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High-Throughput Thermodynamics of Drug-Membrane Interactions from Multiscale Simulations
140a
Sunday, February 12, 2017
arrangement; the adjacent peptides are held together by strong salt-bridges. In contrast, the positive and negative ELKs diffuse to the head group surface, do not form effective scaffolds, and fail to stabilize the discoidal assembly. Hence, the simulations provide a structural rational for the experimental observations, and provide an avenue for computer based design of apolipoprotein mimetic peptides. 688-Pos Board B453 Computational and Experimental Studies of Gold Nanoparticle Templated HDL-Like Nanoparticles for Cholesterol Metabolism Cheng-Tsung Lai1, Wangqiang Sun2, Rohun U. Palekar2, C. Shad Thaxton2, George C. Schatz1. 1 Department of Chemistry, Northwestern University, Evanston, IL, USA, 2 Department of Urology, Northwestern University, Chicago, IL, USA. High-density lipoprotein (HDL) is involved in the transport and metabolism of phospholipids, triglycerides, and cholesterol. Mimics of HDL are being explored as potential therapeutic agents for removing excess cholesterol from arterial plaques. Gold nanoparticles (AuNPs) functionalized with apolipoprotein A-I (apoA-I), and with the lipids 1,2-dipalmitoylsn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (PDP PE) have been demonstrated to be acceptors of cellular cholesterol. However, detailed structural properties of this functionalized HDL AuNP are not well understood. We combined coarse-grained and all-atom MD simulations to simulate the self-assembly of lipids and cholesteryl ester on the AuNP/apoA-I construct to gain insights into its structural properties, and further make comparisons with experimental results. We find that lipids are oriented differently in regions with and without apoA-I. We also find that in this functionalized HDL AuNP, the distribution of cholesteryl ester maintains a reverse concentration gradient that is similar to the gradient found in native HDL. Incubating the HDL AuNP with cholesterol and lecithin: cholesterol acyltransferase (LCAT) demonstrate the HDL AuNP is able to efficiently activate LCAT and form esterified cholesterol from free cholesterol and phospholipid. 689-Pos Board B454 High-Throughput Thermodynamics of Drug-Membrane Interactions from Multiscale Simulations Roberto Menichetti, Kiran Kanekal, Kurt Kremer, Tristan Bereau. Theory Group, Max Planck Institute for Polymer Research, Mainz, Germany. The number of small organic molecules is overwhelmingly large–so large, that most of it remains unexplored. Computer simulations offer an appealing framework to probe many of these compounds without the need to synthesize them in the laboratory. The main hurdles preventing a high-throughput characterization of many small molecules relies on the time investment to parametrize the force field—a process that typically requires significant human intervention—and extensive sampling requirements. We address these issues by first sampling from the coarse-grained Martini model, for which we developed an automated parametrization protocol for small molecules. The resulting potential-of-mean-force (PMF) curves for the insertion of small molecules in lipid membranes show excellent agreement for a number of benchmark cases. Our framework allows us to run high-throughput molecular dynamics simulations and estimate tens of thousands of relative free energies of different small molecules in a lipid bilayer. They provide useful data to explore structure-property relationships from computer simulations.
Optical Microscopy and Super-resolution Imaging: Novel Approaches and Analysis I 690-Pos Board B455 Combining Expansion Microscopy and STED Nanoscopy for the Study of Cellular Organization Isotta Cainero1,2, Michele Oneto1,2, Luca Pesce1,2, Giulia Zanini1,2, Luca Lanzano`1,2, Alberto Diaspro1,2, Paolo Bianchini1,2. 1 Nanophysics, Istituto Italiano di Tecnologia, Genoa, Italy, 2University of Genoa, Genoa, Italy. Expansion microscopy (ExM) is a novel method that allows super-resolution imaging with conventional microscopes(1, 2). It consists in soaking the cells with a polymer, inducing the polymerization to form a dense meshwork throughout the cell, cross-linking the fluorophores to the polymer and, after digestion of cellular protein, rehydrating of the sample. The swelling of the polymer gel led to a fourfold isotropic stretching of the sample. Therefore, it increases the distance between two objects that otherwise couldnot be seen as
two different things with an ordinary microscope. One of the drawback of such a technique is the long preparation made of several stages, i.e. immunostaining, gelation, digestion and expansion. They are really crucial steps for a good imaging post-expansion. In our work we present a comparison between ExM and stimulated emission depletion (STED) nanoscopy(3). Our aim is to study the e possible combination of STED and ExM as a method to further enhance the final resolution achievable. We will in particularly take advantage of the use of separation of photons by lifetime tuning (SPLIT) STED (4). We show application of these methods from single fixed cells to slices of fixed mouse retina tissue. We are also interested in applying the approach to high-density compartments like the cell nucleus to decipher the high-order structure organization of chromatin-DNA. 1. Chen, F., P.W. Tillberg, and E.S. Boyden. 2015. Optical imaging. Expansion microscopy. Science. 347: 543-548. 2. Chozinski, T.J., A.R. Halpern, H. Okawa, H.-J. Kim, G.J. Tremel, R.O.L. Wong, and J.C. Vaughan. 2016. Expansion microscopy with conventional antibodies and fluorescent proteins. Nat Meth. 13: 485-488. 3. Bianchini, P., C. Peres, M. Oneto, S. Galiani, G. Vicidomini, and A. Diaspro. 2015. STED nanoscopy: a glimpse into the future. Cell Tissue Res. 360: 143-150. 4. Lanzano, L., I. Coto Hernandez, M. Castello, E. Gratton, A. Diaspro, and G. Vicidomini. 2015. Encoding and decoding spatio-temporal information for super-resolution microscopy. Nature Communications. 6: 6701. 691-Pos Board B456 Quantitative Microstructure Analysis of Casein Network Dynamics using STED Microscopy with Relation to Macroscopic Gel Properties Zachary J. Glover, Adam Cohen Simonsen, Jonathan Brewer. MEMPHYS, FKF, University of Southern Denmark, Odense, Denmark. Casein proteins are the main structural element in many fermented dairy products. Understanding how protein functionality is affected during modern food processing, such as filtration and spray drying, is key to product and process optimization. Advances in Super Resolution Microscopy have provided novel tools to investigate protein microstructures. Herein, Stimulated Emission Depletion (STED) Microscopy has been used to image casein micelle aggregation, gel formation and network coarsening over time. Protein structures have been resolved to > 100 nm. Native skim milk and reconstituted skim milk powder were used to produce gelled systems. Gelation was induced either through rennet addition and acidification with GDL. Images have quantitatively analyzed to monitor the development of casein network over time, and the fractal dimensions of the different systems have been determined. Label free Coherent Anti-Stokes Raman Spectroscopy (CARS) Microscopy was used to verify that the presence of fluorophore did not interfere with the gelation process during STED microscopy. Image data has been directly correlated with dynamic texture analysis. We demonstrate the ability to resolve casein micelle structures on the scale of a single micelle, and extract valuable information that directly relates to the macroscopic properties of a gelled system. This work provides the basis for the use of STED microscopy to analyze other colloidal structures. We present a framework to extract meaningful information that directly relates to the macroscopic properties and functionality of the system. 692-Pos Board B457 A Novel STED Microscope with Nanometer Axial Sectioning Iva´n Coto Herna´ndez1, Siddharth Sivankutty2, Nicolas Bourg3, Sandrine Le´cart3, Guillaume Dupuis3, Sandrine Le´veˆque-Fort1. 1 Univ. Paris-Sud, Universite´ Paris-Saclay, CNRS, Institut des Sciences Mole´culaires d’Orsay (ISMO), Paris, France, 2Aix-Marseille Universite´, CNRS, Institut Fresnel, Marseille, France, 3Universite´ Paris-Sud, Centre de Photonique BioMe´dicale (CPBM), Paris, France. The axial resolution of conventional Stimulated emission depletion (STED) microscopy is limited by diffraction to about 500 nm. Overcoming this limitation usually comes at the cost of complex instrumentation, as 3D confinement usually implies to combine two STED beams. By trying to combine with TIRF excitation this decrease the lateral performances. In this work we reported a straightforward approach to restrict the axial extension of the STED microscope by only modifying the detection path, thus keeping optimal lateral resolution. This new membrane imaging takes advantage of the supercritical angle (SAF) emission of fluorophore that allows one to discriminate their position regarding the glass coverslip. Indeed only
Sunday, February 12, 2017
arrangement; the adjacent peptides are held together by strong salt-bridges. In contrast, the positive and negative ELKs diffuse to the head group surface, do not form effective scaffolds, and fail to stabilize the discoidal assembly. Hence, the simulations provide a structural rational for the experimental observations, and provide an avenue for computer based design of apolipoprotein mimetic peptides. 688-Pos Board B453 Computational and Experimental Studies of Gold Nanoparticle Templated HDL-Like Nanoparticles for Cholesterol Metabolism Cheng-Tsung Lai1, Wangqiang Sun2, Rohun U. Palekar2, C. Shad Thaxton2, George C. Schatz1. 1 Department of Chemistry, Northwestern University, Evanston, IL, USA, 2 Department of Urology, Northwestern University, Chicago, IL, USA. High-density lipoprotein (HDL) is involved in the transport and metabolism of phospholipids, triglycerides, and cholesterol. Mimics of HDL are being explored as potential therapeutic agents for removing excess cholesterol from arterial plaques. Gold nanoparticles (AuNPs) functionalized with apolipoprotein A-I (apoA-I), and with the lipids 1,2-dipalmitoylsn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (PDP PE) have been demonstrated to be acceptors of cellular cholesterol. However, detailed structural properties of this functionalized HDL AuNP are not well understood. We combined coarse-grained and all-atom MD simulations to simulate the self-assembly of lipids and cholesteryl ester on the AuNP/apoA-I construct to gain insights into its structural properties, and further make comparisons with experimental results. We find that lipids are oriented differently in regions with and without apoA-I. We also find that in this functionalized HDL AuNP, the distribution of cholesteryl ester maintains a reverse concentration gradient that is similar to the gradient found in native HDL. Incubating the HDL AuNP with cholesterol and lecithin: cholesterol acyltransferase (LCAT) demonstrate the HDL AuNP is able to efficiently activate LCAT and form esterified cholesterol from free cholesterol and phospholipid. 689-Pos Board B454 High-Throughput Thermodynamics of Drug-Membrane Interactions from Multiscale Simulations Roberto Menichetti, Kiran Kanekal, Kurt Kremer, Tristan Bereau. Theory Group, Max Planck Institute for Polymer Research, Mainz, Germany. The number of small organic molecules is overwhelmingly large–so large, that most of it remains unexplored. Computer simulations offer an appealing framework to probe many of these compounds without the need to synthesize them in the laboratory. The main hurdles preventing a high-throughput characterization of many small molecules relies on the time investment to parametrize the force field—a process that typically requires significant human intervention—and extensive sampling requirements. We address these issues by first sampling from the coarse-grained Martini model, for which we developed an automated parametrization protocol for small molecules. The resulting potential-of-mean-force (PMF) curves for the insertion of small molecules in lipid membranes show excellent agreement for a number of benchmark cases. Our framework allows us to run high-throughput molecular dynamics simulations and estimate tens of thousands of relative free energies of different small molecules in a lipid bilayer. They provide useful data to explore structure-property relationships from computer simulations.
Optical Microscopy and Super-resolution Imaging: Novel Approaches and Analysis I 690-Pos Board B455 Combining Expansion Microscopy and STED Nanoscopy for the Study of Cellular Organization Isotta Cainero1,2, Michele Oneto1,2, Luca Pesce1,2, Giulia Zanini1,2, Luca Lanzano`1,2, Alberto Diaspro1,2, Paolo Bianchini1,2. 1 Nanophysics, Istituto Italiano di Tecnologia, Genoa, Italy, 2University of Genoa, Genoa, Italy. Expansion microscopy (ExM) is a novel method that allows super-resolution imaging with conventional microscopes(1, 2). It consists in soaking the cells with a polymer, inducing the polymerization to form a dense meshwork throughout the cell, cross-linking the fluorophores to the polymer and, after digestion of cellular protein, rehydrating of the sample. The swelling of the polymer gel led to a fourfold isotropic stretching of the sample. Therefore, it increases the distance between two objects that otherwise couldnot be seen as
two different things with an ordinary microscope. One of the drawback of such a technique is the long preparation made of several stages, i.e. immunostaining, gelation, digestion and expansion. They are really crucial steps for a good imaging post-expansion. In our work we present a comparison between ExM and stimulated emission depletion (STED) nanoscopy(3). Our aim is to study the e possible combination of STED and ExM as a method to further enhance the final resolution achievable. We will in particularly take advantage of the use of separation of photons by lifetime tuning (SPLIT) STED (4). We show application of these methods from single fixed cells to slices of fixed mouse retina tissue. We are also interested in applying the approach to high-density compartments like the cell nucleus to decipher the high-order structure organization of chromatin-DNA. 1. Chen, F., P.W. Tillberg, and E.S. Boyden. 2015. Optical imaging. Expansion microscopy. Science. 347: 543-548. 2. Chozinski, T.J., A.R. Halpern, H. Okawa, H.-J. Kim, G.J. Tremel, R.O.L. Wong, and J.C. Vaughan. 2016. Expansion microscopy with conventional antibodies and fluorescent proteins. Nat Meth. 13: 485-488. 3. Bianchini, P., C. Peres, M. Oneto, S. Galiani, G. Vicidomini, and A. Diaspro. 2015. STED nanoscopy: a glimpse into the future. Cell Tissue Res. 360: 143-150. 4. Lanzano, L., I. Coto Hernandez, M. Castello, E. Gratton, A. Diaspro, and G. Vicidomini. 2015. Encoding and decoding spatio-temporal information for super-resolution microscopy. Nature Communications. 6: 6701. 691-Pos Board B456 Quantitative Microstructure Analysis of Casein Network Dynamics using STED Microscopy with Relation to Macroscopic Gel Properties Zachary J. Glover, Adam Cohen Simonsen, Jonathan Brewer. MEMPHYS, FKF, University of Southern Denmark, Odense, Denmark. Casein proteins are the main structural element in many fermented dairy products. Understanding how protein functionality is affected during modern food processing, such as filtration and spray drying, is key to product and process optimization. Advances in Super Resolution Microscopy have provided novel tools to investigate protein microstructures. Herein, Stimulated Emission Depletion (STED) Microscopy has been used to image casein micelle aggregation, gel formation and network coarsening over time. Protein structures have been resolved to > 100 nm. Native skim milk and reconstituted skim milk powder were used to produce gelled systems. Gelation was induced either through rennet addition and acidification with GDL. Images have quantitatively analyzed to monitor the development of casein network over time, and the fractal dimensions of the different systems have been determined. Label free Coherent Anti-Stokes Raman Spectroscopy (CARS) Microscopy was used to verify that the presence of fluorophore did not interfere with the gelation process during STED microscopy. Image data has been directly correlated with dynamic texture analysis. We demonstrate the ability to resolve casein micelle structures on the scale of a single micelle, and extract valuable information that directly relates to the macroscopic properties of a gelled system. This work provides the basis for the use of STED microscopy to analyze other colloidal structures. We present a framework to extract meaningful information that directly relates to the macroscopic properties and functionality of the system. 692-Pos Board B457 A Novel STED Microscope with Nanometer Axial Sectioning Iva´n Coto Herna´ndez1, Siddharth Sivankutty2, Nicolas Bourg3, Sandrine Le´cart3, Guillaume Dupuis3, Sandrine Le´veˆque-Fort1. 1 Univ. Paris-Sud, Universite´ Paris-Saclay, CNRS, Institut des Sciences Mole´culaires d’Orsay (ISMO), Paris, France, 2Aix-Marseille Universite´, CNRS, Institut Fresnel, Marseille, France, 3Universite´ Paris-Sud, Centre de Photonique BioMe´dicale (CPBM), Paris, France. The axial resolution of conventional Stimulated emission depletion (STED) microscopy is limited by diffraction to about 500 nm. Overcoming this limitation usually comes at the cost of complex instrumentation, as 3D confinement usually implies to combine two STED beams. By trying to combine with TIRF excitation this decrease the lateral performances. In this work we reported a straightforward approach to restrict the axial extension of the STED microscope by only modifying the detection path, thus keeping optimal lateral resolution. This new membrane imaging takes advantage of the supercritical angle (SAF) emission of fluorophore that allows one to discriminate their position regarding the glass coverslip. Indeed only