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Journal of Biomolecular NMR

, Volume 35, Issue 4, pp 285–293 | Cite as

Trans and surface membrane bound zervamicin IIB: 13C-MAOSS-NMR at high spinning speed

  • J. Raap
  • J. Hollander
  • T. V. Ovchinnikova
  • N. V. Swischeva
  • D. Skladnev
  • S. Kiihne
Article

Abstract

Interactions between 15N-labelled peptides or proteins and lipids can be investigated using membranes aligned on a thin polymer film, which is rolled into a cylinder and inserted into the MAS-NMR rotor. This can be spun at high speed, which is often useful at high field strengths. Unfortuantely, substrate films like commercially available polycarbonate or PEEK produce severe overlap with peptide and protein signals in 13C-MAOSS NMR spectra. We show that a simple house hold foil support allows clear observation of the carbonyl, aromatic and Cα signals of peptides and proteins as well as the ester carbonyl and choline signals of phosphocholine lipids. The utility of the new substrate is validated in applications to the membrane active peptide zervamicin IIB. The stability and macroscopic ordering of thin PC10 bilayers was compared with that of thicker POPC bilayers, both supported on the household foil. Sidebands in the 31P-spectra showed a high degree of alignment of both the supported POPC and PC10 lipid molecules. Compared with POPC, the PC10 lipids are slightly more disordered, most likely due to the increased mobilities of the shorter lipid molecules. This mobility prevents PC10 from forming stable vesicles for MAS studies. The 13C-peptide peaks were selectively detected in a 13C-detected 1H-spin diffusion experiment. Qualitative analysis of build-up curves obtained for different mixing times allowed the transmembrane peptide in PC10 to be distinguished from the surface bound topology in POPC. The 13C-MAOSS results thus independently confirms previous findings from 15N spectroscopy [Bechinger, B., Skladnev, D.A., Ogrel, A., Li, X., Rogozhkina, E.V., Ovchinnikova, T.V., O’Neil, J.D.J. and Raap, J. (2001) Biochemistry, 40, 9428–9437]. In summary, application of house hold foil opens the possibility of measuring high resolution 13C-NMR spectra of peptides and proteins in well ordered membranes, which are required to determine the secondary and supramolecular structures of membrane active peptides, proteins and aggregates.

Key words

peptide antibiotic solid state 1H-, 13C-, 31P-NMR substrate supported PC10 and POPC bilayers 

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Notes

Acknowledgements

We are grateful to Dr A.A. Tagaev and Dr T.I. Kostromina (Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia) for their contribution to the preparation of [U-13C]-labelled zervamicin IIB. We thank Prof Dr Mei Hong for providing the spin diffusion simulation program and Dr Xiaolan Yao for her helpful hints to use this program. This work was granted by the Netherlands Organisation for Scientific Research (NWO No. 047.014.017).

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Copyright information

© Springer 2006

Authors and Affiliations

  • J. Raap
    • 1
  • J. Hollander
    • 1
  • T. V. Ovchinnikova
    • 2
  • N. V. Swischeva
    • 2
  • D. Skladnev
    • 3
  • S. Kiihne
    • 1
  1. 1.Leiden Institute of ChemistryLeiden UniversityLeidenThe Netherlands
  2. 2.Shemyakin & Ovchinnikov Institute of Bioorganic ChemistryMoscowRussia
  3. 3.State Research Institute of Genetics and Selection of Industrial MicroorganismsMoscowRussia

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