CRYSTALLOGRAPHIC STUDIES ON P21(H-RAS) USING THE SYNCHROTRON LAUE METHOD - IMPROVEMENT OF CRYSTAL QUALITY AND MONITORING OF THE GTPASE REACTION AT DIFFERENT TIME POINTS

The parameters affecting the crystal quality of complexes between p21H -ras and caged GTP have been investigated. The use of pure diastereome rs of caged GTP complexed to the more stable p21(G12P)' mutant of p21 and the addition of n-octyl-beta-D-glucopyranoside improved the reprod ucibility and decreased the mosaicity of the crystals significantly. F urthermore, the crystallization technique was changed from the batch m ethod to the sitting-drop technique. With the availability of a larger yield of well ordered crystals, it was possible to extend the time-re solved crystallographic investigations on p21H-ras. A structure of p21 (G12P)':GTP could be obtained 2 min after photolytic removal of the ca ge group and led to the identification of a previously unidentified co nformation for the so-called catalytically active loop L4. The refinem ent of five data sets collected within 2 min at different times (2-4, 11-13, 20-22, 30-32 and 90-92 min) after the initiation of the intrins ic GTPase reaction of the protein indicates that the synchrotron Laue method can be used to detect small structural changes and alternative conformations, but is presently limited in the analysis of larger rear rangements since these produce diffuse and broken electron density.