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
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Abstract 15N relaxation rates of amide moieties provide insight both into global as well as local backbone dynamics of peptides and proteins. As the differences in the relaxation rates in general are small, their accurate determination is of prime importance. One potential source of error is fast amide exchange. It is well known that in its presence the effects of saturation transfer and H/D exchange may result in erroneous apparent relaxation rates R 1 and R 2. Here, the extent of...
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The stereochemistry of compounds is assigned very often with proton - proton NOE's by applying the 2D NOESY technique or the 1D selective gradient NOESY technique. These methods fail, however when the distance between protons is too large to measure an NOE. When faced with this situation, it may be possible to measure long range proton - carbon coupling constants which are able to provide the necessary information. Three-bond carbon - proton couplings follow a Karplus relationship where the magnitude of the coupling constant is related to...
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Effect of Freezing Conditions on Distances and Their Distributions Derived from Double Electron Electron Resonance (DEER): A Study of Doubly-Spin-Labeled T4 Lysozyme
Publication year: 2012
Source: Journal of Magnetic Resonance, Available online 24 January 2012</br>
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Pulsed dipolar ESR spectroscopy, DEER and DQC, require frozen samples. An important issue in the biological application of this technique is how the freezing rate and concentration of cryoprotectant could possibly affect the...
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Measurement of rate constants for homodimer subunit exchange using double electronâ??electron resonance and paramagnetic relaxation enhancements
Linear Mode
