Conformational states of human H-Ras detected by high-field EPR, ENDOR, and 31P NMR spectroscopy.
Ras is a central constituent of the intracellular signal transduction that switches between its inactive state with GDP bound and its active state with GTP bound. A number of different X-ray structures are available. Different magnetic resonance techniques were used to characterise the conformational states of the protein and are summarised here. 31P NMR spectroscopy was used as probe for the environment of the phosphate groups of the bound nucleotide. It shows that in liquid solution additional conformational states in the GDP as well as in the GTP forms coexist which are not detected by X-ray crystallography. Some of them can also be detected by solid-state NMR in the micro crystalline state. EPR and ENDOR spectroscopy were used to probe the environment of the divalent metal ion (Mg2+ was replaced by Mn2+) bound to the nucleotide in the protein. Here again different states could be observed. Substitution of normal water by 17O-enriched water allowed the determination of the number of water molecules in the first coordination sphere of the metal ion. In liquid solution, they indicate again the existence of different conformational states. At low temperatures in the frozen state ENDOR spectroscopy suggests that only one state exists for the GDP- and GTP-bound form of Ras, respectively.
Spoerner
Michael
M
Institute for Biophysics and Physical Biochemistry, University of Regensburg, Germany.
Prisner
Thomas F
TF
Bennati
Marina
M
Hertel
Melanie M
MM
Weiden
Norbert
N
Schweins
Thomas
T
Kalbitzer
Hans Robert
HR
D004578:Electron Spin Resonance Spectroscopy; D005615:Freezing; D006801:Humans; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008968:Molecular Conformation; D015394:Molecular Structure; D009154:Mutation / Q000235:genetics; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism; D010759:Phosphorus Isotopes; D012996:Solutions; D018631:ras Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D008024:Ligands; D009711:Nucleotides; D010759:Phosphorus Isotopes; D012996:Solutions; D018631:ras Proteins