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Pulsed ELDOR Measurement of the Distance Between a Spin-Label and Copper (II) Centre in the Copper Loaded R48C Mutant of N. gonorrhoeae Ferric Binding Protein

Matt Bawn, Justin Bradley, Fraser MacMillan
doi: https://doi.org/10.1101/248054
Matt Bawn
1Henry Wellcome Unit for Biological EPR, University of East Anglia, Norwich, NR4 7TJ, U.K.
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Justin Bradley
1Henry Wellcome Unit for Biological EPR, University of East Anglia, Norwich, NR4 7TJ, U.K.
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Fraser MacMillan
1Henry Wellcome Unit for Biological EPR, University of East Anglia, Norwich, NR4 7TJ, U.K.
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Abstract

Distance determination in proteins and biomolecules using pulsed EPR (electron paramagnetic resonance) techniques is becoming an increasingly popular and accessible technique. PELDOR (pulsed electron-electron double resonance), is a technique designed for distance determination over a nanoscopic scale. Here, ferric binding protein (Fbp) is used to demonstrate the practicability of this technique to Cu (II) Metalloproteins. PELDOR is usually applied to bi-radicals or endogenous radicals, and distance determination using pulsed EPR of metal containing centres in biomolecules has been restricted to relaxation experiments. PELDOR distance measurements between a Cu (II) ion and a nitroxide have previously only been reported for model compounds [1, 2].

Fbp as the name suggests usually, contains a Fe (III) ion centre. For the purposes of this investigation the Fe (III) ion was removed and replaced by a Cu (II) ion, after a nitroxide spin-label was added to the Fbp using of site directed spin-labelling (SDSL). PELDOR was then applied to measure the distance between the two centres.

Simulation methods were then employed to fully investigate these data and allow a quantitative interpretation of the copper nitroxide PELDOR data. The observed PELDOR time traces were analysed using DEER analysis[3].

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted January 16, 2018.
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Pulsed ELDOR Measurement of the Distance Between a Spin-Label and Copper (II) Centre in the Copper Loaded R48C Mutant of N. gonorrhoeae Ferric Binding Protein
Matt Bawn, Justin Bradley, Fraser MacMillan
bioRxiv 248054; doi: https://doi.org/10.1101/248054
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Pulsed ELDOR Measurement of the Distance Between a Spin-Label and Copper (II) Centre in the Copper Loaded R48C Mutant of N. gonorrhoeae Ferric Binding Protein
Matt Bawn, Justin Bradley, Fraser MacMillan
bioRxiv 248054; doi: https://doi.org/10.1101/248054

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