Reverse Micelle NMR |
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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. |
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Will it work? Expectations and
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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. |
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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.
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