High pressure 31P NMR spectroscopy on guanine nucleotides

The P-31 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-31 NMR chemical shifts of the phosphate groups of GMP, GDP, and GTP as well as the commonly used GTP analogs GppNHp, GppCH(2)p and GTP gamma S was measured in the absence and presence of Mg2+-ions within a pressure range up to 200 MPa. The pressure dependence of chemical shifts is clearly non-linear. For all nucleotides a negative first order pressure coefficient B (1) was determined indicating an upfield shift of the resonances with pressure. With exception of the alpha-phosphate group of Mg2+center dot GMP and Mg2+center dot GppNHp the second order pressure coefficients are positive. To describe the data of Mg2+center dot GppCH(2)p and GTP gamma S a Taylor expansion of 3rd order is required. For distinguishing pH effects from pressure effects a complete pH titration set is presented for GMP, as well as GDP and GTP in absence and presence of Mg2+ ions using indirect referencing to DSS under identical experimental conditions. By a comparison between high pressure P-31 NMR data on free Mg2+-GDP and Mg2+-GDP in complex with the proto-oncogene Ras we demonstrate that pressure induced changes in chemical shift are clearly different between both forms