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Selective deuteration of tryptophan and methionine residues in maltose binding protein: a model system for neutron scattering

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Abstract

We describe methods that have been developed within the ILL-EMBL Deuteration Laboratory for the production of maltose binding protein (MBP) that has been selectively labelled either with deuterated tryptophan or deuterated methionine (single labelling), or both (double labelling). MBP is used as an important model system for biophysical studies, and selective labelling can be helpful in the analysis of small-angle neutron scattering (SANS) data, neutron reflection (NR) data, and high-resolution neutron diffraction data. The selective labelling was carried out in E. coli high-cell density cultures using auxotrophic mutants in minimal medium containing the required deuterated precursors. Five types of sample were prepared and studied: (1) unmodified hydrogenated MBP (H-MBP), (2) perdeuterated MBP (D-MBP), (3) singly labelled MBP with the tryptophan residues deuterated (D-trp MBP), (4) singly labelled MBP with methionine residues deuterated (D-met MBP) and (5) doubly labelled MBP with both tryptophan and methionine residues deuterated (D-trp/met MBP). Labelled samples were characterised by size exclusion chromatography, gel electrophoresis, light scattering and mass spectroscopy. Preliminary small-angle neutron scattering (SANS) experiments have also been carried out and show measurable differences between the SANS data recorded for the various labelled analogues. More detailed SANS experiments using these labelled MBP analogues are planned; the degree to which such data could enhance structure determination by SANS is discussed.

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Acknowledgments

This work was supported by the European Union under contract RII3-CT-2003-505925. It has also benefited from previous development work carried out under HPRI-2001-50065. We acknowledge the EPSRC for support under grants EP/C015452/1 and GR/R47950/01 and the Institut de Biologie Structurale in Grenoble for the use of their mass spectroscopy service. The authors are also very grateful for advice and assistance from all members of the ILL-EMBL Deuteration Laboratory, in particular Martine Moulin.

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Authors and Affiliations

  1. ILL-EMBL Deuteration Laboratory, Partnership for Structural Biology, Institut Laue Langevin, 38042, Grenoble Cedex 9, France

    Valerie Laux, Phil Callow, Peter A. Timmins, V. Trevor Forsyth & Michael Haertlein

  2. EPSAM/ISTM Research Institutes, Keele University, Staffordshire, ST5 5BG, UK

    Phil Callow & V. Trevor Forsyth

  3. European Molecular Biology Laboratory, Hamburg Outstation, Notkestraße 85, 22603, Hamburg, Germany

    Dmitri I. Svergun

  4. Institute of Crystallography, Russian Academy of Sciences, Leninsky pr. 59, 117333, Moscow, Russia

    Dmitri I. Svergun

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  1. Valerie Laux
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Correspondence to Michael Haertlein.

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Laux, V., Callow, P., Svergun, D.I. et al. Selective deuteration of tryptophan and methionine residues in maltose binding protein: a model system for neutron scattering. Eur Biophys J 37, 815–822 (2008). https://doi.org/10.1007/s00249-008-0280-5

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  • DOI: https://doi.org/10.1007/s00249-008-0280-5

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