title: Measurement of rate constants for homodimer subunit exchange using double electron-electron resonance and paramagnetic relaxation enhancements.
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 (15)N-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/(15)N-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.
authors.affiliation: Department of Chemistry and Biochemistry, and Northeast Structural Genomics Consortium (NESG), Miami University, Oxford, OH 45056, USA.
authors.forename: Yunhuang
authors.initials: Y
authors.lastname: Yang
authors.forename: Theresa A
authors.initials: TA
authors.lastname: Ramelot
authors.forename: Shuisong
authors.initials: S
authors.lastname: Ni
authors.forename: Robert M
authors.initials: RM
authors.lastname: McCarrick
authors.forename: Michael A
authors.initials: MA
authors.lastname: Kennedy
mesh_terms: D000465:Algorithms; D003198:Computer Simulation; D004578:Electron Spin Resonance Spectroscopy; D019906:Nuclear Magnetic Resonance, Biomolecular; D055503:Protein Multimerization; D021122:Protein Subunits / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*
chemical_list: D021122:Protein Subunits; D011506:Proteins