Journal of Biomolecular NMR

, Volume 55, Issue 1, pp 47–58 | Cite as

Measurement of rate constants for homodimer subunit exchange using double electron–electron resonance and paramagnetic relaxation enhancements

  • Yunhuang Yang
  • Theresa A. Ramelot
  • Shuisong Ni
  • Robert M. McCarrick
  • Michael A. Kennedy
Article

Abstract

Here, we report novel methods to measure rate constants for homodimer subunit exchange using double electron–electron resonance (DEER) electron paramagnetic resonance spectroscopy measurements and nuclear magnetic resonance spectroscopy based paramagnetic relaxation enhancement (PRE) measurements. The techniques were demonstrated using the homodimeric protein Dsy0195 from the strictly anaerobic bacterium Desulfitobacterium hafniense Y51. At specific times following mixing site-specific MTSL-labeled Dsy0195 with uniformly 15N-labeled Dsy0195, the extent of exchange was determined either by monitoring the decrease of MTSL-labeled homodimer from the decay of the DEER modulation depth or by quantifying the increase of MTSL-labeled/15N-labeled heterodimer using PREs. Repeated measurements at several time points following mixing enabled determination of the homodimer subunit dissociation rate constant, k−1, which was 0.037 ± 0.005 min−1 derived from DEER experiments with a corresponding half-life time of 18.7 min. These numbers agreed with independent measurements obtained from PRE experiments. These methods can be broadly applied to protein–protein and protein-DNA complex studies.

Keywords

DEER PRE EPR NMR Protein Homodimer Rate constants Subunit exchange 

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Copyright information

© Springer Science+Business Media Dordrecht 2012

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 Northeast Structural Genomics Consortium (NESG)Miami UniversityOxfordUSA
  2. 2.Department of Chemistry and BiochemistryMiami UniversityOxfordUSA

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