| High field solid state NMR is a promising method for elucidating protein structure, particularly in the case of proteins that are not easily studied by X-ray crystallography or solution state NMR. Since the application of solid state NMR to biological samples is relatively new, methods development work is required in order to realize the full potential of this technique. This work describes the design and construction of a triple resonance probe with variable temperature capability, as well as a novel method for preparing nanocrystalline protein samples for NMR experiments.; At high frequency, standard design methods for NMR probes using commercial capacitors prove difficult to implement. A triply tuned 13C, 15N and 1H probe for use at 18.8 T was built using variable transmission line segments as tunable reactances. The compact geometry of this design allows three channels with high power handling capability to fit in a medium bore (63 mm) magnet. Extended time variable temperature operation is integral to the mechanical design, enabling the temperature control necessary for investigation of biological macromolecules. Representative data illustrating the efficiency, RF homogeneity, and signal to noise factor of the probe are presented, as are data illustrating the interaction of the probe electronics with conductive hydrated samples as a function of temperature.; A simple procedure for preparing nanocrystalline protein samples for study by solid state NMR spectroscopy that applies general principles for the crystallization of macromolecules is described. It is shown that nanocrystals prepared by rapid batch crystallization yield equivalent 13C solid state NMR spectra to those of larger X-ray diffraction quality crystals. Single crystal and powder X-ray diffraction measurements were made to compare the degree of order present in polycrystalline, nanocrystalline, and lyophilized ubiquitin.; High resolution natural abundance 13C spectra of lysozyme, ribonuclease A, streptavidin, and cytochrome c nanocrystals demonstrate that this technique is generally applicable. Natural abundance protein nanocrystals can be used diagnostically to optimize conditions for the preparation of isotopically labeled samples. Since the nanocrystals are easily and rapidly produced, they may provide a means by which proteins that have eluded structural determination can be studied. |
