Observation of slow dynamic exchange processes in RAS protein crystals by 31P solid state NMR spectroscopy
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 0.3mm(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.
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The conformational flexibility of RAS proteins revealed by atomistic simulations is consistent with 31P-NMR spectroscopy studies.
“This conformational flexibility revealed through atomistic simulations is consistent with 31P-NMR spectroscopy studies of RAS proteins [21,52]”
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- Authors
- Stumber, M., Geyer, M., Graf, R., Robert Kalbitzer, H., Scheffzek, K., & Haeberlen, U.
- Affiliations
- Max Planck Institute for Medical Research (DE)
- Max Planck Society (DE)
- Corresponding author
- Geyer, Matthias
- Venue
- Journal of Molecular Biology
- Publisher
- Elsevier BV
- Published
- 2002-11
- Volume
- 323
- Issue
- 5
- Pages
- 899-907
- ISSN
- 0022-2836 (ISSN-L)
- OpenAlex
- W2088629679
- Cited by
- 22
- References
- 40
- Citations by year
- 2022: 1 · 2018: 1 · 2015: 1 · 2014: 1 · 2013: 1
- FWCI
- 1.70
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Journal of Molecular Biology (publishedVersion)
- PubMed (publishedVersion)
- University of Regensburg Publication Server (University of Regensburg) (acceptedVersion)
- Max Planck Institute for Plasma Physics (submittedVersion)
- University of Regensburg Publication Server (University of Regensburg) (submittedVersion)
- MPG.PuRe (Max Planck Society) (submittedVersion)
- Funders
- Peter und Traudl Engelhorn Stiftung
- Topics
- Physical Sciences › Chemistry › Spectroscopy › Advanced NMR Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Electron Spin Resonance Studies
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
- Keywords
- Ras GTPase, protein dynamics, conformational heterogeneity, single-crystal NMR, 31P solid-state NMR, magic angle spinning, protein crystallization
- MeSH terms
- Binding Sites, Crystallization, Guanylyl Imidodiphosphate, Models, Molecular, Phosphorus, Protein Conformation, Thermodynamics, Crystallography, X-Ray, ras Proteins, Nuclear Magnetic Resonance, Biomolecular
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