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High pressure 31P NMR spectroscopy on guanine nucleotides

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Abstract

The 31P 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 31P NMR chemical shifts of the phosphate groups of GMP, GDP, and GTP as well as the commonly used GTP analogs GppNHp, GppCH2p and GTPγS 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 α-phosphate group of Mg2+·GMP and Mg2+·GppNHp the second order pressure coefficients are positive. To describe the data of Mg2+·GppCH2p 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 Mg2+ ions using indirect referencing to DSS under identical experimental conditions. By a comparison between high pressure 31P 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.

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Abbreviations

GMP:

Guanosine-5′-mono-phosphate

GDP:

Guanosine-5′-di-phosphate

GTP:

Guanosine-5′-tri-phosphate

GTPγS:

Guanosine-5′-O-(γ-thio)triphosphate)

GppNHp:

Guanosine-5′-(β,γ-imido)triphosphate

GppCH2p:

Guanosine-5′-(β,γ-methylene)triphosphate

Ras:

Protein product of the proto-oncogen rat sarcoma

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Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft (FOR1979), the Human Frontiers Science Program Organisation (HFSPO) and the Bayerische Forschungsstiftung.

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Authors and Affiliations

  1. Centre of Magnetic Resonance in Chemistry and Biomedicine, Institute of Biophysics and Physical Biochemistry, University of Regensburg, Universitätsstraße 31, 93053, Regensburg, Germany

    Michael Spoerner, Matthias Karl, Pedro Lopes, Marcus Hoering, Karoline Loeffel, Andrea Nuehs, Joseph Adelsberger, Werner Kremer & Hans Robert Kalbitzer

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  1. Michael Spoerner
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  2. Matthias Karl
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  3. Pedro Lopes
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  4. Marcus Hoering
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  5. Karoline Loeffel
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Correspondence to Hans Robert Kalbitzer.

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Spoerner, M., Karl, M., Lopes, P. et al. High pressure 31P NMR spectroscopy on guanine nucleotides. J Biomol NMR 67, 1–13 (2017). https://doi.org/10.1007/s10858-016-0079-0

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