Applications of NMR-Based PRE and EPR-Based DEER Spectroscopy to Homodimer Chain Exchange Characterization and Structure Determination

  • Yunhuang Yang
  • Theresa A. Ramelot
  • Shuisong Ni
  • Robert M. McCarrick
  • Michael A. Kennedy
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1091)

Abstract

The success of homodimer structure determination by conventional solution NMR spectroscopy relies greatly on interchain distance restraints (less than 6 Å) derived from nuclear Overhauser effects (NOEs) obtained from 13C-edited, 12C-filtered NOESY experiments. However, these experiments may fail when the mixed 13C-/12C-homodimer is never significantly populated due to slow homodimer chain exchange. Thus, knowledge of the homodimer chain exchange kinetics can be put to practical use in preparing samples using the traditional NMR method. Here, we described detailed procedures for using paramagnetic resonance enhancements (PREs) and EPR spectroscopy to measure homodimer chain exchange kinetics. In addition, PRE and EPR methods can be combined to provide mid-range (<30 Å) and long-range (17–80 Å) interchain distance restraints for homodimer structure determination as a supplement to short-range intrachain and interchain distance restraints (less than 6 Å) typically obtained from 1H-1H NOESY experiments. We present a summary of how to measure these distances using NMR-based PREs and EPR-based double electron electron resonance (DEER) measurements and how to include them in homodimer structure calculations.

Key words

NMR EPR Spectroscopy Homodimer Chain exchange Structure determination 

Notes

Acknowledgments

This work was supported by the National Institute of General Medical Sciences, Grant Number: U54-GM074958; National Science Foundation, Grant Number CHE-0645709; BrukerBiospin, Miami University and Ohio Board of Reagents.

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Copyright information

© Springer Science+Business Media, LLC 2014

Authors and Affiliations

  • Yunhuang Yang
    • 1
  • Theresa A. Ramelot
    • 1
  • Shuisong Ni
    • 2
  • Robert M. McCarrick
    • 2
  • Michael A. Kennedy
    • 1
  1. 1.Department of Chemistry and Biochemistry and the Northeast Structural Genomics ConsortiumMiami UniversityOxfordUSA
  2. 2.Department of Chemistry and BiochemistryMiami UniversityOxfordUSA

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