Studies of P2X4 receptor dynamics upon agonist activation by High-Speed Atomic Force Microscopy and Molecular Dynamics simulations

Membrane proteins are surrounded by a myriad of other biomolecules and hence most of the experiments conducted in native conditions are highly complex to interpret at the single molecule level. P2X membrane receptors, an ATP-triggered channel family, are involved in calcium signaling and inflammation. However, to date, there is limited information about its dynamical biophysical behavior in a well-known lipid environment. In particular, receptor diffusion and pore size should depend on agonist activation, lipid bilayer composition and vary throughout time. Suitable experimental methodologies to tackle them have just recently emerged, such as HSAFM where topographic features of the membrane proteins can be recorded at second resolution. In addition, combining this with all-atom MD simulations, it would be possible to provide mechanistic understanding. Therefore, the goal of this thesis is to characterize the P2X4 receptor diffusion and pore dilation on reconstituted liposomes via HS-AFM imaging and MD simulations. Our HS-AFM results indicate that HOLO state of the P2X4 receptor decreased their mean displacements by 22% compared to the APO state and our MD results shows that interaction of protein-water and protein-lipid interaction fluctuate 9% and 43% higher in the APO state respectively. Also, the HOLO state showed a dilation of its extracellular domain compared to the APO state via HS-AFM imaging; however, MD showed us only a dilation of the internal profile of the P2X4 receptor but not an external broadening. This discrepancy may arise due to lipidic composition in MD which only had neutral lipids and experiments were carried with a proportion of 3:1 of neutral:negatively charged lipids. Moreover, each ATP molecule was calculated to have a high interaction with the two adjacent subunits that is in contact and therefore it may force the subunits to stay close at that point, working as a hinge. Taken together, combining HS-AFM and MD can lead to novel insights into the dynamical behavior of individual receptors. Since our experiments were performed on receptors supported on mica, future work could be to suspend lipid bilayers on porous alumina membranes to emulate the conditions observed in cell membranes where extra and intracellular sides are present.Membrane proteins are surrounded by a myriad of other biomolecules and hence most of the experiments conducted in native conditions are highly complex to interpret at the single molecule level. P2X membrane receptors, an ATP-triggered channel family, are involved in calcium signaling and inflammation. However, to date, there is limited information about its dynamical biophysical behavior in a well-known lipid environment. In particular, receptor diffusion and pore size should depend on agonist activation, lipid bilayer composition and vary throughout time. Suitable experimental methodologies to tackle them have just recently emerged, such as HSAFM where topographic features of the membrane proteins can be recorded at second resolution. In addition, combining this with all-atom MD simulations, it would be possible to provide mechanistic understanding. Therefore, the goal of this thesis is to characterize the P2X4 receptor diffusion and pore dilation on reconstituted liposomes via HS-AFM imaging and MD simulations. Our HS-AFM results indicate that HOLO state of the P2X4 receptor decreased their mean displacements by 22% compared to the APO state and our MD results shows that interaction of protein-water and protein-lipid interaction fluctuate 9% and 43% higher in the APO state respectively. Also, the HOLO state showed a dilation of its extracellular domain compared to the APO state via HS-AFM imaging; however, MD showed us only a dilation of the internal profile of the P2X4 receptor but not an external broadening. This discrepancy may arise due to lipidic composition in MD which only had neutral lipids and experiments were carried with a proportion of 3:1 of neutral:negatively charged lipids. Moreover, each ATP molecule was calculated to have a high interaction with the two adjacent subunits that is in contact and therefore it may force the subunits to stay close at that point, working as a hinge. Taken together, combining HS-AFM and MD can lead to novel insights into the dynamical behavior of individual receptors. Since our experiments were performed on receptors supported on mica, future work could be to suspend lipid bilayers on porous alumina membranes to emulate the conditions observed in cell membranes where extra and intracellular sides are present.Membrane proteins are surrounded by a myriad of other biomolecules and hence most of the experiments conducted in native conditions are highly complex to interpret at the single molecule level. P2X membrane receptors, an ATP-triggered channel family, are involved in calcium signaling and inflammation. However, to date, there is limited information about its dynamical biophysical behavior in a well-known lipid environment. In particular, receptor diffusion and pore size should depend on agonist activation, lipid bilayer composition and vary throughout time. Suitable experimental methodologies to tackle them have just recently emerged, such as HSAFM where topographic features of the membrane proteins can be recorded at second resolution. In addition, combining this with all-atom MD simulations, it would be possible to provide mechanistic understanding. Therefore, the goal of this thesis is to characterize the P2X4 receptor diffusion and pore dilation on reconstituted liposomes via HS-AFM imaging and MD simulations. Our HS-AFM results indicate that HOLO state of the P2X4 receptor decreased their mean displacements by 22% compared to the APO state and our MD results shows that interaction of protein-water and protein-lipid interaction fluctuate 9% and 43% higher in the APO state respectively. Also, the HOLO state showed a dilation of its extracellular domain compared to the APO state via HS-AFM imaging; however, MD showed us only a dilation of the internal profile of the P2X4 receptor but not an external broadening. This discrepancy may arise due to lipidic composition in MD which only had neutral lipids and experiments were carried with a proportion of 3:1 of neutral:negatively charged lipids. Moreover, each ATP molecule was calculated to have a high interaction with the two adjacent subunits that is in contact and therefore it may force the subunits to stay close at that point, working as a hinge. Taken together, combining HS-AFM and MD can lead to novel insights into the dynamical behavior of individual receptors. Since our experiments were performed on receptors supported on mica, future work could be to suspend lipid bilayers on porous alumina membranes to emulate the conditions observed in cell membranes where extra and intracellular sides are present.Membrane proteins are surrounded by a myriad of other biomolecules and hence most of the experiments conducted in native conditions are highly complex to interpret at the single molecule level. P2X membrane receptors, an ATP-triggered channel family, are involved in calcium signaling and inflammation. However, to date, there is limited information about its dynamical biophysical behavior in a well-known lipid environment. In particular, receptor diffusion and pore size should depend on agonist activation, lipid bilayer composition and vary throughout time. Suitable experimental methodologies to tackle them have just recently emerged, such as HSAFM where topographic features of the membrane proteins can be recorded at second resolution. In addition, combining this with all-atom MD simulations, it would be possible to provide mechanistic understanding. Therefore, the goal of this thesis is to characterize the P2X4 receptor diffusion and pore dilation on reconstituted liposomes via HS-AFM imaging and MD simulations. Our HS-AFM results indicate that HOLO state of the P2X4 receptor decreased their mean displacements by 22% compared to the APO state and our MD results shows that interaction of protein-water and protein-lipid interaction fluctuate 9% and 43% higher in the APO state respectively. Also, the HOLO state showed a dilation of its extracellular domain compared to the APO state via HS-AFM imaging; however, MD showed us only a dilation of the internal profile of the P2X4 receptor but not an external broadening. This discrepancy may arise due to lipidic composition in MD which only had neutral lipids and experiments were carried with a proportion of 3:1 of neutral:negatively charged lipids. Moreover, each ATP molecule was calculated to have a high interaction with the two adjacent subunits that is in contact and therefore it may force the subunits to stay close at that point, working as a hinge. Taken together, combining HS-AFM and MD can lead to novel insights into the dynamical behavior of individual receptors. Since our experiments were performed on receptors supported on mica, future work could be to suspend lipid bilayers on porous alumina membranes to emulate the conditions observed in cell membranes where extra and intracellular sides are present.Membrane proteins are surrounded by a myriad of other biomolecules and hence most of the experiments conducted in native conditions are highly complex to interpret at the single molecule level. P2X membrane receptors, an ATP-triggered channel family, are involved in calcium signaling and inflammation. However, to date, there is limited information about its dynamical biophysical behavior in a well-known lipid environment. In particular, receptor diffusion and pore size should depend on agonist activation, lipid bilayer composition and vary throughout time. Suitable experimental methodologies to tackle them have just recently emerged, such as HSAFM where topographic features of the membrane proteins can be recorded at second resolution. In addition, combining this with all-atom MD simulations, it would be possible to provide mechanistic understanding. Therefore, the goal of this thesis is to characterize the P2X4 receptor diffusion and pore dilation on reconstituted liposomes via HS-AFM imaging and MD simulations. Our HS-AFM results indicate that HOLO state of the P2X4 receptor decreased their mean displacements by 22% compared to the APO state and our MD results shows that interaction of protein-water and protein-lipid interaction fluctuate 9% and 43% higher in the APO state respectively. Also, the HOLO state showed a dilation of its extracellular domain compared to the APO state via HS-AFM imaging; however, MD showed us only a dilation of the internal profile of the P2X4 receptor but not an external broadening. This discrepancy may arise due to lipidic composition in MD which only had neutral lipids and experiments were carried with a proportion of 3:1 of neutral:negatively charged lipids. Moreover, each ATP molecule was calculated to have a high interaction with the two adjacent subunits that is in contact and therefore it may force the subunits to stay close at that point, working as a hinge. Taken together, combining HS-AFM and MD can lead to novel insights into the dynamical behavior of individual receptors. Since our experiments were performed on receptors supported on mica, future work could be to suspend lipid bilayers on porous alumina membranes to emulate the conditions observed in cell membranes where extra and intracellular sides are present.Membrane proteins are surrounded by a myriad of other biomolecules and hence most of the experiments conducted in native conditions are highly complex to interpret at the single molecule level. P2X membrane receptors, an ATP-triggered channel family, are involved in calcium signaling and inflammation. However, to date, there is limited information about its dynamical biophysical behavior in a well-known lipid environment. In particular, receptor diffusion and pore size should depend on agonist activation, lipid bilayer composition and vary throughout time. Suitable experimental methodologies to tackle them have just recently emerged, such as HSAFM where topographic features of the membrane proteins can be recorded at second resolution. In addition, combining this with all-atom MD simulations, it would be possible to provide mechanistic understanding. Therefore, the goal of this thesis is to characterize the P2X4 receptor diffusion and pore dilation on reconstituted liposomes via HS-AFM imaging and MD simulations. Our HS-AFM results indicate that HOLO state of the P2X4 receptor decreased their mean displacements by 22% compared to the APO state and our MD results shows that interaction of protein-water and protein-lipid interaction fluctuate 9% and 43% higher in the APO state respectively. Also, the HOLO state showed a dilation of its extracellular domain compared to the APO state via HS-AFM imaging; however, MD showed us only a dilation of the internal profile of the P2X4 receptor but not an external broadening. This discrepancy may arise due to lipidic composition in MD which only had neutral lipids and experiments were carried with a proportion of 3:1 of neutral:negatively charged lipids. Moreover, each ATP molecule was calculated to have a high interaction with the two adjacent subunits that is in contact and therefore it may force the subunits to stay close at that point, working as a hinge. Taken together, combining HS-AFM and MD can lead to novel insights into the dynamical behavior of individual receptors. Since our experiments were performed on receptors supported on mica, future work could be to suspend lipid bilayers on porous alumina membranes to emulate the conditions observed in cell membranes where extra and intracellular sides are present.Membrane proteins are surrounded by a myriad of other biomolecules and hence most of the experiments conducted in native conditions are highly complex to interpret at the single molecule level. P2X membrane receptors, an ATP-triggered channel family, are involved in calcium signaling and inflammation. However, to date, there is limited information about its dynamical biophysical behavior in a well-known lipid environment. In particular, receptor diffusion and pore size should depend on agonist activation, lipid bilayer composition and vary throughout time. Suitable experimental methodologies to tackle them have just recently emerged, such as HSAFM where topographic features of the membrane proteins can be recorded at second resolution. In addition, combining this with all-atom MD simulations, it would be possible to provide mechanistic understanding. Therefore, the goal of this thesis is to characterize the P2X4 receptor diffusion and pore dilation on reconstituted liposomes via HS-AFM imaging and MD simulations. Our HS-AFM results indicate that HOLO state of the P2X4 receptor decreased their mean displacements by 22% compared to the APO state and our MD results shows that interaction of protein-water and protein-lipid interaction fluctuate 9% and 43% higher in the APO state respectively. Also, the HOLO state showed a dilation of its extracellular domain compared to the APO state via HS-AFM imaging; however, MD showed us only a dilation of the internal profile of the P2X4 receptor but not an external broadening. This discrepancy may arise due to lipidic composition in MD which only had neutral lipids and experiments were carried with a proportion of 3:1 of neutral:negatively charged lipids. Moreover, each ATP molecule was calculated to have a high interaction with the two adjacent subunits that is in contact and therefore it may force the subunits to stay close at that point, working as a hinge. Taken together, combining HS-AFM and MD can lead to novel insights into the dynamical behavior of individual receptors. Since our experiments were performed on receptors supported on mica, future work could be to suspend lipid bilayers on porous alumina membranes to emulate the conditions observed in cell membranes where extra and intracellular sides are present.