Introduction

See Nucci et al. (2014) J. Magn. Reson. for a recent review of this topic.

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.

How are reverse micelles prepared in alkanes that are liquid at STP:

Water Loading (W0)
Most reverse literature utilizes the term "water loading" or W0. This refers to the molar ratio of water to surfactants. Generally, this excludes the co-surfactant hexanol. We find it best to explicitly define the absolute concentration of all elements of the reverse micelle solution.

Preparation of Proteins
Your protein needs to be very pure. Contaminants often ruin the phase diagram for the reverse mcielle solution. It is very iportant that you pay close attention to ionic strength. You must be careful to take care of the Donnan equilibrium when setting the ionic strength. A surprisingly large amount of ions can be carried with your protein during purification and concentration. This is the most common source of failure or irreproducibility. Generally you want to keep your ionic strength below 150 mM but this is an adjustable parameter. pH is also a very important adjustable parameter but note that setting the pH is so simple as deciding on the buffer to prepare you protein in. See the sections on Protein Encapsulation and Adjusting the pH.

Direct injection
Most reverse micelle samples in the lab are prepared via inject injection. To make an RM sample via direct injection first make sure to calculate all of your surfactant ratios, how much water to add for your desired water loading, and how concentrated your protein must be to reach the desired sample concentration. For example, for a 500 uL sample made of 75mM surfactant and 80uM protein and a water-loading of 15 you must add 10.15 uL of 4 mM protein solution. A sheet to help with the measurements can be found here: [[1]]. The process is as follows:

This method requires that that your protein can be made to high enough concentrations so that may be directly injected into the surfactant mixture to produce samples of high enough total concentration at the desired water loading. This is often in the range of 4-8 mM, which is too high for many proteins. It should be noted that perfect solutions are not required. Other methods must be used if you cannot concentrate your protein that high due to insolubility & aggregationissues.

Evaporation/injection
The evaporatio/injection approach is one of the most common methods in the lab for concentrating proteins in reverse micelle solutions. It also works well to change water loading or to keep water loading constant upon the addition of a ligand. It entails repetitive injection followed by partial evaporation of the bulk alkane and then a rebalancing of surfactants and water to the desired concentrations. You begin by using a reasonably concentrated solution of your protein (say ~1mM). You injection the volume appropriate for the desired water loading. Approximately half of the organic solvent is then evaporating by a gentle nitrogen stream. Some water will go with it. At this stage, you add more solvent and an additional injection of your 1 mM protein solution. Your surfactant ratios, water loading, and bulk solvent will stay the same every round, but your protein concentration but will increase after every round of evaporating and injection. Be careful when using this method to check your protein both by eye and by NMR after every round or two of evaporation injection. You will want to make sure that you are indeed removing enough water so that your final waterloading is your target water-loading. You also want to make sure that your sample remains clear when reconsititued to make sure it remains stable. In general, this is a great method for proteins that stably encapsulate but cannot be concentrated high enough for the direct injection method to work. A paper describing this method in more detail is here.

Phase transfer

Phase transfer methods are a bit more uncommon but is useful when you don’t know what your optimal waterloading will be, or if you can’t concentrate your protein very much.

The goal is that the protein will enter the reverse micelles with the most stable quantity of water. It reduces uncertainty with water loading and as amenable for proteins that cannot be concentrated for direct injection.

Lyophilization/reconstitution
This is the least commonly used method in the lab, but has been used with some success (e.g. ubiquitin). It is worth a shot, but does not always prove successful. In this method you use lyophilized protein and add that directly to the surfactant/organic mixture. Then add a small volume of just your buffer. vortex well, and hope that your protein dissolves and enters the micelle.