Hochdruck-Perturbation des Ras-Signalwegs
Final Report Abstract
Adaptation of proteins to external chemical and physical stimuli is of utmost importance in maintaining the cycle of life. Ras proteins attached to the plasma membrane frequently encounter mechanical stresses, from extensive actin mesh-like structures, hemodynamic flow, up to high hydrostatic pressure (HHP) stresses. The present study explored the effect of HHP on a membrane-associated signaling module, specifically Ras-membrane association, dissociation and spontaneous intervesicle transfer, to reveal the associated kinetic and volumetric parameters underlying the membrane partitioning and intervesicle transport process. FRET-based assays revealed a biphasic nature of Ras membrane binding, where the first step is the initial docking, reorientation, and subsequent high affinity insertion of the protein into the membranes. The second step stems from a lateral reorganization of Ras proteins, which eventually leads to cluster formation as confirmed by AFM data. In addition, HHP can be inferred as a positive regulator of N-Ras clustering, in particular in heterogeneous membranes. The susceptibility of membrane interaction to pressure raises the idea of a role of lipidated signaling molecules as mechanosensors, transducing mechanical stimuli to chemical signals by regulating their membrane binding and dissociation. Finally, our results provided first insights into the influence of pressure on membrane-associated Ras-controlled signaling events in organisms living under extreme environmental conditions such as those that are encountered in the deep sea and sub-seafloor environments, where pressures reach the kbar (100 MPa) range. In future studies, it is planned to extend the use of the pressure perturbation approach for the modulation of reaction networks and identification of protein connectivity patterns also to in vivo systems.
Publications
- "Temperature-Pressure Phase Diagram of a Heterogeneous Anionic Model Biomembrane System: Results from a Combined Calorimetry, Spectroscopy and Microscopy Study", Biochim. Biophys. Acta - Biomembranes 1808 (2011) 1187- 1195
S. Kapoor, A. Werkmüller, C. Denter, Y. Zhai, J. Markgraf, K. Weise, N. Opitz, and R. Winter
- "Revealing Conformational Substates of Lipidated N-Ras Protein by Pressure Modulation", Proc. Natl. Acad. Sci. U.S.A. 109 (2012) 460-465
S. Kapoor, G. Triola, I. R. Vetter, M. Erlkamp, H. Waldmann, and R. Winter
(See online at https://doi.org/10.1073/pnas.1110553109) - "Pressure Modulation of Ras-Membrane Interactions and Intervesicle Transfer", J. Am. Chem. Soc. 135 (2013) 6149-6156
S. Kapoor, A. Werkmüller, R. S. Goody, H. Waldmann, and R. Winter
(See online at https://doi.org/10.1021/ja312671j) - "Packing Effects of N-Ras Binding to a DOPC Membrane – a Neutron Reflectivity and TIRF Spectroscopy High-Pressure Study", Z. Phys. Chem. 228 (2014) 969-986
M. Erlkamp, J.-F. Moulin, R. Winter, and C. Czeslik
(See online at https://doi.org/10.1515/zpch-2014-0546) - "Thermodynamic, Dynamic and Solvational Properties of PDEδ Binding to Farnesylated Cystein: A Model Study for Uncovering the Molecular Mechanism of PDEδ Interaction with Prenylated Proteins", J. Phys. Chem. B 118 (2014) 966-975
S. Suladze, S. Ismail, and R. Winter
(See online at https://doi.org/10.1021/jp411466r) - "Exploring the Structure and Phase Behavior of Plasma Membrane Vesicles under Extreme Environmental Conditions", Phys. Chem. Chem. Phys. 17 (2015) 7507-7513
J. Seeliger, N. Erwin, C. Rosin, M. Kahse, K. Weise, and R. Winter
(See online at https://doi.org/10.1039/c4cp05845c)
