- Email: [email protected]
Simulations of Membrane Disrupting Peptides Pores Versus Surface Binding
Tuesday, February 14, 2017
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even just a collateral effect. Additionally, AMP interactions with non-lipid cell envelope components of bacteria may be important in modifying how well AMPs are able to disrupt the lipid membrane. In order to connect studies of AMPs in model lipid systems to the more complex real bacterial cell envelopes, we have deuterium-labeled the membranes of the gram-positive bacteria Bacillus subtilis and used 2H NMR to study how lipid acyl chain order in its membranes is affected by treatment with AMPs. We have also observed 2H NMR spectra from Bacillus subtilis in which the peptidoglycan layer has been disrupted. This has allowed us to investigate how disruption of the peptidoglycan layer affects bacterial lipid chain order and the AMP/bacteria interaction.
structure and dynamics of model membrane systems. In particular, we investigated the effects of the antimicrobial peptides gramicidin and alamethecin on the lipid bilayer structure using small angle x-ray and neutron scattering. The structural studies were complemented by NSE experiments to probe the collective bending and thickness fluctuation dynamics in these model systems. Notably, the NSE results revealed enhanced thickness fluctuation dynamics in lipid bilayers containing low concentration of gramicidin that were dampened with increasing peptide concentration. An enhancement in dynamics was not seen in bilayers containing alamethicin, suggesting that the dynamics not only depend on peptide concentration, but also peptide orientation within the membrane
1880-Pos Board B200 Lipid Clustering by Antimicrobial Polymers and Lectins Anja Stulz1, Winfried Ro¨mer2, Karen Lienkamp3, Heiko Heerklotz1,2, Maria Hoernke1,2. 1 Pharmaceutical Technology and Biopharmacy, Albert-Ludwigs-Universit€at, Freiburg i.Br., Germany, 2BIOSS Centre for Biological Signalling Studies, Albert-Ludwigs-Universit€at, Freiburg i.Br., Germany, 3IMTEK Chemistry and Physics of Interfaces, Albert-Ludwigs-Universit€at, Freiburg i.Br., Germany. The current urge to understand the role of lipids in defense against pathogens is driven by two strategies: killing pathogens and protecting the cell from infection. Antimicrobial peptides and antimicrobial polymers offer a promising alternative to classical antibiotics through their action on membrane integrity. In principle, antimicrobial peptides and polymers are able to cluster lipids through lipid selection and recruitment from a mixed membrane. We show that antimicrobial polymers induce leakage in lipid vesicles by transient defects rather than defined pores. The positively charged polymers efficiently cluster negatively charged lipids from mixed model membranes. Larger domains are formed (in the order of 500 lipids). The binding of the polymers to the vesicles is exothermic. Our findings correlate with the polymers activity against bacteria. Similarly, lectins, carbohydrate binding proteins, can recognize certain glycolipids in mixed membranes, bind them and cluster them. By a yet unknown mechanism, the membrane is locally bent. Thus bacterial lectins can initiate uptake of pathogens into host cells and are promising targets for drug development.
1883-Pos Board B203 Mode of Action of Antimicrobial Peptides: Long and Short Amphipathic Alpha-Helixes Use Different Mechanisms Erik Strandberg1, Ariadna Grau-Campistany2, Hector Zamora-Carreras3, Marie-Claude Gagnon4, Philipp M€uhlh€auser1, Parvesh Wadhwani1, Jochen B€urck1, Johannes Reichert1, Michele Auger4, Jean-Francois Paquin4, M. Angeles Jime´nez3, Marta Bruix3, Francesc Rabanal2, Anne S. Ulrich1. 1 Institute for Biological Interfaces, Karlsruhe Institute of Technology, Karlsruhe, Germany, 2Universitat de Barcelona, Barcelona, Spain, 3Instituto de Quı´mica Fı´sica ‘‘Rocasolano’’, CSIC, Madrid, Spain, 4Universite´ Laval, Quebec, QC, Canada. We have studied the membrane structure and orientation of cationic amphipathic a-helical antimicrobial peptides (AMPs) using circular dichroism and solid-state NMR, combined with activity studies. For a series of model peptides, called KIA peptides, a clear length-dependent activity is found, as only peptides long enough to span the hydrophobic thickness of the membrane could induce leakage in vesicles. There is also a clear threshold length for peptides able to kill bacteria [1]. Using another series of KIA-like peptides of different length (from 14 up to 28 residues) but with a constant charge revealed that the length, but not the charge, is the critical factor. In membrane systems with a positive spontaneous curvature, the peptides get inserted into the membrane in a transmembrane orientation. All results indicate that these peptides act by forming proper oligomeric pores in the lipid bilayer. If the peptide is just long enough to span the membrane, it is aligned perfectly upright, but longer peptides can tilt cooperatively in the pore like an iris [1,2]. On the other hand, BP100, a highly helical peptides of only 11 amino acids, is clearly too short to form a transmembrane pore, but it is still strongly active against bacteria. From solid-state 2H-, 15N- and 19F-NMR studies, this peptide is found to dip into the membrane and to show high mobility within the amphiphilic surface layer. This way, it most likely disturbs the lipid order and thereby induces permeability, which suggests a carpet-like mechanism of action [3]. The structural results on these long and short AMPs clearly demonstrate that peptides with similar structural characteristics can act by very different mechanisms. References: [1] Grau-Campistany et al., (2015) Sci Rep 5, 9388. [2] Grau-Campistany et al., (2016) J Phys Chem Lett 7, 1116-1120. [3] Zamora-Carreras et al., (2016). Biochim Biophys Acta 1858, 1328-1338.
1881-Pos Board B201 How Antimicrobial Peptides Permeabilize Membranes with and without Pore Formation Jakob P. Ulmschneider. Institute of Natural Sciences, Shanghai Jiao Tong University, Shanghai, China. One of the biggest enigmas of antimicrobial peptides (AMPs), which protect all forms of life against pathogens, is why few structures of membrane pores have been found despite clear evidence of membrane leakage and antimicrobial activity. We provide a surprisingly simple explanation: For some AMPs such as PGLa (charge þ5), pores are not needed to explain both leakage and peptide translocation. Fully converged, unbiased multi-microsecond equilibrium simulations at all-atomistic level reveal that peptides spontaneously translocate across the membrane individually on a timescale of tens of microseconds, without forming pores. These findings explain why, despite vesicular leakage no channel has been identified for PGLa. However, similar simulations on other, lesser charged AMPs like maculatin clearly show pore formation. The results suggest that for some specific antimicrobial peptides, pore formation may not have to be invoked at all to explain both peptide translocation and membrane permeabilization. 1882-Pos Board B202 The Synergistic Effects of Lipids and Peptides on Membrane Dynamics Elizabeth Kelley1, Andrea Woodka1, Paul Butler1,2, Michihiro Nagao1,3. 1 Center for Neutron Research, NIST, Gaithersburg, MD, USA, 2Chemical and Biomolecular Engineering, University of Delaware, Newark, DE, USA, 3 Center for Exploration of Energy and Matter, Indiana University, Bloomington, IN, USA. There is a growing appreciation that the membrane physical properties are essential to cell and protein function. Simply altering the thickness of model membranes has been shown to influence the biological activity of several proteins, while incorporating peptides into lipid membranes also is known to affect the bilayer structural properties. Clearly there is a synergy in lipidprotein interactions in determining the membrane properties; however, the nature of these interactions are not well understood. Here we use a combination of small angle scattering techniques and neutron spin echo spectroscopy (NSE) to investigate the effects of incorporating a small peptide on both the
1884-Pos Board B204 Simulations of Membrane Disrupting Peptides Pores Versus Surface Binding B. Scott Perrin Jr.1, Riqiang Fu2, Myriam L. Cotten3, Richard W. Pastor1. 1 Laboratory of Computational Biology, NHLBI/NIH, Rockville, MD, USA, 2 National High Magnetic Field Laboratory, Tallahassee, FL, USA, 3 Department of Applied Science, College of William & Mary, Williamsburg, VA, USA. Peptides that disrupt biological membranes are a source of new antibiotic and antiviral therapeutics. Here, the relationships between peptide primary sequences, membrane bound structures, and abilities to disrupt membranes are investigated using all-atom molecular dynamics simulations. First, the archetype barrel-stave alamethicin (alm) pore in a 1,2-dioleoylsn-glycero-3-phosphocholine bilayer at 313 K indicates that ~7 ms is required for equilibration of a preformed 6-peptide pore; the pore remains stable for the duration of the remaining 7 ms of the trajectory, and the structure factors agree well with experiment. A 5 ms simulation of 10 surface-bound alm peptides shows significant peptide unfolding and some unbinding, but no insertion. Simulations at 363 and 413 K with a 0.2 V electric field yield peptide insertion in 1 ms. Insertion is initiated by the folding of residues 3-11 into an a-helix, and mediated by membrane water or by previously inserted peptides. The stability of five alm pore peptides at 413 K with a 0.2 V electric field demonstrates a significant preference for a transmembrane orientation. In contrast, a hypothesis that the antimicrobial peptide piscidin 1 (p1) forms toroidal pores is tested. The
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primary simulation was initialized with 20 peptides in four barrel-stave pores in a fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol bilayer. The four pores relaxed to toroidal by 200 ns, only one pore-like structure containing two transmembrane helices remained at 26 ms, and none of the 18 peptides released to the surface reinserted to form pores. The simulation was repeated at 413 K with an applied electric field and all peptides were surface-bound by 200 ns. Trajectories of surface-bound piscidin with and without applied fields at 313 and 413 K and totaling 6 ms show transient distortions of the bilayer/water interface (consistent with 31P NMR), but no insertion to transmembrane or pore states. 15 N chemical shifts confirm a fully surface-bound conformation. Taken together, the simulation and experimental results imply that transient defects rather than stable pores are responsible for membrane disruption by p1, and likely other AMPs. 1885-Pos Board B205 Simulation and Database-Guided Antimicrobial Peptide Evolution Charles H. Chen1,2, Charles G. Starr3, Gregory Wiedman1, William C. Wimley3, Jakob P. Ulmschneider4, Martin B. Ulmschneider1,2. 1 Whiting School of Engineering, Johns Hopkins University, Baltimore, MD, USA, 2Institute for NanoBiotechnology, Johns Hopkins University, Baltimore, MD, USA, 3Department of Biochemistry and Molecular Biology, Tulane University School of Medicine, New Orleans, LA, USA, 4Institute of Natural Sciences, Shanghai Jiao Tong University, Shanghai, China. Antimicrobial peptides (AMPs) are powerful and ubiquitous components of the innate immune defense in all domains of life. AMPs are amphiphilic peptides that selectively target and kill a wide variety of microbial pathogens at low micro-molar concentrations. These peptides vary widely in size, sequence, and secondary structure, and no common activity motif has been discovered to date. Here, we report several powerful new synthetic AMPs that are developed using a combination of molecular dynamics (MD) simulations and database screening. First, we use MD simulations to study the mechanisms of AMP folding and pore assembly in a bacterial model membrane. The atomic detail information of how pores form was used to develop a novel AMP: LDKA, which is a template sequence based on atomic detail structural information from the simulations. This simple peptide is composed a small number of amino acids and shows powerful pore-forming properties. Second, we apply statistical analysis of several thousand AMPs to optimize the LDKA template sequence. Finally, we apply a high-throughput screen using dye leakage assays to both bacterial and mammalian membrane model vesicles. This allows us to fine-tune the pore size and the binding selectivity for different membrane types. From the screen, we have identified 9 different LDKA analogues with different membrane selectivity and pore sizes. Our work confirms that the combination of MD simulations, database optimization, and theory/computation guided high-throughput screening is a powerful strategy to design potent new antimicrobial peptides for different biomedical targets and applications. 1886-Pos Board B206 Discovering Novel Antimicrobial Peptides using High-Throughput Screening in the Presence of Human Erythrocytes Charles G. Starr, Jing He, William C. Wimley. Department of Biochemistry and Molecular Biology, Tulane University, New Orleans, LA, USA. Antimicrobial peptides (AMPs) have long been considered excellent candidates for development as antibiotic agents to combat the threat of drug resistant bacterial pathogens. Yet, the promise AMPs have displayed in the laboratory has not yielded correlative clinical success. We hypothesized that a major reason for the disparity between outcomes at the bench and in the clinic is AMP interaction with and toxicity towards host cells. Here, we validate this theory using human red blood cells as model eukaryotic cells. Indeed, the potency of several well-studied antimicrobial peptides decreases from minimum sterilizing concentrations (MSC) of 1-5 mM to >30 mM in the presence of RBCs at 1x109 cells/ml; equivalent to 20% of the human physiological concentration. To address this issue, we have developed a high-throughput method to screen a combinatorial peptide library for antimicrobial activity in the presence of concentrated human cells. The screen consists of assays that assess the effect of RBCs on antimicrobial activity as well as toxicity toward eukaryotic cells. We present data on peptides identified and characterized from the high-throughput library screen and show that these new peptides were not as effective as they initially appeared during the screen. We demonstrate that the difference between observed and expected results is due to unforeseen proteolytic activity sequestered within the cytosol of the red blood cells. Finally, we show that a library consensus sequence synthesized using all d-amino acids outperforms all library isolates as well as the template sequence in terms of
microbicidal activity in the presence of RBCs. Ultimately, our results suggest that combining the power of combinatorial library screening with the nuance of rational sequence engineering is an effective approach to engineering novel antimicrobial peptides that retain activity under physiological conditions. 1887-Pos Board B207 Development of Refined Bacterial Spheroplast Analyses to Characterize Hybrid Antimicrobial Peptides Heidi M. Wade1, Louise E.O. Darling2, Donald E. Elmore1. 1 Department of Chemistry and Biochemistry Program, Wellesley College, Wellesley, MA, USA, 2Department of Biological Sciences and Biochemistry Program, Wellesley College, Wellesley, MA, USA. Antimicrobial peptides (AMPs) provide a promising alternative treatment for infectious bacteria resistant to antibiotics. AMPs kill bacterial cells through a variety of diverse mechanisms. While some AMPs, such as parasin, induce membrane permeabilization, others like buforin II (BF2) and DesHDAP1 enter the cell with little disruption to the membrane and kill bacteria by interacting with intracellular components. Previous work in our labs demonstrated the potential of using bacterial spheroplasts to better visualize peptide localization via confocal microscopy. Here, we build upon our previous spheroplast work in two key ways. First, we take advantage of the improved image quality to perform more quantitative analyses to measure the cellular entry or membrane localization of peptides. We then utilized these more systematic approaches to characterize the mechanisms of designed hybrid peptides. Recently, there has been increased interest in designing AMPs by combining two distinct AMPs into a single peptide. It has been shown that such hybrid AMPs are often more potent than their individual AMP components. However, to date all studied hybrid AMPs have combined together two AMPs that work via membrane permeabilization. Thus, we focused on characterizing hybrid peptides that combine one permeabilizing AMP (parasin) and one translocating AMP (DesHDAP1 or BF2) with different orientations and linkers. Our results have shown that the permeabilizing peptide typically dominates the mechanism of action when combined with the translocating peptide. This work is an important step in determining if any trends can be generalized to other hybrids made of engineered permeabilizing-translocating peptide pairs. 1888-Pos Board B208 Real-Time Characterization of an Antimicrobial Mechanism-of-Action with Nonlinear Optical Scattering Michael J. Wilhelm1, Bruk Mensa2, Mohammad Sharifian Gh.1, William F. DeGrado2, Hai-Lung Dai1. 1 Department of Chemistry, Temple University, Philadelphia, PA, USA, 2 Department of Pharmaceutical Chemistry and the Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA, USA. Antimicrobial resistance is an emerging threat that demands continuous development of new classes of antibiotic agents. In the interest of efficiently optimizing viable chemical targets, it is imperative to have experimental techniques capable of characterizing not only the efficacy of an antibiotic, but also the specific mechanism-of-action (MoA). We have recently demonstrated the surface-sensitive nonlinear optical technique, second-harmonic generation light scattering (SHS), for quantifying transmembrane molecular transport in living cells(1). We now show that SHS can be applied as a sensitive probe of membrane permeability in living bacteria. Specifically, by monitoring the uptake response of an SHS-active probe molecule following administration of an antibacterial agent, perturbations in the measured transport response reveal time- and concentration-dependent changes in the permeability of the bacterial membranes, thus permitting real-time characterization of the MoA. As an initial proof-of-principle, we apply SHS for deducing the sequential MoA of the antimicrobial, Bricilidin (Bn). Bn is a synthetic arylamide foldamer exhibiting amphiphilic topology similar to that of cell-penetrating peptides, and was designed to disrupt bacterial membranes(2). Using malachite green (MG) as an SHS-active probe, we characterize the MoA of Bn in Escherichia coli. In general, for low concentrations (<1uM) and short interaction periods (%15min), Bn is observed to primarily affect the bacterial outer membrane, resulting in enhanced permeability. However, for higher concentrations (R5uM) and longer interaction periods (>15min), Bn begins to disrupt the CM, resulting in an apparent decreased permeability, likely indicating depolarization of the membrane. REFERENCES: 1. Wilhelm, M.J., M. Sharifian Gh., and H.-L. Dai. 2015. Chemically Induced Changes to Membrane Permeability in Living Cells Probed by Nonlinear Light Scattering. Biochemistry. 54: 4427–4430. 2. Mensa, B., G.L. Howell, R. Scott, and W.F. DeGrado. 2014. Comparative mechanistic studies of brilacidin, daptomycin, and the antimicrobial peptide LL16. Antimicrob. Agents Chemother. 58: 5136–5145.
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even just a collateral effect. Additionally, AMP interactions with non-lipid cell envelope components of bacteria may be important in modifying how well AMPs are able to disrupt the lipid membrane. In order to connect studies of AMPs in model lipid systems to the more complex real bacterial cell envelopes, we have deuterium-labeled the membranes of the gram-positive bacteria Bacillus subtilis and used 2H NMR to study how lipid acyl chain order in its membranes is affected by treatment with AMPs. We have also observed 2H NMR spectra from Bacillus subtilis in which the peptidoglycan layer has been disrupted. This has allowed us to investigate how disruption of the peptidoglycan layer affects bacterial lipid chain order and the AMP/bacteria interaction.
structure and dynamics of model membrane systems. In particular, we investigated the effects of the antimicrobial peptides gramicidin and alamethecin on the lipid bilayer structure using small angle x-ray and neutron scattering. The structural studies were complemented by NSE experiments to probe the collective bending and thickness fluctuation dynamics in these model systems. Notably, the NSE results revealed enhanced thickness fluctuation dynamics in lipid bilayers containing low concentration of gramicidin that were dampened with increasing peptide concentration. An enhancement in dynamics was not seen in bilayers containing alamethicin, suggesting that the dynamics not only depend on peptide concentration, but also peptide orientation within the membrane
1880-Pos Board B200 Lipid Clustering by Antimicrobial Polymers and Lectins Anja Stulz1, Winfried Ro¨mer2, Karen Lienkamp3, Heiko Heerklotz1,2, Maria Hoernke1,2. 1 Pharmaceutical Technology and Biopharmacy, Albert-Ludwigs-Universit€at, Freiburg i.Br., Germany, 2BIOSS Centre for Biological Signalling Studies, Albert-Ludwigs-Universit€at, Freiburg i.Br., Germany, 3IMTEK Chemistry and Physics of Interfaces, Albert-Ludwigs-Universit€at, Freiburg i.Br., Germany. The current urge to understand the role of lipids in defense against pathogens is driven by two strategies: killing pathogens and protecting the cell from infection. Antimicrobial peptides and antimicrobial polymers offer a promising alternative to classical antibiotics through their action on membrane integrity. In principle, antimicrobial peptides and polymers are able to cluster lipids through lipid selection and recruitment from a mixed membrane. We show that antimicrobial polymers induce leakage in lipid vesicles by transient defects rather than defined pores. The positively charged polymers efficiently cluster negatively charged lipids from mixed model membranes. Larger domains are formed (in the order of 500 lipids). The binding of the polymers to the vesicles is exothermic. Our findings correlate with the polymers activity against bacteria. Similarly, lectins, carbohydrate binding proteins, can recognize certain glycolipids in mixed membranes, bind them and cluster them. By a yet unknown mechanism, the membrane is locally bent. Thus bacterial lectins can initiate uptake of pathogens into host cells and are promising targets for drug development.
1883-Pos Board B203 Mode of Action of Antimicrobial Peptides: Long and Short Amphipathic Alpha-Helixes Use Different Mechanisms Erik Strandberg1, Ariadna Grau-Campistany2, Hector Zamora-Carreras3, Marie-Claude Gagnon4, Philipp M€uhlh€auser1, Parvesh Wadhwani1, Jochen B€urck1, Johannes Reichert1, Michele Auger4, Jean-Francois Paquin4, M. Angeles Jime´nez3, Marta Bruix3, Francesc Rabanal2, Anne S. Ulrich1. 1 Institute for Biological Interfaces, Karlsruhe Institute of Technology, Karlsruhe, Germany, 2Universitat de Barcelona, Barcelona, Spain, 3Instituto de Quı´mica Fı´sica ‘‘Rocasolano’’, CSIC, Madrid, Spain, 4Universite´ Laval, Quebec, QC, Canada. We have studied the membrane structure and orientation of cationic amphipathic a-helical antimicrobial peptides (AMPs) using circular dichroism and solid-state NMR, combined with activity studies. For a series of model peptides, called KIA peptides, a clear length-dependent activity is found, as only peptides long enough to span the hydrophobic thickness of the membrane could induce leakage in vesicles. There is also a clear threshold length for peptides able to kill bacteria [1]. Using another series of KIA-like peptides of different length (from 14 up to 28 residues) but with a constant charge revealed that the length, but not the charge, is the critical factor. In membrane systems with a positive spontaneous curvature, the peptides get inserted into the membrane in a transmembrane orientation. All results indicate that these peptides act by forming proper oligomeric pores in the lipid bilayer. If the peptide is just long enough to span the membrane, it is aligned perfectly upright, but longer peptides can tilt cooperatively in the pore like an iris [1,2]. On the other hand, BP100, a highly helical peptides of only 11 amino acids, is clearly too short to form a transmembrane pore, but it is still strongly active against bacteria. From solid-state 2H-, 15N- and 19F-NMR studies, this peptide is found to dip into the membrane and to show high mobility within the amphiphilic surface layer. This way, it most likely disturbs the lipid order and thereby induces permeability, which suggests a carpet-like mechanism of action [3]. The structural results on these long and short AMPs clearly demonstrate that peptides with similar structural characteristics can act by very different mechanisms. References: [1] Grau-Campistany et al., (2015) Sci Rep 5, 9388. [2] Grau-Campistany et al., (2016) J Phys Chem Lett 7, 1116-1120. [3] Zamora-Carreras et al., (2016). Biochim Biophys Acta 1858, 1328-1338.
1881-Pos Board B201 How Antimicrobial Peptides Permeabilize Membranes with and without Pore Formation Jakob P. Ulmschneider. Institute of Natural Sciences, Shanghai Jiao Tong University, Shanghai, China. One of the biggest enigmas of antimicrobial peptides (AMPs), which protect all forms of life against pathogens, is why few structures of membrane pores have been found despite clear evidence of membrane leakage and antimicrobial activity. We provide a surprisingly simple explanation: For some AMPs such as PGLa (charge þ5), pores are not needed to explain both leakage and peptide translocation. Fully converged, unbiased multi-microsecond equilibrium simulations at all-atomistic level reveal that peptides spontaneously translocate across the membrane individually on a timescale of tens of microseconds, without forming pores. These findings explain why, despite vesicular leakage no channel has been identified for PGLa. However, similar simulations on other, lesser charged AMPs like maculatin clearly show pore formation. The results suggest that for some specific antimicrobial peptides, pore formation may not have to be invoked at all to explain both peptide translocation and membrane permeabilization. 1882-Pos Board B202 The Synergistic Effects of Lipids and Peptides on Membrane Dynamics Elizabeth Kelley1, Andrea Woodka1, Paul Butler1,2, Michihiro Nagao1,3. 1 Center for Neutron Research, NIST, Gaithersburg, MD, USA, 2Chemical and Biomolecular Engineering, University of Delaware, Newark, DE, USA, 3 Center for Exploration of Energy and Matter, Indiana University, Bloomington, IN, USA. There is a growing appreciation that the membrane physical properties are essential to cell and protein function. Simply altering the thickness of model membranes has been shown to influence the biological activity of several proteins, while incorporating peptides into lipid membranes also is known to affect the bilayer structural properties. Clearly there is a synergy in lipidprotein interactions in determining the membrane properties; however, the nature of these interactions are not well understood. Here we use a combination of small angle scattering techniques and neutron spin echo spectroscopy (NSE) to investigate the effects of incorporating a small peptide on both the
1884-Pos Board B204 Simulations of Membrane Disrupting Peptides Pores Versus Surface Binding B. Scott Perrin Jr.1, Riqiang Fu2, Myriam L. Cotten3, Richard W. Pastor1. 1 Laboratory of Computational Biology, NHLBI/NIH, Rockville, MD, USA, 2 National High Magnetic Field Laboratory, Tallahassee, FL, USA, 3 Department of Applied Science, College of William & Mary, Williamsburg, VA, USA. Peptides that disrupt biological membranes are a source of new antibiotic and antiviral therapeutics. Here, the relationships between peptide primary sequences, membrane bound structures, and abilities to disrupt membranes are investigated using all-atom molecular dynamics simulations. First, the archetype barrel-stave alamethicin (alm) pore in a 1,2-dioleoylsn-glycero-3-phosphocholine bilayer at 313 K indicates that ~7 ms is required for equilibration of a preformed 6-peptide pore; the pore remains stable for the duration of the remaining 7 ms of the trajectory, and the structure factors agree well with experiment. A 5 ms simulation of 10 surface-bound alm peptides shows significant peptide unfolding and some unbinding, but no insertion. Simulations at 363 and 413 K with a 0.2 V electric field yield peptide insertion in 1 ms. Insertion is initiated by the folding of residues 3-11 into an a-helix, and mediated by membrane water or by previously inserted peptides. The stability of five alm pore peptides at 413 K with a 0.2 V electric field demonstrates a significant preference for a transmembrane orientation. In contrast, a hypothesis that the antimicrobial peptide piscidin 1 (p1) forms toroidal pores is tested. The
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Tuesday, February 14, 2017
primary simulation was initialized with 20 peptides in four barrel-stave pores in a fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol bilayer. The four pores relaxed to toroidal by 200 ns, only one pore-like structure containing two transmembrane helices remained at 26 ms, and none of the 18 peptides released to the surface reinserted to form pores. The simulation was repeated at 413 K with an applied electric field and all peptides were surface-bound by 200 ns. Trajectories of surface-bound piscidin with and without applied fields at 313 and 413 K and totaling 6 ms show transient distortions of the bilayer/water interface (consistent with 31P NMR), but no insertion to transmembrane or pore states. 15 N chemical shifts confirm a fully surface-bound conformation. Taken together, the simulation and experimental results imply that transient defects rather than stable pores are responsible for membrane disruption by p1, and likely other AMPs. 1885-Pos Board B205 Simulation and Database-Guided Antimicrobial Peptide Evolution Charles H. Chen1,2, Charles G. Starr3, Gregory Wiedman1, William C. Wimley3, Jakob P. Ulmschneider4, Martin B. Ulmschneider1,2. 1 Whiting School of Engineering, Johns Hopkins University, Baltimore, MD, USA, 2Institute for NanoBiotechnology, Johns Hopkins University, Baltimore, MD, USA, 3Department of Biochemistry and Molecular Biology, Tulane University School of Medicine, New Orleans, LA, USA, 4Institute of Natural Sciences, Shanghai Jiao Tong University, Shanghai, China. Antimicrobial peptides (AMPs) are powerful and ubiquitous components of the innate immune defense in all domains of life. AMPs are amphiphilic peptides that selectively target and kill a wide variety of microbial pathogens at low micro-molar concentrations. These peptides vary widely in size, sequence, and secondary structure, and no common activity motif has been discovered to date. Here, we report several powerful new synthetic AMPs that are developed using a combination of molecular dynamics (MD) simulations and database screening. First, we use MD simulations to study the mechanisms of AMP folding and pore assembly in a bacterial model membrane. The atomic detail information of how pores form was used to develop a novel AMP: LDKA, which is a template sequence based on atomic detail structural information from the simulations. This simple peptide is composed a small number of amino acids and shows powerful pore-forming properties. Second, we apply statistical analysis of several thousand AMPs to optimize the LDKA template sequence. Finally, we apply a high-throughput screen using dye leakage assays to both bacterial and mammalian membrane model vesicles. This allows us to fine-tune the pore size and the binding selectivity for different membrane types. From the screen, we have identified 9 different LDKA analogues with different membrane selectivity and pore sizes. Our work confirms that the combination of MD simulations, database optimization, and theory/computation guided high-throughput screening is a powerful strategy to design potent new antimicrobial peptides for different biomedical targets and applications. 1886-Pos Board B206 Discovering Novel Antimicrobial Peptides using High-Throughput Screening in the Presence of Human Erythrocytes Charles G. Starr, Jing He, William C. Wimley. Department of Biochemistry and Molecular Biology, Tulane University, New Orleans, LA, USA. Antimicrobial peptides (AMPs) have long been considered excellent candidates for development as antibiotic agents to combat the threat of drug resistant bacterial pathogens. Yet, the promise AMPs have displayed in the laboratory has not yielded correlative clinical success. We hypothesized that a major reason for the disparity between outcomes at the bench and in the clinic is AMP interaction with and toxicity towards host cells. Here, we validate this theory using human red blood cells as model eukaryotic cells. Indeed, the potency of several well-studied antimicrobial peptides decreases from minimum sterilizing concentrations (MSC) of 1-5 mM to >30 mM in the presence of RBCs at 1x109 cells/ml; equivalent to 20% of the human physiological concentration. To address this issue, we have developed a high-throughput method to screen a combinatorial peptide library for antimicrobial activity in the presence of concentrated human cells. The screen consists of assays that assess the effect of RBCs on antimicrobial activity as well as toxicity toward eukaryotic cells. We present data on peptides identified and characterized from the high-throughput library screen and show that these new peptides were not as effective as they initially appeared during the screen. We demonstrate that the difference between observed and expected results is due to unforeseen proteolytic activity sequestered within the cytosol of the red blood cells. Finally, we show that a library consensus sequence synthesized using all d-amino acids outperforms all library isolates as well as the template sequence in terms of
microbicidal activity in the presence of RBCs. Ultimately, our results suggest that combining the power of combinatorial library screening with the nuance of rational sequence engineering is an effective approach to engineering novel antimicrobial peptides that retain activity under physiological conditions. 1887-Pos Board B207 Development of Refined Bacterial Spheroplast Analyses to Characterize Hybrid Antimicrobial Peptides Heidi M. Wade1, Louise E.O. Darling2, Donald E. Elmore1. 1 Department of Chemistry and Biochemistry Program, Wellesley College, Wellesley, MA, USA, 2Department of Biological Sciences and Biochemistry Program, Wellesley College, Wellesley, MA, USA. Antimicrobial peptides (AMPs) provide a promising alternative treatment for infectious bacteria resistant to antibiotics. AMPs kill bacterial cells through a variety of diverse mechanisms. While some AMPs, such as parasin, induce membrane permeabilization, others like buforin II (BF2) and DesHDAP1 enter the cell with little disruption to the membrane and kill bacteria by interacting with intracellular components. Previous work in our labs demonstrated the potential of using bacterial spheroplasts to better visualize peptide localization via confocal microscopy. Here, we build upon our previous spheroplast work in two key ways. First, we take advantage of the improved image quality to perform more quantitative analyses to measure the cellular entry or membrane localization of peptides. We then utilized these more systematic approaches to characterize the mechanisms of designed hybrid peptides. Recently, there has been increased interest in designing AMPs by combining two distinct AMPs into a single peptide. It has been shown that such hybrid AMPs are often more potent than their individual AMP components. However, to date all studied hybrid AMPs have combined together two AMPs that work via membrane permeabilization. Thus, we focused on characterizing hybrid peptides that combine one permeabilizing AMP (parasin) and one translocating AMP (DesHDAP1 or BF2) with different orientations and linkers. Our results have shown that the permeabilizing peptide typically dominates the mechanism of action when combined with the translocating peptide. This work is an important step in determining if any trends can be generalized to other hybrids made of engineered permeabilizing-translocating peptide pairs. 1888-Pos Board B208 Real-Time Characterization of an Antimicrobial Mechanism-of-Action with Nonlinear Optical Scattering Michael J. Wilhelm1, Bruk Mensa2, Mohammad Sharifian Gh.1, William F. DeGrado2, Hai-Lung Dai1. 1 Department of Chemistry, Temple University, Philadelphia, PA, USA, 2 Department of Pharmaceutical Chemistry and the Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA, USA. Antimicrobial resistance is an emerging threat that demands continuous development of new classes of antibiotic agents. In the interest of efficiently optimizing viable chemical targets, it is imperative to have experimental techniques capable of characterizing not only the efficacy of an antibiotic, but also the specific mechanism-of-action (MoA). We have recently demonstrated the surface-sensitive nonlinear optical technique, second-harmonic generation light scattering (SHS), for quantifying transmembrane molecular transport in living cells(1). We now show that SHS can be applied as a sensitive probe of membrane permeability in living bacteria. Specifically, by monitoring the uptake response of an SHS-active probe molecule following administration of an antibacterial agent, perturbations in the measured transport response reveal time- and concentration-dependent changes in the permeability of the bacterial membranes, thus permitting real-time characterization of the MoA. As an initial proof-of-principle, we apply SHS for deducing the sequential MoA of the antimicrobial, Bricilidin (Bn). Bn is a synthetic arylamide foldamer exhibiting amphiphilic topology similar to that of cell-penetrating peptides, and was designed to disrupt bacterial membranes(2). Using malachite green (MG) as an SHS-active probe, we characterize the MoA of Bn in Escherichia coli. In general, for low concentrations (<1uM) and short interaction periods (%15min), Bn is observed to primarily affect the bacterial outer membrane, resulting in enhanced permeability. However, for higher concentrations (R5uM) and longer interaction periods (>15min), Bn begins to disrupt the CM, resulting in an apparent decreased permeability, likely indicating depolarization of the membrane. REFERENCES: 1. Wilhelm, M.J., M. Sharifian Gh., and H.-L. Dai. 2015. Chemically Induced Changes to Membrane Permeability in Living Cells Probed by Nonlinear Light Scattering. Biochemistry. 54: 4427–4430. 2. Mensa, B., G.L. Howell, R. Scott, and W.F. DeGrado. 2014. Comparative mechanistic studies of brilacidin, daptomycin, and the antimicrobial peptide LL16. Antimicrob. Agents Chemother. 58: 5136–5145.