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Dissolution Dynamic Nuclear Polarization Instrumentation for Real-time Enzymatic Reaction Rate Measurements by NMR
Published on: February 23, 2016
1H dynamic nuclear polarization based on an endogenous radical.
Thorsten Maly1, Dongtao Cui, Robert G Griffin
1Francis Bitter Magnet Laboratory and Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
The Journal of Physical Chemistry. B
|April 5, 2012
Summary
Dynamic nuclear polarization (DNP) using naturally occurring radicals in proteins like flavodoxin enhances solid-state NMR signals up to 15-fold. Deuteration significantly improves signal enhancement and build-up times for biochemical studies.
Area of Science:
- Biophysics
- Structural Biology
- Nuclear Magnetic Resonance (NMR) Spectroscopy
Background:
- Solid-state NMR is crucial for studying biomolecular structures.
- Dynamic Nuclear Polarization (DNP) can enhance NMR signals but often requires exogenous radicals.
- Naturally occurring radicals in biological systems offer a potential alternative for DNP.
Purpose of the Study:
- To demonstrate DNP enhancement of solid-state NMR signals using an endogenous radical in a protein.
- To investigate the effect of protein isotopic composition on DNP enhancement.
- To assess the feasibility of DNP with endogenous radicals for biochemical applications.
Main Methods:
- Dynamic Nuclear Polarization (DNP) experiments on flavodoxin protein.
- Utilized the flavin mononucleotide (FMN) semiquinone as a naturally occurring radical.
- Varied the isotopic composition of the protein (deuteration).
- Measured EPR signals, DNP enhancement factors, and build-up time constants (τ(B)).
Main Results:
- Achieved a 15-fold enhancement of solid-state NMR signals using the endogenous FMN semiquinone radical.
- Identified the solid effect as the dominant DNP mechanism.
- Deuteration of the protein increased signal enhancement and build-up times.
- Observed a 2-fold higher maximal enhancement in deuterated versus proteated protein.
Conclusions:
- DNP using endogenous protein radicals is feasible and significantly enhances NMR signals.
- Protein deuteration optimizes DNP performance by slowing polarization dissipation.
- This approach could enable MAS NMR characterization of complex biological systems and provide additional information.
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