All-Atoms Md Simulation of Protein Translocation through α-Hemolysin Nanopore: Implications for...
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4048-Pos Board B776 Molecular Dynamics Simulations of Colloids in Single Solid-State Nanopores Nazar Ileri 1 , Matthew Davenport 1,2 , Sonia E. Letant 1 , Joseph W. Tringe 1 . 1 Lawrence Livermore National Lab, Livermore, CA, USA, 2 University of California Irvine, Irvine, CA, USA. The transport of biomolecules and small particles such as viruses through constrained geometries is critical to understand for efficient molecular separa- tions, detection and selective binding for biomedical and biodefense applica- tions. To develop improved capabilities for isolating and controlling both particles and molecules, there is a need for models developed by computational techniques and matched by well-integrated experiments. Here, we use molec- ular dynamics simulations to study the flow of colloidal silica particles (diam- eter ~50-100 nm) through cylindrical nanopores with diameter 200nm, and lengths 50-100 nm. Particle translocation is investigated in the presence of an applied electric field. We demonstrate the molecular origin of the pore resis- tance change that occurs once the colloid is inside the pore. The rate at which particles transit the nanopore is highly dependent on the dispersion forces and the electric field, which is in turn a function of both the applied voltage and the pore geometry. Models are supported by experimental findings obtained with resistive pulse sensing. This work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52- 07NA27344, LLNL-ABS-644260. 4049-Pos Board B777 Exploring Dynamic Events of Bacterial Microcompartment Shell Pores Sunny Chun 1 , Jiyong Park 1 , Michael C. Thompson 1 , Changsun Eun 2 , J Andrew McCammon 2 , Kendall N. Houk 1 , Todd O. Yeates 1 . 1 University of California, Los Angeles, Los Angeles, CA, USA, 2 University of California, San Diego, San Diego, CA, USA. Bacterial Microcompartments (BMCs) are proteinaceous organelles that sequester key metabolic reactions to increase enzymatic efficiency and prevent the loss of volatile or cytotoxic intermediates. 7 distinct BMC-type operons have been identified in 20-25% of bacteria, including BMCs involved in carbon fixation, propanediol catabolism, and ethanolamine catabolism. Ranging from 100 to 150 nm in width, BMCs are encapsulated in a protein shell consisting of hexagonal tiles of the conserved BMC-fold proteins. The BMC-fold pro- teins homo-oligomerize into hexameric or pseudo-hexameric assemblies with unique functionalities. High resolution X-ray determined structures have previ- ously elucidated the organization and tiling interactions of several shell proteins from BMCs. However, it remains unknown how small molecule metabolites may enter or exit the BMC, passing through the protein shell; nor how intermediates may be trapped inside. Here, we present initial findings using current methods in Molecular Dynamics to investigate BMC shell pores, including: (1) Metadynamics and Umbrella Sampling to probe the free energy profile of small molecule metabolites at predicted routes through BMC shell protein hexamer pores; (2) Accelerated MD and Targeted MD to observe the conformational change between BMC shell protein pseudo-hexamers that have been solved in open and closed pore conformations. These experiments shed insight on BMC shell pore dynamics and function, furthering our under- standing of BMC systems. 4050-Pos Board B778 All-Atoms Md Simulation of Protein Translocation through a-Hemolysin Nanopore: Implications for Protein Sequence/Structural Analyses Daniele Di Marino 1 , Anna Tramontano 1,2 , Mauro Chinappi 2 . 1 University of Rome "La Sapienza", Department of Physics, Rome, Italy, 2 Center for Life Nano Science @Sapienza, Istituto Italiano di Tecnologia, Rome, Italy. The cell isolation from the external environment and the cellular compartmen- talization is due to the presence of membranes composed by phospholipidic bilayers. Different proteins that play numerous biological functions are present in the membrane. These proteins are mainly involved in the communication between the cell and the external environment and between the different cellular compartments. Some of them form pores into the lipid bilayer that allow the translocation of molecules of different size and with different chemical-physical properties. The transport of these molecules across the mem- brane is crucial for the cellular survival. This feature of the membrane proteins has been extensively studied in order to use them as nanopores in biochemical and industrial fields. During the last decades nanopores have been exploited in the development of new techniques for nucleic acids sequencing. Much less effort has been dedi- cated to protein and polypeptide analysis using nanopores. Only in the last years pioneering studies appeared in the literature suggesting potentially revo- lutionary applications in the study of protein sequence and structure. Recent experimental data have demonstrated that, in order to be translocated across nanopores, proteins must be unfolded, with the unfolding process occurring through a multi-step mechanism. It has been observed, for instance, that the transport of the thioredoxin through the a-hemolysin nanopore occurs via a four steps process. In this work, all atom molecular dynamics simulations of the thioredoxin trans- location across the a-hemolysin nanopore have been performed. The data obtained allowed the description of the molecular mechanisms at the basis of the protein transport at the atomic level. Our results allowed us to gain a deeper knowledge regarding the translocation of protein through nanopores, useful for the development of nanopore based applications for several crucial biochemical analyses. 4051-Pos Board B779 Coarse-Grained Modeling of DNA-Vesicle Systems with the Martini Force Field Jaakko Uusitalo 1,2 , Helgi I. Ingo ´lfsson 1,2 , Parisa Akhshi 3 , D. Peter Tieleman 3 , Bert Poolman 1,2 , Andreas Herrmann 2 , Siewert J. Marrink 1,2 . 1 Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, Netherlands, 2 Zernike Institute for Advanced Materials, University of Groningen, Groningen, Netherlands, 3 University of Calgary, Calgary, AB, Canada. Computational modeling of DNA is a prime example of both recent advances in force field development as well as lingering difficulties in MD simulations. Coarse-grained models reduce the number of particles in the system and in- crease the available time and length scales of MD simulations [1]. However, most of the coarse-grained DNA models handle just DNA or, at most, only a few other classes of biomolecules which limits the range of applications for which they can be used. We are developing a coarse-grained model for DNA that is compatible with the Martini force field [2] and, thus, allows simulation studies of various DNA/biomolecular systems. We present the implementation of the current Martini DNA model and compare it to atomistic simulations and experimental results. We applied our model to study the behavior of DNA- copolymers in lipid membranes and vesicles. This work complements the work of our experimental collaborators working on selective fusion of vesicles, based on block copolymers containing single-stranded DNA that are placed on the surfaces of the vesicles. The selectivity in fusion is based on the hybridiza- tion of complementary DNA strands. Our Martini simulations provide near- atomic detail on the DNA-polymer-membrane interactions that is used to guide the experimental work. [1] H. Ingolfsson, C.A. Lopez, J.J. Uusitalo, D.H. de Jong, S. Gopal, X. Periole, S.J. Marrink. The power of coarse-graining in biomolecular simulations. WIREs Comput. Mol. Sci., in press, 2013. DOI:10.1002/wcms.1169. [2] S.J. Marrink, D.P. Tieleman. Perspective on the Martini model. Chem. Soc. Rev., 42, 6801-6822, 2013. 4052-Pos Board B780 Refinement of Multisite Ion Model through Simulation of Osmotic Pressure: Applications on Ion-Mediated Calculations of SsRNA Akansha Saxena, Angel E. Garcia. Physics,Applied Physics & Astronomy, Rensselaer Polytechnic Institute, Troy, NY, USA. Accurate force field parameters for ions are essential for meaningful simulation studies of proteins and nucleic acids. Currently accepted models of ions, espe- cially for divalent ions, do not necessarily reproduce the right physiological behavior. In our previous work, we found a model, called the multisite-ion model, where instead of treating the ions as an isolated sphere, we split the charge into multiple sites (Saxena et al, 2013). With this new model, we were not only able to achieve accurate coordination geometries around the ion, but were also able to predict better free energies for proteins and nucleic acids. Here, we further refine the model by focusing on the behavior of divalent ions in concentrated electrolyte solutions. With several ions present in water, it is important that there are no artifacts such as unusual ion-ion pairing or crys- tallization. Recently, Luo and Roux showed that osmotic pressure could be suc- cessfully used for refinement of ion parameters in NaCl and KCl solutions. We use the same method to test and calibrate the multisite-model of Mg 2þ and Ca 2þ in concentrated solution with Cl-. We find that after refinement the solu- tion gets rid of direct ion-pairs, matching the experimentally observed behavior. Subsequently, we use the refined parameters to observe the depen- dence of counterions on the flexibility of ssRNA. We compare our results to smFRET experiments by Chen et al, which show that Mg 2þ has more charge screening efficiency than Na þ on a 40-mer uridylate (rU40). Saxena, A., & Sept, D. (2013). JCTC, 9(8), 3538-3542. Wednesday, February 19, 2014 803a
Transcript of All-Atoms Md Simulation of Protein Translocation through α-Hemolysin Nanopore: Implications for...
Wednesday, February 19, 2014 803a
4048-Pos Board B776Molecular Dynamics Simulations of Colloids in Single Solid-StateNanoporesNazar Ileri1, Matthew Davenport1,2, Sonia E. Letant1, Joseph W. Tringe1.1Lawrence Livermore National Lab, Livermore, CA, USA, 2University ofCalifornia Irvine, Irvine, CA, USA.The transport of biomolecules and small particles such as viruses throughconstrained geometries is critical to understand for efficient molecular separa-tions, detection and selective binding for biomedical and biodefense applica-tions. To develop improved capabilities for isolating and controlling bothparticles and molecules, there is a need for models developed by computationaltechniques and matched by well-integrated experiments. Here, we use molec-ular dynamics simulations to study the flow of colloidal silica particles (diam-eter ~50-100 nm) through cylindrical nanopores with diameter 200nm, andlengths 50-100 nm. Particle translocation is investigated in the presence ofan applied electric field. We demonstrate the molecular origin of the pore resis-tance change that occurs once the colloid is inside the pore. The rate at whichparticles transit the nanopore is highly dependent on the dispersion forces andthe electric field, which is in turn a function of both the applied voltage and thepore geometry. Models are supported by experimental findings obtained withresistive pulse sensing.This work was performed under the auspices of the U.S. Department of Energyby Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344, LLNL-ABS-644260.
4049-Pos Board B777Exploring Dynamic Events of Bacterial Microcompartment Shell PoresSunny Chun1, Jiyong Park1, Michael C. Thompson1, Changsun Eun2,J Andrew McCammon2, Kendall N. Houk1, Todd O. Yeates1.1University of California, Los Angeles, Los Angeles, CA, USA, 2Universityof California, San Diego, San Diego, CA, USA.Bacterial Microcompartments (BMCs) are proteinaceous organelles thatsequester key metabolic reactions to increase enzymatic efficiency and preventthe loss of volatile or cytotoxic intermediates. 7 distinct BMC-type operonshave been identified in 20-25% of bacteria, including BMCs involved in carbonfixation, propanediol catabolism, and ethanolamine catabolism. Ranging from100 to 150 nm in width, BMCs are encapsulated in a protein shell consistingof hexagonal tiles of the conserved BMC-fold proteins. The BMC-fold pro-teins homo-oligomerize into hexameric or pseudo-hexameric assemblies withunique functionalities. High resolution X-ray determined structures have previ-ously elucidated the organization and tiling interactions of several shellproteins from BMCs. However, it remains unknown how small moleculemetabolites may enter or exit the BMC, passing through the protein shell;nor how intermediates may be trapped inside. Here, we present initial findingsusing current methods in Molecular Dynamics to investigate BMC shell pores,including: (1) Metadynamics and Umbrella Sampling to probe the free energyprofile of small molecule metabolites at predicted routes through BMC shellprotein hexamer pores; (2) Accelerated MD and Targeted MD to observe theconformational change between BMC shell protein pseudo-hexamers thathave been solved in open and closed pore conformations. These experimentsshed insight on BMC shell pore dynamics and function, furthering our under-standing of BMC systems.
4050-Pos Board B778All-Atoms Md Simulation of Protein Translocation through a-HemolysinNanopore: Implications for Protein Sequence/Structural AnalysesDaniele Di Marino1, Anna Tramontano1,2, Mauro Chinappi2.1University of Rome "La Sapienza", Department of Physics, Rome, Italy,2Center for Life Nano Science @Sapienza, Istituto Italiano di Tecnologia,Rome, Italy.The cell isolation from the external environment and the cellular compartmen-talization is due to the presence of membranes composed by phospholipidicbilayers. Different proteins that play numerous biological functions are presentin the membrane. These proteins are mainly involved in the communicationbetween the cell and the external environment and between the differentcellular compartments. Some of them form pores into the lipid bilayer thatallow the translocation of molecules of different size and with differentchemical-physical properties. The transport of these molecules across the mem-brane is crucial for the cellular survival. This feature of the membrane proteinshas been extensively studied in order to use them as nanopores in biochemicaland industrial fields.During the last decades nanopores have been exploited in the development ofnew techniques for nucleic acids sequencing. Much less effort has been dedi-cated to protein and polypeptide analysis using nanopores. Only in the lastyears pioneering studies appeared in the literature suggesting potentially revo-
lutionary applications in the study of protein sequence and structure. Recentexperimental data have demonstrated that, in order to be translocated acrossnanopores, proteins must be unfolded, with the unfolding process occurringthrough a multi-step mechanism. It has been observed, for instance, that thetransport of the thioredoxin through the a-hemolysin nanopore occurs via afour steps process.In this work, all atom molecular dynamics simulations of the thioredoxin trans-location across the a-hemolysin nanopore have been performed. The dataobtained allowed the description of the molecular mechanisms at the basis ofthe protein transport at the atomic level. Our results allowed us to gain a deeperknowledge regarding the translocation of protein through nanopores, useful forthe development of nanopore based applications for several crucial biochemicalanalyses.
4051-Pos Board B779Coarse-Grained Modeling of DNA-Vesicle Systems with the Martini ForceFieldJaakko Uusitalo1,2, Helgi I. Ingolfsson1,2, Parisa Akhshi3,D. Peter Tieleman3, Bert Poolman1,2, Andreas Herrmann2,Siewert J. Marrink1,2.1Groningen Biomolecular Sciences and Biotechnology Institute, Universityof Groningen, Groningen, Netherlands, 2Zernike Institute for AdvancedMaterials, University of Groningen, Groningen, Netherlands, 3University ofCalgary, Calgary, AB, Canada.Computational modeling of DNA is a prime example of both recent advances inforce field development as well as lingering difficulties in MD simulations.Coarse-grained models reduce the number of particles in the system and in-crease the available time and length scales of MD simulations [1]. However,most of the coarse-grained DNA models handle just DNA or, at most, only afew other classes of biomolecules which limits the range of applications forwhich they can be used. We are developing a coarse-grained model for DNAthat is compatible with the Martini force field [2] and, thus, allows simulationstudies of various DNA/biomolecular systems. We present the implementationof the current Martini DNA model and compare it to atomistic simulations andexperimental results. We applied our model to study the behavior of DNA-copolymers in lipid membranes and vesicles. This work complements thework of our experimental collaborators working on selective fusion of vesicles,based on block copolymers containing single-stranded DNA that are placed onthe surfaces of the vesicles. The selectivity in fusion is based on the hybridiza-tion of complementary DNA strands. Our Martini simulations provide near-atomic detail on the DNA-polymer-membrane interactions that is used to guidethe experimental work.[1] H. Ingolfsson, C.A. Lopez, J.J. Uusitalo, D.H. de Jong, S. Gopal, X. Periole,S.J. Marrink. The power of coarse-graining in biomolecular simulations.WIREs Comput. Mol. Sci., in press, 2013. DOI:10.1002/wcms.1169.[2] S.J. Marrink, D.P. Tieleman. Perspective on the Martini model. Chem. Soc.Rev., 42, 6801-6822, 2013.
4052-Pos Board B780Refinement of Multisite Ion Model through Simulation of OsmoticPressure: Applications on Ion-Mediated Calculations of SsRNAAkansha Saxena, Angel E. Garcia.Physics,Applied Physics & Astronomy, Rensselaer Polytechnic Institute,Troy, NY, USA.Accurate force field parameters for ions are essential for meaningful simulationstudies of proteins and nucleic acids. Currently accepted models of ions, espe-cially for divalent ions, do not necessarily reproduce the right physiologicalbehavior. In our previous work, we found a model, called the multisite-ionmodel, where instead of treating the ions as an isolated sphere, we split thecharge into multiple sites (Saxena et al, 2013). With this new model, wewere not only able to achieve accurate coordination geometries around theion, but were also able to predict better free energies for proteins and nucleicacids. Here, we further refine the model by focusing on the behavior of divalentions in concentrated electrolyte solutions. With several ions present in water, itis important that there are no artifacts such as unusual ion-ion pairing or crys-tallization. Recently, Luo and Roux showed that osmotic pressure could be suc-cessfully used for refinement of ion parameters in NaCl and KCl solutions. Weuse the same method to test and calibrate the multisite-model of Mg2þ andCa2þ in concentrated solution with Cl-. We find that after refinement the solu-tion gets rid of direct ion-pairs, matching the experimentally observedbehavior. Subsequently, we use the refined parameters to observe the depen-dence of counterions on the flexibility of ssRNA. We compare our results tosmFRET experiments by Chen et al, which show that Mg2þ has more chargescreening efficiency than Naþ on a 40-mer uridylate (rU40).Saxena, A., & Sept, D. (2013). JCTC, 9(8), 3538-3542.
