Characterization of rare conformational states of proteins by combination of high-pressure X-raycrystallography with high-pressure NMR spectroscopy. Application to the small GproteinRas, its oncogenic mutants and its drug complexes.

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High pressure NMR spectroscopy (HPNMR) represents a powerful, still developing method to detect and characterize rare 'excited' conformational states. These states play an essential role in function and folding of proteins. HPNMR provides their thermodynamic parameters but gives structural information with limited resolution only. High pressure macromolecular x-ray crystallography (HPMX) is a complementary new method in protein science that basically can provide highly resolved spatial structures at high pressures. As component of an important and well-characterized signal transduction system we will concentrate on the small G-Protein Ras (rat sarcoma) protein (Ras mutations are found mutated in more than 30 % of all human cancers). It serves as a well-characterized model for method development in the proposed project, but also novel thermodynamic and structural insights are from high medical importance since they may help with the development of allosteric Ras inhibitors. By HPNMR of Ras in complex with the GTP analog GppNHp four different states 1(0), 1(T), 2(T), and 3(T) could be thermodynamically characterized that coexist at ambient pressure. Small compounds that preferentially bind to state 1(T) can allosterically inhibit the effector interaction and thus in principle can suppress the proliferative effect of oncogenic Ras mutants. The main goals of this project are (1) a thorough quantitative analysis of the rare conformational states of H- and K-Ras and their oncogenic mutants in complex with GDP and GppNHp by HPNMR supported by alternative NMR methods such as CEST and relaxation dispersion measurements, (2) to prove that HPMX can efficiently trap high-energy conformers of proteins identified in solution by high-pressure NMR spectroscopy that were identified in solution by HPNMR, (3) to correlate the observed pressure response of NMR parameters with structural changes detected by HPMX, and (4) to show that in principle the obtained high-pressure crystallographic structures can be used to design specific allosteric inhibitors binding to rare states. For showing if the thermodynamically similar state sin crystal and solution are also structurally similar, population-weighted NOESY spectra are back calculated from the X-ray structures and are compared with the experimental solution state NOESY spectra at different pressures in detail. As preliminary experiments show high pressure soaking with small state specific compounds may represent a novel method to access rare conformational states by crystallography. The dynamics under pressure of the different states will be studied by 2D transient P-jump experiments on isotope enriched Ras protein with the high-pressure jump system developed by us. The study will be complemented by relaxation dispersion and chemical exchange saturation transfer (CEST) experiments at different pressures.