Reverse Micelle Encapsulation of Proteins for NMR Spectroscopy

Abstract

Reverse micelle (RM) encapsulation of proteins for NMR spectroscopy has many advantages over standard NMR methods such as enhanced tumbling and improved sensitivity. It has opened many otherwise difficult lines of investigation including the study of membrane associated proteins, large soluble proteins, unstable protein states, and the study of protein surface hydration dynamics. Recent technological developments have extended the ability of RM encapsulation with high structural fidelity for nearly all proteins and thereby allow high-quality state-of-the-art NMR spectroscopy. Optimal conditions are achieved using a streamlined screening protocol, which is described here. Commonly studied proteins spanning a range of molecular weights are used as examples. Very low-viscosity alkane solvents, such as propane or ethane, are useful for studying very large proteins but require the use of specialized equipment to permit preparation and maintenance of well-behaved solutions under elevated pressure. The procedures for the preparation and use of solutions of reverse micelles in liquefied ethane and propane are described. The focus of this chapter is to provide procedures to optimally encapsulate proteins in reverse micelles for modern NMR applications.

Sustainable low liquor ratio dyeing of cotton with C.I. Reactive Blue 21 using dioctyl sodium sulfosuccinate

“…The chemical structure of AOT is made up of a moiety of sulfonate groups as the hydrophilic head and twin aliphatic chains as the hydrophobic tail covalently connected at the head group. 32 These surfactant micelles may exert anti-agglomeration effects on copper phthalocyanine dyes. The purpose of this study is to reduce the liquor ratio by using AOT to generate micelles of phthalocyanine reactive dyes.…”

Optimization of Biocompatibility for a Hydrophilic Biological Molecule Encapsulation System

“…The data presented here represent the first effort to adapt the 10MAG/LDAO system for applications in which biocompatibility is important. Previous uses of this surfactant system have focused on structural studies of proteins and other biopolymers [ 17 , 21 ]. 10MAG/LDAO has proven to be the most versatile system for encapsulating proteins in reverse micelles without disrupting their native fold.…”

“…Water-in-oil-in-water (W/O/W) nanoparticles are comprised of aqueous pockets that are encapsulated by an oil layer stabilized by surfactants that make the whole particle water-soluble [ 15 ]. Surfactants present in the interior layer form reverse micelles (RM), i.e., water-in-oil microemulsions, capable of hydrophilic drug encapsulation [ 17 ]. Hydrophilic drugs can be loaded into these systems without high temperatures or pressures that might negatively impact the structural integrity of protein-based drugs.…”

Protein conformational entropy is not slaved to water

“…The ubiquitin reverse micelle sample was prepared 42 in 75 mM bis-2-ethylhexylsulfosuccinate (AOT) at a W 0 (molar water:surfactant ratio) of 10 in perdeuterated hexane. When encapsulation of each protein is appropriately optimized 25 , the chemical shifts of backbone and methyl resonances of the encapsulated protein are statistically indistinguishable from the protein in bulk solution indicating that the native state is maintained. Indeed, the explicitly determined structure of encapsulated ubiquitin is essentially that same as that in bulk solution and in the crystal 42 .…”

“…We then employed the unique properties of proteins encapsulated within the water core of reverse micelles to further investigate the influence of the dynamics of the hydration shell on internal protein motion. Under the water-limited conditions employed here, the reverse micelle encapsulates a single protein molecule surrounded by several thousand waters composing a 10–15 Å hydration layer 25 . The sign of nuclear Overhauser effects arising from protein-water 1 H– 1 H dipolar interactions 26 indicates that the effective correlation time of water adjacent to the protein in the reverse micelle is slowed by two orders of magnitude relative to bulk water 27 .…”