Journal of Biomolecular NMR

, Volume 50, Issue 3, pp 229–236 | Cite as

A systematic mutagenesis-driven strategy for site-resolved NMR studies of supramolecular assemblies

  • Carlos Amero
  • M. Asunción Durá
  • Marjolaine Noirclerc-Savoye
  • Arnaud Perollier
  • Benoit Gallet
  • Michael J. Plevin
  • Thierry Vernet
  • Bruno Franzetti
  • Jérôme Boisbouvier
Article

Abstract

Obtaining sequence-specific assignments remains a major bottleneck in solution NMR investigations of supramolecular structure, dynamics and interactions. Here we demonstrate that resonance assignment of methyl probes in high molecular weight protein assemblies can be efficiently achieved by combining fast NMR experiments, residue-type-specific isotope-labeling and automated site-directed mutagenesis. The utility of this general and straightforward strategy is demonstrated through the characterization of intermolecular interactions involving a 468-kDa multimeric aminopeptidase, PhTET2.

Keywords

NMR spectroscopy High molecular weight proteins Assignment Site-specific mutagenesis Methyl-group labeling 

Supplementary material

10858_2011_9513_MOESM1_ESM.pdf (991 kb)
Supplementary material 1 (PDF 990 kb)

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Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Carlos Amero
    • 1
    • 2
  • M. Asunción Durá
    • 1
  • Marjolaine Noirclerc-Savoye
    • 3
  • Arnaud Perollier
    • 1
  • Benoit Gallet
    • 3
  • Michael J. Plevin
    • 1
  • Thierry Vernet
    • 1
    • 3
    • 4
  • Bruno Franzetti
    • 1
    • 3
    • 4
  • Jérôme Boisbouvier
    • 1
    • 3
    • 4
    • 5
  1. 1.Institut de Biologie Structurale Jean-Pierre EbelCNRSGrenobleFrance
  2. 2.Centro de Investigaciones QuímicasUniversidad Autónoma Del Estado de MorelosCuernavacaMéxico
  3. 3.Institut de Biologie Structurale Jean-Pierre EbelCEAGrenobleFrance
  4. 4.Institut de Biologie StructuraleUniversite Joseph FourierGrenobleFrance
  5. 5.Institut de Biologie StructuraleJean-Pierre Ebel CNRS/CEA/UJFGrenoble CedexFrance

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