Solid-state 31P NMR spectroscopy of microcrystals of the Ras protein and its effector loop mutants: comparison between crystalline and solution state.
Cycling between a GTP bound "on" state and a GDP bound "off" state, guanine nucleotide-binding (GNB) proteins act as molecular switches. The switching process and the interaction with effectors, GTPase-activating proteins, and guanosine nucleotide-exchange factors is accompanied by pronounced conformational changes of the switch regions of the GNB proteins. The aim of the present contribution is to correlate conformational changes observed by liquid-state NMR with solid-state (31)P NMR data and with the results of X-ray crystallography. Crystalline wild-type Ras complexed with GTP analogs such as GppCH(2)p and GppNHp could be prepared. At low temperatures, two different signals were found for the gamma-phosphate group of GppNHp bound to wild-type Ras. This behavior indicates the existence of two different conformations of the molecule in the crystalline state as it is found in solution but not by X-ray crystallography. In contrast to the GppNHp complex, the two separate gamma-phosphate signals could not be observed for GppCH(2)p bound to wild-type Ras. However, an increasing linewidth at low temperature indicates the presence of an exchange process. The results obtained for the wild-type protein are compared with the behavior of GppNHp complexes of the effector loop mutants Ras(T35S) and Ras(T35A). These mutants prefer a conformation similar to the GDP bound "off" state.
Iuga
Adriana
A
Universität Regensburg, Institut für Biophysik und physikalische Biochemie, Postfach, D-93040 Regensburg, Germany.
Spoerner
Michael
M
Kalbitzer
Hans Robert
HR
Brunner
Eike
E
D001665:Binding Sites; D003460:Crystallization; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism; D066298:In Vitro Techniques; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D010758:Phosphorus / Q000737:chemistry; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012996:Solutions; D018631:ras Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D011994:Recombinant Proteins; D012996:Solutions; D006153:Guanosine Diphosphate; D010758:Phosphorus; D006165:Guanylyl Imidodiphosphate; C004805:5'-guanylylmethylenebisphosphonate; D006160:Guanosine Triphosphate; D018631:ras Proteins