMR-CDT. Stacey Bell and Janet Lovett thank EPSRC Grant Number EP/LO22044/1. Janet Lovett also thanks the Royal Society for a University Research Fellowship and Research Grant RG120645. Sabine Van Doorslaer acknowledges the Research Foundation – Flanders (FWO) for financial support (grant G.0687.13). The W-band instrument was developed under the UK Research Council’s Basic Technology Program (grant EP/F039034/1). We also thank the Wellcome Trust (grant 099149/Z/12/Z).
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