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J Mol Biol. 2004 Sep 17;342(3):1033-40.

Solid-state 31P NMR spectroscopy of microcrystals of the Ras protein and its effector loop mutants: comparison between crystalline and solution state.

Author information

1
Universität Regensburg, Institut für Biophysik und physikalische Biochemie, Postfach, D-93040 Regensburg, Germany.

Abstract

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.

PMID:
15342254
DOI:
10.1016/j.jmb.2004.07.077
[Indexed for MEDLINE]

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