tailieunhanh - Báo cáo khoa học: Slow conformational dynamics of the guanine nucleotide-binding protein Ras complexed with the GTP analogue GTPcS

The guanine nucleotide-binding protein Ras occurs in solution in two different conformational states, state 1 and state 2 with an equilibrium constant K12 of , when the GTP analogue guanosine-5¢-(b,c-imido)tri-phosphate or guanosine-5¢-(b,c-methyleno)triphosphate is bound to the active centre. | ỊFEBS Journal Slow conformational dynamics of the guanine nucleotide-binding protein Ras complexed with the GTP analogue GTPyS Michael Spoerner1 Andrea Nuehs1 Christian Herrmann2 Guido Steiner1 and Hans Robert Kalbitzer1 1 Universitat Regensburg Institut fur Biophysik und physikalische Biochemie Germany 2 Ruhr Universitat Bochum Physikalische Chemie I Germany Keywords conformationalequilibria GTP analog GTPyS Ras Correspondence H. R. Kalbitzer Institut fur Biophysik und physikalische Biochemie UniversitatsstraBe 31 Regensburg D-93040 Germany Fax 49 941 943 2479 Tel 49 941 943 2595 E-mail . Received 28 July 2006 revised 13 November 2006 accepted 8 January 2007 doi The guanine nucleotide-binding protein Ras occurs in solution in two different conformational states state 1 and state 2 with an equilibrium constant K12 of when the GTP analogue guanosine-5 - P y-imido tri-phosphate or guanosine-5 - P y-methyleno triphosphate is bound to the active centre. State 2 is assumed to represent a strong binding state for effectors with a conformation similar to that found for Ras complexed to effectors. In the other state state 1 the switch regions of Ras are most probably dynamically disordered. Ras variants that exist predominantly in state 1 show a drastically reduced affinity to effectors. In contrast Ras wt bound to the GTP analogue guanosine-5 -O- 3-thiotriphosphate GTPyS leads to 31P NMR spectra that indicate the prevalence of only one conformational state with K12 10. Titration with the Ras-binding domain of Raf-kinase Raf-RBD shows that this state corresponds to effector binding state 2. In the GTPcS complex of the effector loop mutants Ras T35S and Ras T35A two conformational states different to state 2 are detected which interconvert over a millisecond time scale. Binding studies with Raf-RBD suggest that both mutants exist mainly in low-affinity states 1a and 1b. From line-shape .