New insight into the dynamic properties and the active site architecture of H-Ras p21 revealed by X-ray crystallography at very high resolution.
BACKGROUND
In kinetic crystallography, the usually static method of X-ray diffraction is expanded to allow time-resolved analysis of conformational rearrangements in protein structures. To achieve this, reactions have to be triggered within the protein crystals of interest, and optical spectroscopy can be used to monitor the reaction state. For this approach, a modified form of H-Ras p21 was designed which allows reaction initiation and fluorescence readout of the initiated GTPase reaction within the crystalline state. Rearrangements within the crystallized protein due to the progressing reaction and associated heterogeneity in the protein conformations have to be considered in the subsequent refinement processes.


RESULTS
X-ray diffraction experiments on H-Ras p21 in different states along the reaction pathway provide detailed information about the kinetics and mechanism of the GTPase reaction. In addition, a very high data quality of up to 1.0 Å resolution allowed distinguishing two discrete subconformations of H-Ras p21, expanding the knowledge about the intrinsic flexibility of Ras-like proteins, which is important for their function. In a complex of H-Ras•GppNHp (guanosine-5'-(β,γ-imido)-triphosphate), a second Mg2+ ion was found to be coordinated to the γ-phosphate group of GppNHp, which positions the hydrolytically active water molecule very close to the attacked γ-phosphorous atom.


CONCLUSIONS
For the structural analysis of very high-resolution data we have used a new 'two-chain-isotropic-refinement' strategy. This refinement provides an alternative and easy to interpret strategy to reflect the conformational variability within crystal structures of biological macromolecules. The presented fluorescent form of H-Ras p21 will be advantageous for fluorescence studies on H-Ras p21 in which the use of fluorescent nucleotides is not feasible.
Department of Biophysics, Division of Structural Biology, Saarland University, Homburg/Saar, Germany.
Björn U
BU
Klink
Axel J
AJ
Scheidig
D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics*; D018360:Crystallography, X-Ray; D006868:Hydrolysis; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D011487:Protein Conformation; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry* / Q000235:genetics; D014961:X-Ray Diffraction
D016283:Proto-Oncogene Proteins p21(ras)