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Christian Braun - One of the best experts on this subject based on the ideXlab platform.
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Structure/function correlates and Protein/Lipid Interaction of the viral potassium channel KcvNTS
2014Co-Authors: Christian BraunAbstract:Ion channels are present in every domain of life. They catalyze the rapid and selective flux of ions across membranes. It is well established that mutations or dysfunctions of ion channels often cause severe diseases. To understand the molecular mechanisms behind these so-called channelopathies it is necessary to understand the structure/function correlates and Protein/Lipid Interaction of channels at the single-Protein level. Planar Lipid bilayer techniques, the oldest and most reduced systems for a functional characterization of ion channels, are well suited to examine basic structure/function relations in a defined Lipid environment. Here we improved the performance of the conventional planar Lipid bilayer technique. An air-bubble functions as a tool for the rapid creation and stabilization of bilayers and even more important for reducing the number of active channels in the bilayer for real single-channel recordings. A further technical improvement is the establishment of an in vitro (cell-free) expression system for ion channels, which supports a rapid Protein production and a contamination free reconstitution. With these systems we performed a detailed single-channel analysis of the viral K+ channel KcvNTS, one of the smallest potassium channels known so far. The data show that the Protein has a very high selectivity for K+ over Na+; it is permeable for Rb+ although the unitary conductance is lower than that of K+. When Cs+ is the only cation present in the buffer the channel conducts it, albeit with a low conductance. If Cs+ is present together with K+ even at a low concentration it will block the K+ inward current in a side specific and voltage dependent manner. The Cs+ block increases in strength over several minutes suggesting a slow conformational change in the Protein. A further characteristic feature of the KcvNTS is a distinct pH dependency of the open probability. The latter decreases from a value close to 90% with acidification of the buffer down to ca. 10%. This pH dependent open probability is correlated with an increase in the voltage dependency of the channel suggesting a titratable amino acid in the Protein, which acquires the function of a voltage sensor and presumably of a gate. Because of its small size with only 82 aa per monomer the KcvNTS Protein is quasi fully embedded in the membrane. A functional test in planar Lipid bilayers of different thickness, which are made from Lipids with different acyl chain length (C14 - C16/18) or by adding solvents or cholesterol, shows that the channel is functional under all conditions. While the unitary conductance is not affected by the thickness the open probability is sensitive to it. With increasing bilayer thickness the channel exhibits more frequently a second gating mode, which is characterized by a voltage dependency. In the latter mode positive voltages cause a decrease in the channel open probability. The question of the molecular mechanism, which is responsible for this unusual gating in a channel without a notable charge in the electrical field, remains unanswered. Also the question why the membrane thickness affects this gating mode remains unsolved. The data however show for the first time that the Lipid environment can have a dramatic effect on the voltage dependency of a Protein. Since the bilayer technique bears the hazard of artifacts from impure Protein isolations and from Lipid pores KcvNTS is as a control also produced and purified from Pichia pastoris and expressed heterologously in HEK293 cells. Comparing the aforementioned data of the in vitro expressed Protein reconstituted in conventional planar Lipid bilayers with those recorded with other methods and in particular with those measured by the conventional patch clamp technique in HEK293 cells show no large difference between the different systems. The results of these experiments stress that the obtained data with the in vitro expressed KcvNTS channel in conventional bilayers is indeed representative for the function of this channel Protein. Collectively the present results show that a Protein as small as the KcvNTS is able to function in a robust manner as a selective K+ channel in different membrane environments. The Protein, which is equivalent to the pore module of more complex K+ channels, has inherent gating properties, which are sensitive to the pH, Cs+ and the membrane thickness. This underscores the view of multiple gates in the pore module of K+ channels.
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structure function correlates and Protein Lipid Interaction of the viral potassium channel kcvnts
2014Co-Authors: Christian BraunAbstract:Ion channels are present in every domain of life. They catalyze the rapid and selective flux of ions across membranes. It is well established that mutations or dysfunctions of ion channels often cause severe diseases. To understand the molecular mechanisms behind these so-called channelopathies it is necessary to understand the structure/function correlates and Protein/Lipid Interaction of channels at the single-Protein level. Planar Lipid bilayer techniques, the oldest and most reduced systems for a functional characterization of ion channels, are well suited to examine basic structure/function relations in a defined Lipid environment. Here we improved the performance of the conventional planar Lipid bilayer technique. An air-bubble functions as a tool for the rapid creation and stabilization of bilayers and even more important for reducing the number of active channels in the bilayer for real single-channel recordings. A further technical improvement is the establishment of an in vitro (cell-free) expression system for ion channels, which supports a rapid Protein production and a contamination free reconstitution. With these systems we performed a detailed single-channel analysis of the viral K+ channel KcvNTS, one of the smallest potassium channels known so far. The data show that the Protein has a very high selectivity for K+ over Na+; it is permeable for Rb+ although the unitary conductance is lower than that of K+. When Cs+ is the only cation present in the buffer the channel conducts it, albeit with a low conductance. If Cs+ is present together with K+ even at a low concentration it will block the K+ inward current in a side specific and voltage dependent manner. The Cs+ block increases in strength over several minutes suggesting a slow conformational change in the Protein. A further characteristic feature of the KcvNTS is a distinct pH dependency of the open probability. The latter decreases from a value close to 90% with acidification of the buffer down to ca. 10%. This pH dependent open probability is correlated with an increase in the voltage dependency of the channel suggesting a titratable amino acid in the Protein, which acquires the function of a voltage sensor and presumably of a gate. Because of its small size with only 82 aa per monomer the KcvNTS Protein is quasi fully embedded in the membrane. A functional test in planar Lipid bilayers of different thickness, which are made from Lipids with different acyl chain length (C14 - C16/18) or by adding solvents or cholesterol, shows that the channel is functional under all conditions. While the unitary conductance is not affected by the thickness the open probability is sensitive to it. With increasing bilayer thickness the channel exhibits more frequently a second gating mode, which is characterized by a voltage dependency. In the latter mode positive voltages cause a decrease in the channel open probability. The question of the molecular mechanism, which is responsible for this unusual gating in a channel without a notable charge in the electrical field, remains unanswered. Also the question why the membrane thickness affects this gating mode remains unsolved. The data however show for the first time that the Lipid environment can have a dramatic effect on the voltage dependency of a Protein. Since the bilayer technique bears the hazard of artifacts from impure Protein isolations and from Lipid pores KcvNTS is as a control also produced and purified from Pichia pastoris and expressed heterologously in HEK293 cells. Comparing the aforementioned data of the in vitro expressed Protein reconstituted in conventional planar Lipid bilayers with those recorded with other methods and in particular with those measured by the conventional patch clamp technique in HEK293 cells show no large difference between the different systems. The results of these experiments stress that the obtained data with the in vitro expressed KcvNTS channel in conventional bilayers is indeed representative for the function of this channel Protein. Collectively the present results show that a Protein as small as the KcvNTS is able to function in a robust manner as a selective K+ channel in different membrane environments. The Protein, which is equivalent to the pore module of more complex K+ channels, has inherent gating properties, which are sensitive to the pH, Cs+ and the membrane thickness. This underscores the view of multiple gates in the pore module of K+ channels.
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minimal viral potassium channels for studying Protein Lipid Interaction
Biophysical Journal, 2014Co-Authors: Christian Braun, Indra Schroeder, Leonhard M Henkes, Cristina Arrigoni, Stefan M Kast, Anna Moroni, Gerhard ThielAbstract:The channel Proteins from Chlorella viruses are miniature versions of K+ channels. They are the structural equivalents of the pore module of complex K+ channels from eukaryotes and they include the selectivity filter, the cavity and gates. The channel KcvNTS is with only 82 amino acids particularly small and molecular dynamics simulations suggest that the channel is quasi fully embedded in a di-myristoylphosphatidylcholin (C14) Lipid bilayer [1]. This close Interaction between the channel Protein and its Lipid environment offers the possibility to examine Lipid/Protein Interaction for a channel pore in a systematic manner. For this purpose we reconstituted the purified channel Protein in Lipid bilayers with different chain lengths, phosphoLipid head groups and in the absence and presence of detergents or cholesterol. The analysis of single channel activity shows that the membrane composition affects channel gating but not the unitary conductance. Most interesting is a voltage dependency of the channel, which is introduced by the presence of cholesterol, detergent or bilayers with long chain length.Reference1. Braun, C.J., et al., Viral potassium channels as a robust model system for studies of membrane-Protein Interaction. Biochim Biophys Acta, 2013.
Gerhard Thiel - One of the best experts on this subject based on the ideXlab platform.
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minimal viral potassium channels for studying Protein Lipid Interaction
Biophysical Journal, 2014Co-Authors: Christian Braun, Indra Schroeder, Leonhard M Henkes, Cristina Arrigoni, Stefan M Kast, Anna Moroni, Gerhard ThielAbstract:The channel Proteins from Chlorella viruses are miniature versions of K+ channels. They are the structural equivalents of the pore module of complex K+ channels from eukaryotes and they include the selectivity filter, the cavity and gates. The channel KcvNTS is with only 82 amino acids particularly small and molecular dynamics simulations suggest that the channel is quasi fully embedded in a di-myristoylphosphatidylcholin (C14) Lipid bilayer [1]. This close Interaction between the channel Protein and its Lipid environment offers the possibility to examine Lipid/Protein Interaction for a channel pore in a systematic manner. For this purpose we reconstituted the purified channel Protein in Lipid bilayers with different chain lengths, phosphoLipid head groups and in the absence and presence of detergents or cholesterol. The analysis of single channel activity shows that the membrane composition affects channel gating but not the unitary conductance. Most interesting is a voltage dependency of the channel, which is introduced by the presence of cholesterol, detergent or bilayers with long chain length.Reference1. Braun, C.J., et al., Viral potassium channels as a robust model system for studies of membrane-Protein Interaction. Biochim Biophys Acta, 2013.
Philip C. Biggin - One of the best experts on this subject based on the ideXlab platform.
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State-dependent Protein-Lipid Interactions of a pentameric ligand-gated ion channel in a neuronal membrane.
PLoS computational biology, 2021Co-Authors: Marc A. Dämgen, Philip C. BigginAbstract:Pentameric ligand-gated ion channels (pLGICs) are receptor Proteins that are sensitive to their membrane environment, but the mechanism for how Lipids modulate function under physiological conditions in a state dependent manner is not known. The glycine receptor is a pLGIC whose structure has been resolved in different functional states. Using a realistic model of a neuronal membrane coupled with coarse-grained molecular dynamics simulations, we demonstrate that some key Lipid-Protein Interactions are dependent on the receptor state, suggesting that Lipids may regulate the receptor's conformational dynamics. Comparison with existing structural data confirms known Lipid binding sites, but we also predict further Protein-Lipid Interactions including a site at the communication interface between the extracellular and transmembrane domain. Moreover, in the active state, cholesterol can bind to the binding site of the positive allosteric modulator ivermectin. These Protein-Lipid Interaction sites could in future be exploited for the rational design of Lipid-like allosteric drugs.
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State-dependent Protein-Lipid Interactions of a pentameric ligand-gated ion channel in a neuronal membrane
2020Co-Authors: Marc A. Dämgen, Philip C. BigginAbstract:AbstractPentameric ligand-gated ion channels (pLGICs) are receptor Proteins that are sensitive to their membrane environment, but the mechanism for how Lipids modulate function under physiological conditions in a state dependent manner is not known. The glycine receptor is a pLGIC whose structure has been resolved in different functional states. Using a realistic model of a neuronal membrane coupled with coarse-grained molecular dynamics simulations, we demonstrate that the Lipid-Protein Interactions are dependent on the receptor state, suggesting that Lipids may regulate the receptor’s conformational dynamics. Comparison with existing structural data confirms known Lipid binding sites, but we also predict further Protein-Lipid Interactions including a site at the communication interface between the extracellular and transmembrane domain. Moreover, in the active state, cholesterol can bind to the binding site of the positive allosteric modulator ivermectin. These Protein-Lipid Interaction sites could in future be exploited for the rational design of Lipid-like allosteric drugs.Author SummaryIon channels are Proteins that control the flow of ions into the cell. The family of ion channels known as the pentameric ligand gated ion channels (pLGICS) open in response to the binding of a neurotransmitter, moving the channel from a resting state to an open state. The glycine receptor is a pLGIC whose structure has been resolved in different functional states. It is also known that the response of pLGICs can also be modified by different types of Lipid found within the membrane itself but exactly how is unclear. Here, we used a realistic model of a neuronal membrane and performed molecular dynamics simulations to show various Lipid-Protein Interactions that are dependent on the channel state. Our work also reveals previously unconsidered Protein-Lipid Interactions at a key junction of the channel known to be critical for the transmission of the opening process. We also demonstrate that cholesterol interacts with the Protein at a site already known to bind to another compound that modulates the channel, called ivermectin. The work should be useful for future drug design.
Rajeshwer S. Sankhala - One of the best experts on this subject based on the ideXlab platform.
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Thermodynamic Analysis of Protein-Lipid Interactions by Isothermal Titration Calorimetry.
Methods in molecular biology (Clifton N.J.), 2019Co-Authors: Musti J. Swamy, Rajeshwer S. Sankhala, Bhanu Pratap SinghAbstract:Isothermal titration calorimetry is a highly sensitive and powerful technique for the study of molecular Interactions. This method can be applied universally for studying the Interaction between moleculeAbstracts, molecular assembles and ions as it measures the heat changes resulting from such Interactions and does not need any probe molecule/moiety to be incorporated into the system under investigation. This method has been applied quite extensively to investigate the Interaction of Proteins with other biomolecules such as small ligands, other Proteins, nucleic acids, Lipid membranes as well as to study the Interaction of antibodies, drugs, metal ions and nanoparticles with target Proteins or antigens, nucleic acids, and membranes. In this chapter, we describe the application of ITC for the investigation of thermodynamics of Protein-Lipid Interaction. A number of important parameters such as enthalpy of binding (ΔH), entropy of binding (ΔS), association constant (Ka), binding stoichiometry (n) and free energy of binding (ΔG) can be obtained from a single calorimetric titration, providing a complete thermodynamic characterization of the Interaction. The method is described in detail taking the major Protein of the bovine seminal plasma, PDC-109, which exhibits a high preference for Interaction with choline-containing Lipids, as an example. The method can be applied to investigate thermodynamic parameters associated with the Interaction of other soluble Proteins with Lipid membranes.
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Probing the thermodynamics of Protein-Lipid Interactions by isothermal titration calorimetry.
Methods in molecular biology (Clifton N.J.), 2012Co-Authors: Musti J. Swamy, Rajeshwer S. SankhalaAbstract:Isothermal titration calorimetry is a highly sensitive technique for the study of molecular Interactions. This method has been applied quite extensively to investigate the Interaction of Proteins with small ligands, other Proteins, and nucleic acids as well as with drugs and metal ions. In this chapter, we describe the application of ITC for the investigation of thermodynamics of Protein-Lipid Interaction. A number of parameters such as enthalpy of binding (ΔH), entropy of binding (ΔS), association constant (K (a)), binding stoichiometry (n), and free energy of binding (ΔG) can be obtained from a single calorimetric titration, providing a complete thermodynamic characterization of the Interaction. The method is described in detail taking the major Protein of the bovine seminal plasma, PDC-109, which exhibits a high preference for Interaction with choline-containing Lipids, as an example. The method can be applied to investigate the thermodynamics of the Interaction of other soluble Proteins with Lipid membranes.
Anne-claude Gavin - One of the best experts on this subject based on the ideXlab platform.
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A protocol for the systematic and quantitative measurement of Protein–Lipid Interactions using the liposome-microarray-based assay
Nature Protocols, 2016Co-Authors: Antoine-emmanuel Saliba, Ivana Vonkova, Samy Deghou, Stefano Ceschia, Christian Tischer, Karl G. Kugler, Peer Bork, Jan Ellenberg, Anne-claude GavinAbstract:Here the authors detail the protocol for their recently developed liposome-microarray-based assay (LiMA) that integrates liposomes, microfluidics and fluorescence microscopy to enable the systematic and quantitative measurement of Protein–Lipid Interactions. Lipids organize the activity of the cell's proteome through a complex network of Interactions. The assembly of comprehensive atlases embracing all Protein–Lipid Interactions is an important challenge that requires innovative methods. We recently developed a liposome-microarray-based assay (LiMA) that integrates liposomes, microfluidics and fluorescence microscopy and which is capable of measuring Protein recruitment to membranes in a quantitative and high-throughput manner. Compared with previous assays that are labor-intensive and difficult to scale up, LiMA improves the Protein–Lipid Interaction assay throughput by at least three orders of magnitude. Here we provide a step-by-step LiMA protocol that includes the following: (i) the serial and generic production of the liposome microarray; (ii) its integration into a microfluidic format; (iii) the measurement of fluorescently labeled Protein (either purified Proteins or from cell lysate) recruitment to liposomal membranes using high-throughput microscopy; (iv) automated image analysis pipelines to quantify Protein–Lipid Interactions; and (v) data quality analysis. In addition, we discuss the experimental design, including the relevant quality controls. Overall, the protocol—including device preparation, assay and data analysis—takes 6–8 d. This protocol paves the way for Protein–Lipid Interaction screens to be performed on the proteome and Lipidome scales.
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A protocol for the systematic and quantitative measurement of Protein-Lipid Interactions using the liposome-microarray-based assay.
Nature protocols, 2016Co-Authors: Antoine-emmanuel Saliba, Ivana Vonkova, Samy Deghou, Stefano Ceschia, Christian Tischer, Karl G. Kugler, Peer Bork, Jan Ellenberg, Anne-claude GavinAbstract:Lipids organize the activity of the cell's proteome through a complex network of Interactions. The assembly of comprehensive atlases embracing all Protein-Lipid Interactions is an important challenge that requires innovative methods. We recently developed a liposome-microarray-based assay (LiMA) that integrates liposomes, microfluidics and fluorescence microscopy and which is capable of measuring Protein recruitment to membranes in a quantitative and high-throughput manner. Compared with previous assays that are labor-intensive and difficult to scale up, LiMA improves the Protein-Lipid Interaction assay throughput by at least three orders of magnitude. Here we provide a step-by-step LiMA protocol that includes the following: (i) the serial and generic production of the liposome microarray; (ii) its integration into a microfluidic format; (iii) the measurement of fluorescently labeled Protein (either purified Proteins or from cell lysate) recruitment to liposomal membranes using high-throughput microscopy; (iv) automated image analysis pipelines to quantify Protein-Lipid Interactions; and (v) data quality analysis. In addition, we discuss the experimental design, including the relevant quality controls. Overall, the protocol-including device preparation, assay and data analysis-takes 6-8 d. This protocol paves the way for Protein-Lipid Interaction screens to be performed on the proteome and Lipidome scales.
