The structural and dynamic aspects of proteins have been at center stage of our understanding of the basis of their function. Nuclear magnetic resonance in solution has contributed significantly to this advancement and the information inherent in the NMR phenomena offers much more. Yet, despite tremendous advances in technology, experimental design and analytical strategies, solution NMR spectroscopy remains fundamentally restricted due to its extraordinary insensitivity. The inability to investigate protens and other biopolymers at well below sub-millimolar concentrations and using sub-micromole amounts presents severe limitations on future applications. Nevertheless, solution NMR offers, in principle, access to information that is very difficult to obtain by other means. Examples include access to dynamics over an enormous range of time scales, to details of ligand binding, to structures in unusual contexts and so on. Thus, it seems important to improve the sensitivity of the solution NMR experiment in order to reduce experiment time, lower the absolute quantities of sample required and open a lower concentration regime where proteins of limited solubility can be accessed. With this in mind there has been a revival of an old phenomenon - dynamic nuclear polarization (DNP). The idea is to use the enormously greater polarization of a radical electron in a magnetic field to polarize nuclei such as hydrogen to a much greater degree than the Boltzmann distribution dictated by the properties of the nuclei themselves. The physics underlying this process can be quite complicated, particularly in the solid state where several mechanisms for polarization are operative. In solution, it is generally thought that such DNP transfer will occur primarily through the Overhauser effect. One can imagine that increases in sensitivity of several hundred folds are accessible. For solution NMR, the basic strategy is to saturate the electronic transition of a stable free radical and transfer this non-equilibrium polarization to the hydrogen spins of water, which will in turn transfer this polarization to the hydrogens of the dissolved macromolecule. Unfortunately, technical aspects of this approach seem to prove fatal to the idea in its current form. The primary reason is that the frequency of the electron transition of suitable radicals lies in the subTHz spectrum where water absorbs strongly. Thus, irradiation results in catastrophic heating of the sample and its destruction. Here we will take advantage of the physical properties of solutions of encapsulated proteins dissolved in low viscosity solvents of suitable dielectric character. Such samples are largely transparent to the subTHz frequencies required and thereby avoid significant heating during saturation of the electronic transition. A variety of proteins ranging from small to large soluble proteins; acidic t basic proteins; integral and anchored membrane proteins; proteins of marginal stability and nucleic acids can be encapsulated with high structural fidelity. Thus the merging of the reverse micelle technology with DNP will provide a significant increase in the sensitivity of the solution NMR spectroscopy of proteins and nucleic acids.
Knowledge of the structure of proteins and nucleic acids at the atomic-scale is central to our understanding of their biological function. Such information is usually derived from crystallography and nuclear magnetic resonance (NMR). Unfortunately, NMR is inherently limited by its poor sensitivity. This project seeks to dramatically increase the sensitivity of solution NMR spectroscopy by enabling the use of dynamic nuclear polarization.
| Valentine, Kathleen G; Mathies, Guinevere; Bédard, Sabrina et al. (2014) Reverse micelles as a platform for dynamic nuclear polarization in solution NMR of proteins. J Am Chem Soc 136:2800-7 |
| Nucci, Nathaniel V; Valentine, Kathleen G; Wand, A Joshua (2014) High-resolution NMR spectroscopy of encapsulated proteins dissolved in low-viscosity fluids. J Magn Reson 241:137-47 |
