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Journal of Biomolecular NMR

, Volume 52, Issue 3, pp 245–256 | Cite as

Redox-dependent conformational changes in eukaryotic cytochromes revealed by paramagnetic NMR spectroscopy

  • Alexander N. Volkov
  • Sophie Vanwetswinkel
  • Karen Van de Water
  • Nico A. J. van Nuland
Article

Abstract

Cytochrome c (Cc) is a soluble electron carrier protein, transferring reducing equivalents between Cc reductase and Cc oxidase in eukaryotes. In this work, we assessed the structural differences between reduced and oxidized Cc in solution by paramagnetic NMR spectroscopy. First, we have obtained nearly-complete backbone NMR resonance assignments for iso-1-yeast Cc and horse Cc in both oxidation states. These were further used to derive pseudocontact shifts (PCSs) arising from the paramagnetic haem group. Then, an extensive dataset comprising over 450 measured PCSs and high-resolution X-ray and solution NMR structures of both proteins were used to define the anisotropic magnetic susceptibility tensor, Δχ. For most nuclei, the PCSs back-calculated from the Δχ tensor are in excellent agreement with the experimental PCS values. However, several contiguous stretches—clustered around G41, N52, and A81—exhibit large deviations both in yeast and horse Cc. This behaviour is indicative of redox-dependent structural changes, the extent of which is likely conserved in the protein family. We propose that the observed discrepancies arise from the changes in protein dynamics and discuss possible functional implications.

Keywords

Cytochrome c Paramagnetic NMR Protein dynamics Pseudocontact shifts 

Notes

Acknowledgments

We thank Dr. Jonathan Worrall and Prof. Marcellus Ubbink for the kind gifts of the Cc expression vectors, Dr. Christophe Schmitz for useful tips on the installation and use of Numbat software, and Dr. Lieven Buts for the help with the data analysis. ANV is an FWO Post-Doctoral Researcher.

Supplementary material

10858_2012_9607_MOESM1_ESM.docx (1.1 mb)
Supplementary material 1 (DOCX 1126 kb)

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Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Alexander N. Volkov
    • 1
    • 2
  • Sophie Vanwetswinkel
    • 1
    • 2
  • Karen Van de Water
    • 1
    • 2
  • Nico A. J. van Nuland
    • 1
    • 2
  1. 1.Jean Jeener NMR Centre, Structural Biology BrusselsVrije Universiteit BrusselBrusselsBelgium
  2. 2.Department of Structural BiologyVIBBrusselsBelgium

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