High Pressure Promotes Binding of the Allosteric Inhibitor Zn-Cyclen in Crystals of Activated H-Ras.

In this work, we experimentally investigate the potency of high pressure to drive a protein toward an excited state where an inhibitor targeted for this state can bind. Ras proteins are small GTPases cycling between active GTP-bound and inactive GDP-bound states. Various states of GTP-bound Ras in active conformation coexist in solution, amongst them, state 2 which binds to effectors, and state 1, weakly populated at ambient conditions, which has a low affinity for effectors.

Equilibria between conformational states of the Ras oncogene protein revealed by high pressure crystallography.

In this work, we experimentally investigate the allosteric transitions between conformational states on the Ras oncogene protein using high pressure crystallography. Ras protein is a small GTPase involved in central regulatory processes occurring in multiple conformational states. Ras acts as a molecular switch between active GTP-bound, and inactive GDP-bound states, controlling essential signal transduction pathways.

Pressure response of P chemical shifts of adenine nucleotides.

High pressure NMR spectroscopy is a powerful method for identifying rare conformational states of proteins from the pressure response of their chemical shifts. Many proteins have bound adenine nucleotides at their active centers, in most cases in a complex with Mg-ions. The P NMR signals of phosphate groups of the nucleotides can be used as probes for conformational transitions in the proteins themselves.

High pressure P NMR spectroscopy on guanine nucleotides.

The P NMR pressure response of guanine nucleotides bound to proteins has been studied in the past for characterizing the pressure perturbation of conformational equilibria. The pressure response of the P NMR chemical shifts of the phosphate groups of GMP, GDP, and GTP as well as the commonly used GTP analogs GppNHp, GppCHp and GTPγS was measured in the absence and presence of Mg-ions within a pressure range up to 200 MPa. The pressure dependence of chemical shifts is clearly non-linear.

Elucidating the mode of action of a typical Ras state 1(T) inhibitor.

The small GTPase Ras is an essential component of signal transduction pathways within the cell, controlling proliferation, differentiation, and apoptosis. Only in the GTP-bound form does Ras interact strongly with effector molecules such as Raf-kinase, thus acting as a molecular switch. In the GTP-bound form, Ras exists in a dynamic equilibrium between at least two distinct conformational states, 1(T) and 2(T), offering different functional properties of the protein.