- Email: [email protected]
Molecular Transport through Large Diameter DNA Origami Channels
416a
Tuesday, February 14, 2017
pathway at physiological temperatures. Whole-cell patch-clamp experiments revealed that SiNPs trigger large currents that could be blocked by the cation channel blocker ruthenium red in a voltage-dependent manner. Analysis of the selectivity properties of this current showed that they are mainly carried by cations. Taken together, our results demonstrate that SiNPs induce Ca2þ-permeable pores in the plasma membrane, and that this phenomenon is enhanced by factors that increase membrane fluidity. We propose that this Ca2þ entry pathway may be relevant for the toxicological properties of SiNPs.
2. Zhou, et al & Guo, P. (2016). Oriented single directional insertion of nanochannel of bacteriophage SPP1 DNA packaging motor into lipid bilayer via polar hydrophobicity. Biomaterials, 105: 222. 3. Wendell, et al & Guo, P. (2009). Translocation of double-stranded DNA through membrane-adapted phi29 motor protein nanopores. Nature nanotechnology, 4:765. 4. Haque, et al & Guo, P. (2012). Real-time sensing and discrimination of single chemicals using the channel of phi29 DNA packaging nanomotor. ACS nano, 6:3251.
2046-Pos Board B366 How to Get Large Drugs through Small Pores? Exploiting the Porins Pathway in Pseudomonas Aeruginosa Susruta Samanta1, Tommaso D’Agostino1, Ishan Ghai2, Monisha Pathania3, Silvia Acosta Gutierrez1, Mariano Andrea Scorciapino1, Igor Bodrenko1, Richard Wagner2, Bert van den Berg3, Mathias Winterhalter2, Matteo Ceccarelli1. 1 Physics, University of Cagliari, Monserrato, Italy, 2Jacobs University, Bremen, Germany, 3University of Newcastle, Newcastle upon Tyne, United Kingdom. The main focus of our study is to identify the structural features responsible for the transport of molecules through substrate-specific channels of Gram negative bacterium Pseudomonas aeruginosa. We present a precise molecular analysis of the structure and dynamics of OccK8 to explain the translocation of natural amino acid residues and antibiotics of two different chemical families through it. We used molecular dynamics simulations to obtain information on the molecule/channel interactions and proposed quantitative structure-function relationships based on them. We found some physical chemical properties of molecules playing an important role in modulating the translocation through OccK8. Molecules with zwitterionic structures have an affinity towards the polar eyelet region and presence of negative charge help interaction with the basic ladder facilitating translocation. A favorable chemical structure helps to widen the otherwise small constriction region helping translocation of relatively large antibiotics. Whenever possible, liposome swelling assays and electrophysiology at single-molecule level were used to confirm our data.
2049-Pos Board B369 Perturbation of Bilayer Surface Tension Differentially Modulates Mechanosensitive Ion Channels Navid Bavi1,2, Charles D. Cox1,2, Omid Bavi3, Boris Martinac1,2. 1 Victor Chang Cardiac Research Institute, Sydney, Australia, 2St. Vincent’s Clinical School, University of New South Wales, Sydney, Australia, 3 Institute for Nanoscience and Nanotechnology, Sharif University of Technology, Tehran, Iran, Islamic Republic of. The lipid composition of biological membranes modulates the activity of integral membrane proteins (1). This is particularly important for mechanosensitive (MS) channels, regardless of their gating paradigm. Here we use the surface active agent 2,2,2-trifluoroethanol (TFE) as a pharmacological tool to study the effect of surface tension perturbations on an array of bacterial and mammalian MS channels including MscL, Piezo1 and TREK-1. We chose TFE (a general anaesthetic) due to the fact that its effect on the bacterial channel MscS has already been studied. In particular TFE facilitates MscS activation from the periplasmic side, while it abolishes MscS current from the cytoplasmic side (2, 3). Here, we demonstrate that 2 % v/v TFE can also facilitate the activation of: (i) MscL if TFE is added to either bilayer leaflet, (ii) Piezo1 only if added to the cytoplasmic side and (iii) TREK-1 only from the extracellular side. Our molecular dynamics simulations revealed TFE increases the surface tension and the first moment of the transbilayer pressure profile markedly and hence facilitates activation of MscL. Using our molecular dynamics, energetic analysis and collective experimental data, we postulate there is a close relationship between MS channel shape and its activation mechanism by surface tension perturbations. The activation curve of MscL, which is a cylindrical protein, was shifted to the left (activated easier) upon addition of TFE from either side. MscS and TREK on the other hand, which are conical, were only facilitated from the extracellular side. The activation curve of Piezo1 was also shifted to left only when TFE was added to the cytoplasmic side. Given surface active drugs are adsorbed onto cell membranes, these findings provide a mechanistic understanding of their non-specific impact on the function of different membrane proteins, particularly MS ion channels. 1. Cantor RS (1998) The lateral pressure profile in membranes: a physical mechanism of general anesthesia. Toxicol Lett 100-101:451-458. 2. Akitake B, Spelbrink RE, Anishkin A, Killian JA, de Kruijff B, & Sukharev S (2007) 2,2,2-Trifluoroethanol changes the transition kinetics and subunit interactions in the small bacterial mechanosensitive channel MscS. Biophys J 92(8):2771-2784. 3. Nomura T, Cox CD, Bavi N, Sokabe M, & Martinac B (2015) Unidirectional incorporation of a bacterial mechanosensitive channel into liposomal membranes. FASEB J 29(10):4334-4345.
2047-Pos Board B367 Molecular Transport through Large Diameter DNA Origami Channels Swati Krishnan, Friedrich Simmel. Physics, TU munich, Munich, Germany. We introduce a large DNA membrane channel with a z 4 nm diameter pore, which spontaneously inserts into flat lipid bilayer membranes using hydrophobic functionalizations or streptavidin linkages between biotinylated channels and lipids. Using a dye influx assay, we demonstrate formation of membrane pores in giant unilamellar vesicles. The versatility of DNA nanotechnology makes these channels promising molecular devices for synthetic biology and biosensing. 2048-Pos Board B368 Fingerprinting and Differentiation of Small Proteins with a Large Channel of Bacteriophage PHI29 DNA Packaging Motor Zhouxiang Ji, Shaoying Wang, Zhengyi Zhao, Zhi Zhou, Farzin Haque, Peixuan Guo. The Ohio State University, Columbus, OH, USA. Insertion of biological nanopores to the lipid membranes has inspired single molecule sensing of peptides and proteins. Most biological membrane nanopores are around 1.2 nm, too small for the folded protein to pass through the nanopore. A well-studied bacteriophage phi29 DNA packaging motor with a large channel has been inserted into the lipid membrane, served as the biosensor of nucleic acid and chemical. Herein, we reported that the large channel of bacteriophage phi29 packaging motor can be used for fingerprinting of peptides or small proteins. Different peptides can be distinguished well with the blockage, dwell time or ionic signature, and the oligomeric states of peptides can be investigated in real time. The translocation was confirmed by single molecule florescence imaging. Further, Ni-NTA nanogold binding assay showed that peptides were translocated from the N-terminal to the C-terminal of the channel. Single direction insertion of the nanopore into the channel was controlled by polar hydrophobicity of the N or C terminal. Our results demonstrate the potential of this nanopore system for the detection of peptide biomarkers related to certain diseases. Reference: 1. Ji, Z. et al & Guo, P. (2016). Fingerprinting of Peptides with a Large Channel of Bacteriophage Phi29 DNA Packaging Motor. Small, 12: 4572.
2050-Pos Board B370 Novel Properties of LRRC8-Mediated VRAC Currents Antonella Gradogna1, He´ctor Gaita´n-Pen˜as2, Lara Laparra-Cuervo3, Carles Solsona4, Vı´ctor Ferna´ndez-Duen˜as5, Alejandro Barrallo-Gimeno2, Francisco Ciruela6, Melike Lakadamyali3, Rau´l Este´vez2, Michael Pusch1. 1 Istituto di Biofisica, Genoa, Italy, 2Unitat de Fisiologı´a, Departament de Cie`ncies Fisiolo`giques II, IDIBELL-Universitat de Barcelona, Barcelona, Spain, 3The Barcelona Institute of Science and Technology, ICFO-Institut de Cie`ncies Foto`niques, Barcelona, Spain, 4Laboratory of Neurobiology, IDIBELL-Universitat de Barcelona, Barcelona, Spain, 5Unitat de Farmacologia, Departament Patologia i Terape`utica Experimenta, IDIBELLUniversitat de Barcelona, Barcelona, Spain, 6Unitat de Farmacologia, Departament Patologia i Terape`utica Experimental, IDIBELL-Universitat de Barcelona, Barcelona, Spain. Heteromers of the LRRC8A protein with other LRRC8 members (B, C, D, E) form the volume regulated anion channel (VRAC). We used the Xenopus oocyte system to study functional and structural properties of these channels. 8A/8C, 8A/8D, and 8A/8E heteromers gave rise to small volume stimulated currents. We discovered that adding fluorescent proteins to the C-terminus resulted in constitutive anion channel activity that was further
Tuesday, February 14, 2017
pathway at physiological temperatures. Whole-cell patch-clamp experiments revealed that SiNPs trigger large currents that could be blocked by the cation channel blocker ruthenium red in a voltage-dependent manner. Analysis of the selectivity properties of this current showed that they are mainly carried by cations. Taken together, our results demonstrate that SiNPs induce Ca2þ-permeable pores in the plasma membrane, and that this phenomenon is enhanced by factors that increase membrane fluidity. We propose that this Ca2þ entry pathway may be relevant for the toxicological properties of SiNPs.
2. Zhou, et al & Guo, P. (2016). Oriented single directional insertion of nanochannel of bacteriophage SPP1 DNA packaging motor into lipid bilayer via polar hydrophobicity. Biomaterials, 105: 222. 3. Wendell, et al & Guo, P. (2009). Translocation of double-stranded DNA through membrane-adapted phi29 motor protein nanopores. Nature nanotechnology, 4:765. 4. Haque, et al & Guo, P. (2012). Real-time sensing and discrimination of single chemicals using the channel of phi29 DNA packaging nanomotor. ACS nano, 6:3251.
2046-Pos Board B366 How to Get Large Drugs through Small Pores? Exploiting the Porins Pathway in Pseudomonas Aeruginosa Susruta Samanta1, Tommaso D’Agostino1, Ishan Ghai2, Monisha Pathania3, Silvia Acosta Gutierrez1, Mariano Andrea Scorciapino1, Igor Bodrenko1, Richard Wagner2, Bert van den Berg3, Mathias Winterhalter2, Matteo Ceccarelli1. 1 Physics, University of Cagliari, Monserrato, Italy, 2Jacobs University, Bremen, Germany, 3University of Newcastle, Newcastle upon Tyne, United Kingdom. The main focus of our study is to identify the structural features responsible for the transport of molecules through substrate-specific channels of Gram negative bacterium Pseudomonas aeruginosa. We present a precise molecular analysis of the structure and dynamics of OccK8 to explain the translocation of natural amino acid residues and antibiotics of two different chemical families through it. We used molecular dynamics simulations to obtain information on the molecule/channel interactions and proposed quantitative structure-function relationships based on them. We found some physical chemical properties of molecules playing an important role in modulating the translocation through OccK8. Molecules with zwitterionic structures have an affinity towards the polar eyelet region and presence of negative charge help interaction with the basic ladder facilitating translocation. A favorable chemical structure helps to widen the otherwise small constriction region helping translocation of relatively large antibiotics. Whenever possible, liposome swelling assays and electrophysiology at single-molecule level were used to confirm our data.
2049-Pos Board B369 Perturbation of Bilayer Surface Tension Differentially Modulates Mechanosensitive Ion Channels Navid Bavi1,2, Charles D. Cox1,2, Omid Bavi3, Boris Martinac1,2. 1 Victor Chang Cardiac Research Institute, Sydney, Australia, 2St. Vincent’s Clinical School, University of New South Wales, Sydney, Australia, 3 Institute for Nanoscience and Nanotechnology, Sharif University of Technology, Tehran, Iran, Islamic Republic of. The lipid composition of biological membranes modulates the activity of integral membrane proteins (1). This is particularly important for mechanosensitive (MS) channels, regardless of their gating paradigm. Here we use the surface active agent 2,2,2-trifluoroethanol (TFE) as a pharmacological tool to study the effect of surface tension perturbations on an array of bacterial and mammalian MS channels including MscL, Piezo1 and TREK-1. We chose TFE (a general anaesthetic) due to the fact that its effect on the bacterial channel MscS has already been studied. In particular TFE facilitates MscS activation from the periplasmic side, while it abolishes MscS current from the cytoplasmic side (2, 3). Here, we demonstrate that 2 % v/v TFE can also facilitate the activation of: (i) MscL if TFE is added to either bilayer leaflet, (ii) Piezo1 only if added to the cytoplasmic side and (iii) TREK-1 only from the extracellular side. Our molecular dynamics simulations revealed TFE increases the surface tension and the first moment of the transbilayer pressure profile markedly and hence facilitates activation of MscL. Using our molecular dynamics, energetic analysis and collective experimental data, we postulate there is a close relationship between MS channel shape and its activation mechanism by surface tension perturbations. The activation curve of MscL, which is a cylindrical protein, was shifted to the left (activated easier) upon addition of TFE from either side. MscS and TREK on the other hand, which are conical, were only facilitated from the extracellular side. The activation curve of Piezo1 was also shifted to left only when TFE was added to the cytoplasmic side. Given surface active drugs are adsorbed onto cell membranes, these findings provide a mechanistic understanding of their non-specific impact on the function of different membrane proteins, particularly MS ion channels. 1. Cantor RS (1998) The lateral pressure profile in membranes: a physical mechanism of general anesthesia. Toxicol Lett 100-101:451-458. 2. Akitake B, Spelbrink RE, Anishkin A, Killian JA, de Kruijff B, & Sukharev S (2007) 2,2,2-Trifluoroethanol changes the transition kinetics and subunit interactions in the small bacterial mechanosensitive channel MscS. Biophys J 92(8):2771-2784. 3. Nomura T, Cox CD, Bavi N, Sokabe M, & Martinac B (2015) Unidirectional incorporation of a bacterial mechanosensitive channel into liposomal membranes. FASEB J 29(10):4334-4345.
2047-Pos Board B367 Molecular Transport through Large Diameter DNA Origami Channels Swati Krishnan, Friedrich Simmel. Physics, TU munich, Munich, Germany. We introduce a large DNA membrane channel with a z 4 nm diameter pore, which spontaneously inserts into flat lipid bilayer membranes using hydrophobic functionalizations or streptavidin linkages between biotinylated channels and lipids. Using a dye influx assay, we demonstrate formation of membrane pores in giant unilamellar vesicles. The versatility of DNA nanotechnology makes these channels promising molecular devices for synthetic biology and biosensing. 2048-Pos Board B368 Fingerprinting and Differentiation of Small Proteins with a Large Channel of Bacteriophage PHI29 DNA Packaging Motor Zhouxiang Ji, Shaoying Wang, Zhengyi Zhao, Zhi Zhou, Farzin Haque, Peixuan Guo. The Ohio State University, Columbus, OH, USA. Insertion of biological nanopores to the lipid membranes has inspired single molecule sensing of peptides and proteins. Most biological membrane nanopores are around 1.2 nm, too small for the folded protein to pass through the nanopore. A well-studied bacteriophage phi29 DNA packaging motor with a large channel has been inserted into the lipid membrane, served as the biosensor of nucleic acid and chemical. Herein, we reported that the large channel of bacteriophage phi29 packaging motor can be used for fingerprinting of peptides or small proteins. Different peptides can be distinguished well with the blockage, dwell time or ionic signature, and the oligomeric states of peptides can be investigated in real time. The translocation was confirmed by single molecule florescence imaging. Further, Ni-NTA nanogold binding assay showed that peptides were translocated from the N-terminal to the C-terminal of the channel. Single direction insertion of the nanopore into the channel was controlled by polar hydrophobicity of the N or C terminal. Our results demonstrate the potential of this nanopore system for the detection of peptide biomarkers related to certain diseases. Reference: 1. Ji, Z. et al & Guo, P. (2016). Fingerprinting of Peptides with a Large Channel of Bacteriophage Phi29 DNA Packaging Motor. Small, 12: 4572.
2050-Pos Board B370 Novel Properties of LRRC8-Mediated VRAC Currents Antonella Gradogna1, He´ctor Gaita´n-Pen˜as2, Lara Laparra-Cuervo3, Carles Solsona4, Vı´ctor Ferna´ndez-Duen˜as5, Alejandro Barrallo-Gimeno2, Francisco Ciruela6, Melike Lakadamyali3, Rau´l Este´vez2, Michael Pusch1. 1 Istituto di Biofisica, Genoa, Italy, 2Unitat de Fisiologı´a, Departament de Cie`ncies Fisiolo`giques II, IDIBELL-Universitat de Barcelona, Barcelona, Spain, 3The Barcelona Institute of Science and Technology, ICFO-Institut de Cie`ncies Foto`niques, Barcelona, Spain, 4Laboratory of Neurobiology, IDIBELL-Universitat de Barcelona, Barcelona, Spain, 5Unitat de Farmacologia, Departament Patologia i Terape`utica Experimenta, IDIBELLUniversitat de Barcelona, Barcelona, Spain, 6Unitat de Farmacologia, Departament Patologia i Terape`utica Experimental, IDIBELL-Universitat de Barcelona, Barcelona, Spain. Heteromers of the LRRC8A protein with other LRRC8 members (B, C, D, E) form the volume regulated anion channel (VRAC). We used the Xenopus oocyte system to study functional and structural properties of these channels. 8A/8C, 8A/8D, and 8A/8E heteromers gave rise to small volume stimulated currents. We discovered that adding fluorescent proteins to the C-terminus resulted in constitutive anion channel activity that was further