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

Abstract

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 (< 25 kDa) 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 100 kDa, 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.

Role of cavities and hydration in the pressure unfolding of T4lysozyme

“…The viscosity of pentane at 1 bar is one fourth that of water. As the reverse micelle particle is approximately four times larger than the protein in free solution, the encapsulated protein tumbles with approximately the same correlation time as aqueous protein at ambient pressure (46). The viscosity of pentane at 2 kbar, however, is approximately the same as that of water, so the tumbling time of the reverse micelle particle increases by a factor of four over the pressure range examined here (47).…”

Defining the Apoptotic Trigger

“…Encapsulation of proteins within the water core of reverse micelles largely eliminates artifacts introduced by hydrogen exchange (18). When combined with heteronuclear NMR methods to provide spectral resolution, the nuclear Overhauser effect can be confidently employed to probe for protein/ water interactions (8,18). Analysis of 1 H-1 H NOEs involving the water resonance allowed the identification of four distinct FIGURE 1.…”

“…To mimic this condition, we employ the interior water core of a reverse micelle to solubilize cytochrome c and use the surrounding surfactant shell as a host for cardiolipin. Under appropriate conditions, proteins can be encapsulated within reverse micelles with high structural fidelity (8). When prepared in solvents of sufficiently low viscosity (9), one can also obtain high resolution solution NMR data to comprehensively characterize the structure of the encapsulated protein (10) and its interaction with ligands embedded in the surfactant wall or dissolved in the aqueous core (11).…”

Biomolecular Crowding Arising from Small Molecules, Molecular Constraints, Surface Packing, and Nano-Confinement

“…After this initial application, Wand and coworkers have extended and optimized this RM encapsulation strategy for many other proteins, including cytochrome c and flavodoxin, and have used this method to investigate ligand binding, among other processes. 61 …”