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 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 ∼-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.

Figure 1

Schematic illustrations of the strategies for introduction of nitroxide spin radicals to reverse micelles. (A) Nitroxide covalently attached to the protein (MTSL). (B) Nitroxide dissolved in the aqueous core (TEMPOL). (C) Nitroxide attached to a carrier embedded in the surfactant shell (TEMPO-PC).

Figure 2

Structural integrity of encapsulated, spin-labeled flavodoxin is maintained. ¹⁵N HSQC spectra of (A) ¹⁵N flavodoxin (C55A), (B) ¹⁵N flavodoxin (C55A, S72C) with ¹⁵N MTSL covalently attached, (C) ¹⁵N flavodoxin C55A with TEMPOL, and (D) ¹⁵N flavodoxin (C55A) with TEMPO-PC. (E) The chemical shift differences (Δ = ((Δδ_Nγ_N/γ_H)² + (Δδ_H)²)^1/2) of backbone amide ¹H–¹⁵N resonances of flavodoxin in free aqueous solution and flavodoxin in 10MAG/LDAO reverse micelles. All residues that could be measured are shown including the site of mutation and ligand attachment (red arrow). Very minor chemical shift perturbations are found (R² = 0.999 and <rmsd> = 0.010), indicating that high structural fidelity is maintained upon encapsulation of the protein with spin label in the three labeling scenarios examined (see Figure 1).

Figure 3

X-band EPR spectra of the nitroxide spin radical in the three labeling scenarios in 10MAG/LDAO reverse micelles. (A) ¹⁵N-flavodoxin in the aqueous core and covalently attached to ¹⁵N-MTSL (B) ¹⁴N-TEMPOL solubilized in the aqueous core with ¹⁵N-flavodoxin. (C) ¹⁵N-flavodoxin and ¹⁴N-TEMPO-PC solubilized in the reverse micelle surfactant shell. The triplet splitting is a result of the spin 1 ¹⁴N-electron hyperfine coupling for ¹⁴N-TEMPO-PC and ¹⁴N-TEMPOL while the doublet splitting arises from the spin 1/2 ¹⁵N-electron hyperfine coupling of the ¹⁵N-MTSL. These spectra were obtained at 25 °C with 4 mm sample tubes. The red arrow indicates the frequency for the application of the microwave power for the DNP experiments.

Figure 4

Dependence of paramagnetic relaxation enhancements of the encapsulated protein on the method of nitroxide incorporation. Color-coded PREs of amide ¹⁵N–¹H correlations of flavodoxin encapsulated in 10MAG/LDAO reverse micelles are mapped onto the 1FLV PDB structure. The FMN moiety is shown in blue. (A) PREs from the MTSL spin label covalently attached at Cys72 (green dot) of encapsulated flavodoxin (C55A, S72C). (B) PREs with TEMPOL in the aqueous core of the reverse micelle with flavodoxin (C55A) encapsulated. (C) PREs with TEMPO-PC solubilized in the surfactant shell of the reverse micelle with flavodoxin (C55A) encapsulated. Structural renderings were generated using PyMol.

Figure 5

Dynamic nuclear polarization in reverse micelles. ¹H NMR spectra (14.7 MHz) of the water core of 10MAG/LDAO reverse micelles with (blue) and without (red) saturation of the 9.4 GHz EPR transition indicated in Figure 3 for (A) ¹⁵N-flavodoxin covalently attached to ¹⁵N-MTSL and dissolved in the aqueous core; (B) ¹⁴N-TEMPOL solubilized in the aqueous core with ¹⁵N-flavodoxin; and (C) ¹⁵N-flavodoxin in the aqueous core and ¹⁴N-TEMPO-PC solubilized in the reverse micelle surfactant shell. Samples were prepared with a W₀ of 20.

Figure 6

¹H spectra of the reverse micelle solution. Overlay of the 14.7 MHz DNP enhanced ¹H spectrum (blue, phase inverted for clarity) onto the 600 MHz ¹H spectrum (black) of 400 μM flavodoxin (C55A), 200 μM TEMPOL solubilized in 100 mM LDAO/10MAG reverse micelles at a W₀ of 20. A 10 Hz exponential apodization function was applied to both FIDs. Integration values for the 600 MHz spectrum are indicated for the water resonance and the region containing the resonances due to surfactants and alkane solvent.

Figure 7

Efficiency of transfer of magnetization between encapsulated protein and the water core at 14 T. (A) Semilog plot of the ratio of the intensity the water NOE cross peak relative to the amide diagonal resonance of a 3D ¹⁵N NOESY HSQC for flavodoxin (C55A) encapsulated in LDAO/10 MAG reverse micelles at pH 8.0 in red and aqueous flavodoxin (C55A) at pH 8.0 in blue The NOE mixing time was 100 ms for both spectra. Approximately 90% of the amide hydrogens of encapsulated flavodoxin show NOEs to water. Fewer sites show NOEs to water in the aqueous condition with the intensity ratio reduced by 1–2 orders of magnitude compared to the reverse micelle spectrum. (B) The ¹H water plane of the 3D ¹⁵N NOESY HSQC spectrum of flavodoxin (C55A) encapsulated in LDAO/10 MAG reverse micelles at pH 8.0. (C) Mapping of the NOE intensity ratio of the water cross peak to the amide diagonal peak onto the three-dimensional structure of flavodoxin (PDB code FLV1). The color bar indicates white through dark blue for stronger NOEs with red indicating resonances that display significant hydrogen exchange with water. These amide hydrogens are located at the edges of secondary structure elements and in loops.