Reverse Micelle
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 a 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 an apparatus capable of making such samples in ethane and using such samples in a modern NMR cryoprobe without modification or risk.
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High Pressure NMR
In the early 1990s, we became very interested in the application of high hydrostatic pressure to problems in protein biophysics. High pressure NMR of biomacromolecules was largely limited to the so-called autoclave design where the entire probe is put under pressure. This approach allowed very high pressures to be achieved but completely compromised the quality of the probe that could be constructed under this constraint. We implemented a high pressure tube approach that enabled NMR at kilobar pressures in a standard state-of-the-art high performance probe. The key feature was the invention of a method for joining the tube to a pressure manifold. We have used hydrostatic pressure NMR in a variety of ways over the past fifteen years. An initial result was to take advantage of the large difference in DV associated with disruption and solvation of ion pairs (large and negative due to electrostriction) as compared to the disruption of a VDW interaction (small).
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Sparse Sampling NMR
The high probability of degenerate frequencies in NMR spectra of complex biopolymers such as proteins presented a great barrier to detailed analysis. The combination of multidimensional NMR spectroscopy and high magnetic field strengths has overcome the resulting resonance assignment problem for proteins less than 50 kDa. Furthermore, recent advances in NMR instrumentation have largely removed sensitivity as a limiting parameter for protein samples in the millimolar concentration range. As a consequence, the orthogonal linear sampling requirements of conventional multidimensional NMR spectroscopy have required longer acquisition times than potentially needed with respect to signal-to-noise. A number of approaches have been introduced to escape the linear sequential sampling requirements of the standard fast Fourier transform that is usually employed to deal with the processing of the time domain NMR signal. Sparse sampling offers tremendous potential for overcoming the time limitations imposed by the traditional Cartesian sampling of indirectly detected dimensions of multidimensional NMR data. Importantly, in many instances, sensitivity rather than time remains of foremost importance when collecting data on protein samples. We are exploring how to optimize the collection of radial sampled multidimensional NMR data to achieve maximal signal-to-noise in a variety of contexts.
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Hydrogen Exchange
Relaxation