Category Archives: Reverse Micelles

High-resolution NMR spectroscopy of encapsulated proteins dissolved in low-viscosity fluids

Some more information on encapsulated proteins, an approach that was shown recently to be a very interesting path for solution-state DNP by the same group.

Nucci, N.V., K.G. Valentine, and A.J. Wand, High-resolution NMR spectroscopy of encapsulated proteins dissolved in low-viscosity fluids. J Magn Reson, 2014. 241(0): p. 137-47.

http://www.ncbi.nlm.nih.gov/pubmed/24656086

High-resolution multi-dimensional solution NMR is unique as a biophysical and biochemical tool in its ability to examine both the structure and dynamics of macromolecules at atomic resolution. Conventional solution NMR approaches, however, are largely limited to examinations of relatively small (<25kDa) molecules, mostly due to the spectroscopic consequences of slow rotational diffusion. Encapsulation of macromolecules within the protective nanoscale aqueous interior of reverse micelles dissolved in low viscosity fluids has been developed as a means through which the ‘slow tumbling problem’ can be overcome. This approach has been successfully applied to diverse proteins and nucleic acids ranging up to 100kDa, considerably widening the range of biological macromolecules to which conventional solution NMR methodologies may be applied. Recent advances in methodology have significantly broadened the utility of this approach in structural biology and molecular biophysics.

Optimized reverse micelle surfactant system for high-resolution NMR spectroscopy of encapsulated proteins and nucleic acids dissolved in low viscosity fluids

Recently the group of Joshua Wand has described the use of reverse micelles as a platform for DNP for solution-state NMR spectroscopy. Although this article is not directly related to DNP it gives more insight into the use of reverse micelles to obtain high resolution NMR spectra of large marcromolecules.

Dodevski, I., et al., Optimized reverse micelle surfactant system for high-resolution NMR spectroscopy of encapsulated proteins and nucleic acids dissolved in low viscosity fluids. J Am Chem Soc, 2014. 136(9): p. 3465-74.

http://www.ncbi.nlm.nih.gov/pubmed/24495164

An optimized reverse micelle surfactant system has been developed for solution nuclear magnetic resonance studies of encapsulated proteins and nucleic acids dissolved in low viscosity fluids. Comprising the nonionic 1-decanoyl-rac-glycerol and the zwitterionic lauryldimethylamine-N-oxide (10MAG/LDAO), this mixture is shown to efficiently encapsulate a diverse set of proteins and nucleic acids. Chemical shift analyses of these systems show that high structural fidelity is achieved upon encapsulation. The 10MAG/LDAO surfactant system reduces the molecular reorientation time for encapsulated macromolecules larger than ~20 kDa leading to improved overall NMR performance. The 10MAG/LDAO system can also be used for solution NMR studies of lipid-modified proteins. New and efficient strategies for optimization of encapsulation conditions are described. 10MAG/LDAO performs well in both the low viscosity pentane and ultralow viscosity liquid ethane and therefore will serve as a general surfactant system for initiating solution NMR studies of proteins and nucleic acids.

Reverse micelles as a platform for dynamic nuclear polarization in solution NMR of proteins

Valentine, K.G., et al., Reverse micelles as a platform for dynamic nuclear polarization in solution NMR of proteins. J Am Chem Soc, 2014. 136(7): p. 2800-7.

http://pubs.acs.org/doi/abs/10.1021/ja4107176

Despite tremendous advances in recent years, solution NMR remains fundamentally restricted due to its inherent insensitivity. Dynamic nuclear polarization (DNP) potentially offers significant improvements in this respect. The basic DNP strategy is to irradiate the EPR transitions of a stable radical and transfer this nonequilibrium polarization to the hydrogen spins of water, which will in turn transfer polarization to the hydrogens of the macromolecule. Unfortunately, these EPR transitions lie in the microwave range of the electromagnetic spectrum where bulk water absorbs strongly, often resulting in catastrophic heating. Furthermore, the residence times of water on the surface of the protein in bulk solution are generally too short for efficient transfer of polarization. Here we take advantage of the properties of solutions of encapsulated proteins dissolved in low viscosity solvents to implement DNP in liquids. Such samples are largely transparent to the microwave frequencies required and thereby avoid significant heating. Nitroxide radicals are introduced into the reverse micelle system in three ways: attached to the protein, embedded in the reverse micelle shell, and free in the aqueous core. Significant enhancements of the water resonance ranging up to approximately -93 at 0.35 T were observed. We also find that the hydration properties of encapsulated proteins allow for efficient polarization transfer from water to the protein. These and other observations suggest that merging reverse micelle encapsulation technology with DNP offers a route to a significant increase in the sensitivity of solution NMR spectroscopy of proteins and other biomolecules.

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