Reverse Micelles As a Platform for Dynamic Nuclear Polarization in Solution NMR of Proteins

Abstract

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 non-equilibrium 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 ~−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 bio-molecules.

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

“…In addition to classic structure determination, the reverse micelle NMR systems offer a range of benefits in biophysical studies of proteins and nucleic acids such as hydration, temperature and pressure dependent phenomena, ligand binding, confined space effects and so on. The recent observation of substantial nitroxide-mediated dynamic nuclear polarization of the reverse micelle water core encourages the development of the reverse micelle NMR method as a generally applicable approach [38]. …”

“…In practice, this provides a two- to three-fold signal-to-noise advantage, which is particularly important in light of the relatively limited concentration of encapsulated protein solutions that can currently be achieved. Looking forward, the recent demonstration that solutions of reverse micelles seem to enable dynamic nuclear polarization in large-volume liquid samples bodes well for overcoming this concentration limitation in the future [38]. …”

“…PRE data are readily obtained for encapsulated proteins using the same methods as applied in aqueous solution. Figure 8 illustrates attenuation of amide hydrogen cross peak intensity in a 15 N-HSQC spectrum in the presence of oxidized (paramagnetic) MTSL versus reduced (diamagnetic) MTSL attached to residue 57 of a double mutant (S57C, C55A) of the 19 kDa protein flavodoxin in 10MAG/LDAO reverse micelles dissolved in liquid pentane [38]. These relaxation enhancements are also mapped to a cartoon of the flavodoxin structure, illustrating the expected distance relationship between the site of the probe and the broadened amide sites.…”

Reverse Micelle Encapsulation of Proteins for NMR Spectroscopy

“…RM samples effectively replace ~98% of the aqueous buffer with alkane, thus eliminating this dielectric penalty, allowing very high salt concentrations to be used in the aqueous RM core (Flynn, Mattiello, Hill, & Wand, 2000). Greatly reduced water content also allows dynamic nuclear polarization (DNP) of proteins in a standard volume solution (Valentine, Mathies, Bédard, Nucci, Dodevski, Stetz et al, 2014) though the general utility of this remains to be demonstrated. In addition, the aqueous core of a RM is a confined space that greatly destabilizes extended or unfolded states and drives proteins to the more compact less extensive state.…”

“…The phase diagram of reverse micelle surfactants is complex and can be manipulated to result in particles with sufficient anisotropic magnetic susceptibility to result in partial alignment and measureable RDCs (Valentine, Pometun, Kielec, Baigelman, Staub, Owens et al, 2006) (Figure 3). Other long distance restraints can also be obtained using paramagnetic relaxation effects (PRE) (Valentine, et al, 2014) and pseudo-contact shifts (PCS) (O’Brien, et al, 2015). High-resolution structures of proteins in different reverse micelle systems have been solved including ubiquitin in AOT reverse micelles (Babu, Flynn, & Wand, 2001), and oxidized horse cytochrome c in 10MAG/LDAO reverse micelles (O’Brien, et al, 2015) (Figure 4).…”