Reverse Micelle NMR

 

About a decade ago we introduced the idea of encapsulating a soluble protein within the protective aqueous core of reverse micelle and then dissolving the entire assembly in a low viscosity fluid. The idea was to use the reduced viscosity to make the protein effectively tumble faster than it otherwise would in free aqueous solution. Aside from the practical issue of placing a protein within such a particle with high structural fidelity, one has to use a solvent of low enough viscosity to overcome the significant "volume penalty" presented by the simple fact that the protein is now part of a larger particle than the protein itself is in free aqueous solution. As a result there are very few solvents that are of low enough viscosity to enable a significant net reduction in rotational correlation time. We have chosen to focus initially on the short chain alkanes including pentane, butane, propane and ethane. All but pentane are gases at STP and thus must be liquified by pressure. Propane and especially ethane require significant pressure for optimum reverse micelle properties. Thus, we were faced with two significant tasks - creating encapsulation conditions conducive to high performance multidimensional and multinuclear NMR that are useful for soluble proteins in general and building apparatus capable of making such samples in ethane and using such samples in a modern NMR cryoprobe without modification or risk.

The basic idea is encapsulated in the figure on the right (no pun intended) where the soluble protein of interest is placed inside the protective water core of a reverse micelle assembly which in turn is solubilized in a low viscosity solvent.


Will it work?

Expectations and
the volume penalty predicted hydrodynamic performance


(Yes)

The "volume penalty" presented by encapsulation is actually quite severe (figure above). Simulations of the anticipated tumbling time for reverse micelle particles containing minimal water (low molar ratio of water to surfactant also known as "water loading" or W0) suggest that significant gains in effective macromolecular tumbling can be achieved. However, experience has shown that significant pressures are required to maintain high quality (i.e. homogeneous and stable) preparations of encapsulated proteins dissolved in liquid propane and ethane. The deficit is that new apparatus is required - a means to prepare such solutions and a high performance (i.e. good filling factor; high resolution quality) NMR tube that can maintain the desired pressure. Fortunately, we have been working with very high pressure NMR for some time and had considerable experience in this area, which greatly facilitated our progress.

The figure above illustrates the anticipated performance for a protein solubilized in a reverse micelle comprised of AOT surfactant molecules. The red line indicates the anticiapted tumbling time for an encapsulated protein (assuming "infinite" viscosity within the reverse micelle i.e the protein tumbling is determined by the tumbling of the reverse micelle) if the reverse micelle assembling were dissolved in a fluid having the viscosity of water. This is the volume penalty that must be overcome. The straight blue line defines the tumbling time of the protein (plus a 20% hydration shell) in water. We wish to beat that i.e. be below it. The remaining lines are the curves predicted for pentane, butane, propane and ethane. Note there are two lines for ethane, at different pressures. Initially, it seemed that very high pressures were required to maintain solutions of reverse micelles in ethane but later we realized a little trick to reduce the pressure required (see below). The exciting conclusion is that in principle a 100 kDa protein could be made to tumble like a 10 kDa protein and thereby allow for high resolution NMR of proteins using the classic suite of triple resonance experiments without the need for deuteration or the TROSY effect.

Current generation high pressure NMR cell

  • AZO injection molded ceramic tube
  • Fail-safe to 10,000 /25,000 psi
  • 400/200 ┬Ál sample volume
  • Self contained or tethered

Peterson and Wand (2005) Rev. Sci. Inst. 76:094101/1 - 094101/7

 

Current Project Personnel:

Nathaniel Nucci
John Gledhill
Kathy Valentine
Igor Dodveski
Adam Seitz
Sabrina Bedard

 

On-Going & Future Projects

Our current methodological focus of the reverse micelle strategy is a continuing effort to improve the sample i.e. find more efficient ways to sample the surfactant library for optimal solubilization conditions and broaden the library itself. This is particularly important for integral membrane proteins. See structural biology for applications.

 

Useful papers:

Kielec et al. (2009) Structure
Lefebvre et al (2005) JMR
Peterson & Wand (2005) JACS
Peterson & Wand (2005) Rev. Sci. Inst.
Wand et al. (1998) PNAS

Over a period of about a decade we manufactured several different pressure-tolerant tubes capable of "reverse micelle NMR." The final generation of custom made tubes is based on a ceramic NMR tube fitted with our patented method for high pressure sealing to a BeCu or stainless steel pressure manifold or valve that allows transfer of the sample and sealing of the tube. This basic technology has been improved and commercialized by Daedalus Innovations (please see disclosure). We are no longer developing this aspect of the reverse micelle apparatus.

There are several mechanisms for preparing solutions of encapsulated proteins dissolved in low viscosity fluids that require pressurization to maintain a liquid state. All demand that the sample preparation be done under pressure, which in the case of ethane will often exceed 4,000 p.s.i (~265 bar). Again, this apparatus was developed over the past decade and has since been commercialized by Daedalus Innovations. The strategy is quite simple. Solvent gas is liquified by pressurization in a syringe pump. Protein, surfactants and water are combined in a mixing chamber with the solvent. A novel high pressure ram moves the sample to the reverse micelle NMR cell. We now prepare solutions of encapsulated proteins in ethane routinely and completely without incident do NMR spectroscopy in modern triple resonance cryoprobes. Here are an original few spectra:

Ubiquitin at 55MPa (pl=6.6) in 100mM AOT

Cytochrome c at 55MPa (pl=11) in 150mM 70% C12E4 /25% AOT / 5% DTAB

Flavoprotein at 28MPa (pl=4.2) in 100mM CTAB, 8% hexanol

Peterson et al (2005) JACS 127:10176-10177

  • Can prepare and tranfer samples at pressures up to 15,000 psi
  • Novel piston ram for sample transfer
  • Can accomodate multiple mixing stages
  • Can be automated