Observation of Slow Dynamic Exchange Processes in Ras Protein Crystals by 31 P Solid State NMR Spectroscopy Michael Stumber 1 , Matthias Geyer 1 * , Robert Graf 2 Hans Robert Kalbitzer 3 , Klaus Scheffzek 4 and Ulrich Haeberlen 1 1 Max-Planck-Institut fu ¨r medizinische Forschung Jahnstrasse 29, 69120 Heidelberg, Germany 2 Max-Planck-Institut fu ¨r Polymerforschung Ackermannweg 10, 55128 Mainz, Germany 3 Universita ¨t Regensburg Institut fu ¨r Biophysik und Physikalische Biochemie Postfach, 93040 Regensburg Germany 4 European Molecular Biology Laboratory, Structural and Computational Biology Programme, Meyerhofstraße 1 69117 Heidelberg, Germany The folding, structure and biological function of many proteins are inherently dynamic properties of the protein molecule. Often, the respec- tive 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 mm 3 size single- crystal of GTPase Ras bound to the nucleotide GppNHp, that is, the signal from approximately 10 15 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. q 2002 Elsevier Science Ltd. All rights reserved Keywords: solid state NMR; MAS NMR; protein dynamics; dynamics in crystals; Ras *Corresponding author Introduction Guanine nucleotide-binding proteins of the superfamily of small GTPases play a central role in cell signalling events. They function as molecu- lar switches cycling between guanosine tri- phosphate (GTP)-bound “on” and guanosine diphosphate (GDP)-bound “off” states. 1 In the GTP-bound state, they interact with effector pro- teins which trigger a variety of downstream events. The most prominent member of this family is the Ras protein, which relays signals from cell surface receptors to the nucleus to stimulate cell prolifera- tion and differentiation. 2 Specific mutations of Ras leading to inhibition of GTP hydrolysis or to acceleration of GDP release are involved in about 30% of human cancers. 3 Mutation to oncogenic Ras is the most frequently occurring gain-of- function alteration detected in human tumors. 4 The structure of Ras has been determined by X-ray crystallography 5–7 and NMR spectroscopy 8,9 in its diphosphate (GDP) and triphosphate (GppNHp and GppCH 2 p, two slowly hydrolizing GTP analogues, and also with GTP) bound forms. The structures obtained indicate that two regions, called switch 1 (effector loop, residues 30–38) and switch 2 (loop L4, residues 60–76), are crucial to the functioning of Ras as a signalling molecule. Both switch regions are close to the bound 0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved Present address: M. Geyer, Max-Planck-Institut fu ¨r molekulare Physiologie, Abteilung Physikalische Biochemie, Otto-Hahn-Str. 11, 44227 Dortmund, Germany. E-mail address of the corresponding author: geyer@mpimf-heidelberg.mpg.de Abbreviations used: MAS NMR, magic angle spinning NMR. doi:10.1016/S0022-2836(02)01010-0 available online at http://www.idealibrary.com on B w J. Mol. Biol. (2002) 323, 899–907