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

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


Abstract Here, we report novel methods to measure rate constants for homodimer subunit exchange using double electronâ??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 â??1, which was 0.037 ± 0.005 minâ??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 proteinâ??protein and protein-DNA complex studies.

• Content Type Journal Article
• Category Article
• Pages 1-12
• DOI 10.1007/s10858-012-9685-7
• Authors
  • Yunhuang Yang, Department of Chemistry and Biochemistry, and Northeast Structural Genomics Consortium (NESG), Miami University, Oxford, OH 45056, USA
  • Theresa A. Ramelot, Department of Chemistry and Biochemistry, and Northeast Structural Genomics Consortium (NESG), Miami University, Oxford, OH 45056, USA
  • Shuisong Ni, Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, USA
  • Robert M. McCarrick, Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, USA
  • Michael A. Kennedy, Department of Chemistry and Biochemistry, and Northeast Structural Genomics Consortium (NESG), Miami University, Oxford, OH 45056, USA

• • Journal Journal of Biomolecular NMR
  • Online ISSN 1573-5001
  • Print ISSN 0925-2738

Source: Journal of Biomolecular NMR