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Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues
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Published on: November 22, 2014

Sequence-specific and stereospecific assignment of methyl groups using paramagnetic lanthanides.

Michael John1, Christophe Schmitz, Ah Young Park

  • 1Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia.

Journal of the American Chemical Society
|October 13, 2007
PubMed
Summary

This study introduces a rapid method using pseudocontact shifts (PCSs) from lanthanide ions for protein methyl group assignments in known structures. This technique simplifies complex protein analysis without needing prior assignments or complex experiments.

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Methods to Identify the NMR Resonances of the 13C-Dimethyl N-terminal Amine on Reductively Methylated Proteins

Published on: December 12, 2013

Area of Science:

  • Biochemistry
  • Structural Biology
  • Biophysics

Background:

  • Protein structure determination is crucial for understanding function.
  • Assigning resonances for methyl groups in proteins is challenging.
  • Paramagnetic lanthanide ions can induce pseudocontact shifts (PCSs) useful for structural studies.

Purpose of the Study:

  • To develop a fast and efficient method for sequence-specific resonance assignments of protein methyl groups.
  • To utilize PCSs from site-specifically bound lanthanide ions for this purpose.
  • To demonstrate the method's applicability to complex protein systems.

Main Methods:

  • Site-specific binding of paramagnetic lanthanide ions to proteins.
  • Measurement of pseudocontact shifts (PCSs).
  • Development and application of the Possum program for simultaneous 13C-HSQC assignments and PCS measurements.
  • Utilizing methyl Cz-exchange experiments for PCS measurements in exchange situations.

Main Results:

  • Fast, sequence-specific resonance assignments of methyl groups (Val, Leu, Met) were achieved.
  • Stereospecific assignments of Val and Leu methyls were obtained.
  • The method does not require prior assignments of the diamagnetic protein or complex magnetization transfer experiments.
  • Successful demonstration on the Escherichia coli DNA polymerase III subunit complex.

Conclusions:

  • PCSs induced by lanthanide ions offer a powerful and rapid approach for protein methyl group resonance assignment.
  • The developed methods, including the Possum program and methyl Cz-exchange experiments, enhance accessibility to PCS measurements.
  • This technique simplifies structural analysis of complex protein systems.