High pressure 31P NMR spectroscopy on guanine nucleotides

Abstract

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 theP 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. For all nucleotides a negative first order pressure coefficient B was determined indicating an upfield shift of the resonances with pressure. With exception of the α-phosphate group of Mg·GMP and Mg·GppNHp the second order pressure coefficients are positive. To describe the data of Mg·GppCHp and GTPγS 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 Mg ions using indirect referencing to DSS under identical experimental conditions. By a comparison between high pressure P NMR data on free Mg-GDP and Mg-GDP in complex with the proto-oncogene Ras we demonstrate that pressure induced changes in chemical shift are clearly different between both forms.

Direct observation of dynamic protein interactions involving human microtubules using solid-state NMR spectroscopy

“…2b, right) that were well resolved compared to previous results on lyophilized MTs 32 . The observed spectral resolution allowed us to distinguish NMR signals from both molecules (Supplementary Table 1) 32,33 . Integrated peak intensities (Supplementary Table 1) correlated well with the notion that the molar ratio of GDP:GTP was 1:1, which is in agreement with previous studies 4,31 further supporting the idea that our ssNMR preparations were well folded and functional.…”

Structural Analysis of Human Cofilin 2/Filamentous Actin Assemblies: Atomic-Resolution Insights from Magic Angle Spinning NMR Spectroscopy

“…The CP contact time was 5 ms and the CORD mixing time was 50 ms. To rule out the possibility that certain signals in the dREDOR based spectra may be artifacts arising from dynamic residues due to insufficient suppression of 1 H spin polarization, control experiments were conducted as described in our report45.…”

Pressure dependence of side chain 1H and 15N-chemical shifts in the model peptides Ac-Gly-Gly-Xxx-Ala-NH2

“…1 is sufficient in the pressure range studied here (Arnold et al 2002;Koehler et al 2012;Beck Erlach et al 2016. The description of the pressure response of small molecules such as the GTP analog GTPγS sometimes requires a third degree polynomial (Spoerner et al 2017). When the ratio of B 2 /B 1 is negative, an extremum with slope zero would be reached at a pressure P ex with An extremum has not observed for our model peptides in the range up to 200 MP.…”