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Abstract

The folding, structure and biological function of many proteins are inherently dynamic properties of the protein molecule. Often, the respective molecular processes are preserved upon protein crystallization, leading, in X-ray diffraction experiments, to a blurring of the electron density map and reducing the resolution of the derived structure. Nuclear magnetic resonance (NMR) is known to be an alternative method to study molecular structure and dynamics. We designed and built a probe for phosphorus solid state NMR that allows for the first time to study static properties as well as dynamic processes in single-crystals of a protein by NMR spectroscopy. The sensitivity achieved is sufficient to detect the NMR signal from individual phosphorus sites in a 0.3 mm3 size single-crystal of GTPase Ras bound to the nucleotide GppNHp, that is, the signal from approximately 1015 phosphorus nuclei. The NMR spectra obtained are discussed in terms of the conformational variability of the active center of the Ras–nucleotide complex. We conclude that, in the crystal, the protein complex exists in three different conformations. Magic angle spinning (MAS) NMR spectra of a powder sample of Ras–GppNHp show a splitting of one of the phosphate resonances and thus confirm this conclusion. The MAS spectra provide, furthermore, evidence of a slow, temperature-dependent dynamic exchange process in the Ras protein crystal.