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Using Molecular Dynamics Simulations to Characterize the Role Played by Basic Residues in Interactions of HDAPs and Bacterial Lipid Membranes
416a
Tuesday, March 1, 2016
Antimicrobial peptides (AMPs) are a novel class of antibiotics comprised of short peptide sequences made up of 5 to 50 amino acids. Of particular interest are a-helical AMPs that kill bacteria through either membrane lysis or translocation and binding to internal targets. Due to their mechanism of action, the ability of a-helical AMPs to interact with biological membranes selectively is critical to generating AMPs with a wide therapeutic window. Of the many biophysical factors to consider when studying this membrane selectivity, secondary structure has been shown to play a critical role. To elucidate the role of helicity in determining AMP membrane selectivity and potency, we have created a library of AMPs with varying stabilized a-helical structures and determined the degree of peptide folding in the presence of liposomes modeled after bacterial and mammalian membranes using circular dichroism (CD) spectroscopy. By correlating our measurements with data from antimicrobial and hemolytic assays, we found that a threshold point existed above which increasing a-helicity diminishes potency and membrane selectivity. The results from this study will aid us in building a biophysical model that could be used for the computational design of new AMPs that have a better chance of being translated into viable clinical treatments. 2060-Pos Board B204 Adaptation of Escherichia Coli Spheroplasts to the Characterization of Antimicrobial Peptides Lei Wei1, Donald E. Elmore2. 1 Biochemistry Program, Wellesley College, Wellesley, MA, USA, 2 Department of Chemistry and Biochemistry Program, Wellesley College, Wellesley, MA, USA. With activity against a broad range of microorganisms and low susceptibility to the resistance mechanisms developed by multidrug resistant organisms, naturally-occurring antimicrobial peptides (AMPs) have the potential to serve as effective antimicrobial agents to combat the ever-increasing number of antibiotic-resistant illnesses. In addition to various spectroscopic measurements, antimicrobial activity assays, and molecular modeling, the mechanisms of action of novel AMPs can be further characterized using confocal microscopy. This imaging technique utilizes fluorescently-labeled peptides or fluorophore-containing peptide conjugates to examine the localization of antimicrobial peptides in bacterial cells, providing insight into a peptide’s mechanism of action. However, the small size and the rod shape of bacteria make it difficult to acquire clear and conclusive images to determine the localization of the peptide in the bacterial cell. The present study focuses on the adaptation of Escherichia coli spheroplasts, which are spherical and at least four times larger than normal bacteria, as a model for the characterization of the mechanisms of action of AMPs. The larger size of the spheroplasts increases the ease of focusing on the target and allows clearer images with increased resolution and more convincing evidence of peptide localization. The spherical shape of the spheroplasts allows for equivalent images to be taken from any angle of the sample. During the optimization process for this approach, we found that the presence of the stabilizing agent, divalent magnesium cation, affects the translocation of cell-penetrating AMPs. Ongoing work is focused on finding a suitable replacement for magnesium in spheroplast stabilization. The successful adaptation of E. coli spheroplasts protocols with confocal microscopy will allow the system to be used for the further characterization of a wide variety of molecules, including AMPs and other cell-penetrating, non-antimicrobial peptides. 2061-Pos Board B205 The Role of Arginine and Lysine in Histone Derived Antimicrobial Peptides Dania Figueroa1, Carla Perez2, Donald E. Elmore3. 1 Biochemistry Program, Wellesley College, Wellesley, MA, USA, 2 Department of Chemistry, Wellesley College, Wellesley, MA, USA, 3 Department of Chemistry and Biochemistry Program, Wellesley College, Wellesley, MA, USA. To combat the rapid increase of antibiotic resistant bacteria scientists have turned to antimicrobial peptides. Antimicrobial peptides (AMPs) are small proteins within the innate immune system with the ability to kill bacteria, fungi and viruses. Previous research has shown that a greater number of arginine amino acids enhance the antimicrobial activity of some AMPs. This study investigates the role of basic amino acids in three histone derived antimicrobial peptides (HDAPs): buforin II, parasin and DesHDAP1. Variants of all three were made by mutating all lysine residues to arginine or all arginine residues to lysine. We were interested in characterizing the mutant AMPs antimicrobial activity and method of action against a variety of gram positive and gram negative bacterial strains. Using a radial diffusion assay (RDA) we observed increased levels of antimicrobial activity in the arginine mutants and decreased activity in the lysine mutants compared to the wild type AMPs. A propidium iodide assay
(PI) for membrane permeabilization illustrated a similar trend, as permeabilization activity increased in the arginine mutants and decreased in the lysine mutants compared to wild type AMPs. RDA and PI assays also point to potential differences in the activity of mutant AMPs against gram positive and gram negative bacterial membranes. Together, these observations will help promote the rational design of AMPs with enhanced activity. 2062-Pos Board B206 Using Molecular Dynamics Simulations to Characterize the Role Played by Basic Residues in Interactions of HDAPs and Bacterial Lipid Membranes Sung Hyun Lee1, Donald E. Elmore2. 1 Department of Chemistry, Wellesley College, Wellesley, MA, USA, 2 Department of Chemistry and Biochemistry Program, Wellesley College, Wellesley, MA, USA. Antimicrobial peptides are small, cationic peptides that induce bacterial cell death via cell lysis or interactions with intracellular components. Currently, with growing bacterial resistance to existing antimicrobial drugs there is an urgent need to study the potentcy of antimicrobial peptides and find ways to improve their activity. Most AMPs include positively charged amino acids that allow binding to negatively-charged bacterial membranes. Previous studies have shown that an increased composition of arginine versus lysine enhances the activity of two histone-derived antimicrobial peptides (HDAPs), buforin II (BF2) and DesHDAP1. Variants of these peptides with all basic residues modified to arginine had increased antibacterial activity and membrane translocation but unchanged membrane permeabilization. In this study, we use molecular dynamics simulations to study how the arginine-modified peptides interact with model bacterial lipid membranes (1:3 POPG:POPE) differently than wild-type and lysine-modified peptides. Simulations were performed with one and four peptides for wild type BF2 (BF2wt), argininemodified BF2 (BF2R), and lysine-modified BF2 (BF2K). The results of 100ns simulations of each system show that BF2R has increased interactions with the lipid membrane and structural stability compared to the other peptides. The analysis of hydrogen bonding per lipids supports that anionic POPG lipids interact more with the peptides than zwitterionic POPE lipids, further emphasizing the role of the basic residues. Analogous simulations considering the lipid interactions of DesHDAP1 and its arginine and lysine mutants are ongoing. Our findings elucidate mechanistic details of peptidelipid interactions that will become helpful in the development of AMPs with improved activity. 2063-Pos Board B207 Mimicking and Understanding the Agglutination Effect of the Antimicrobial Peptide Thanatin using Model Phospholipid Vesicles E´mile Robert, Thierry Lefe`vre, Matthieu Fillion, Benjamin Martial, Justine Dionne, Miche`le Auger. Chemistry, Universite Laval, Quebec, QC, Canada. Thanatin is a cationic 21-residue antimicrobial and antifongical peptide found in the spined soldier bug Podisus maculiventris. It is believed that it does not permeabilize membranes, but rather induces the agglutination of bacteria and inhibits cellular respiration. To clarify its mode of action, lipid vesicle organization and aggregation propensity as well as peptide secondary structure have been studied using different membrane models. Dynamic light scattering and turbidimetry results show that specific mixtures of negatively charged and zwitterionic phospholipid vesicles are able to mimic the agglutination effect of thanatin observed on G- and Gþ bacterial cells, while mono-constituent (‘‘conventional’’) models cannot reproduce this phenomenon. The model of eukaryotic cell reveals no particular interaction with thanatin, consistently with the literature. Infrared spectroscopy shows that in the conditions where vesicle agglutination occurs, thanatin exhibits a particular spectral pattern in the amide I’ region and in the region associated with Arg side-chains. The data suggest that thanatin mainly retains its hairpin structure, Arg residues being involved in strong interactions with anionic groups of phospholipids. In the absence of vesicle agglutination, the peptide conformation and Arg side-chain environment are similar to those observed in solution. The data show that a negatively charged membrane is required for thanatin to be active, but this condition is insufficient. The activity of thanatin seems to be modulated by charge surface density of membranes and thanatin concentration. 2064-Pos Board B208 2H NMR Studies of Living Bacteria Interacting with Antimicrobial Peptides Nury P. Santisteban1, Michael R. Morrow1, Valerie Booth2. 1 Physics and Physical Oceanography, Memorial University of Newfoundland, St. John’s, NL, Canada, 2Biochemistry, Memorial University of Newfoundland, St. John’s, NL, Canada.
Tuesday, March 1, 2016
Antimicrobial peptides (AMPs) are a novel class of antibiotics comprised of short peptide sequences made up of 5 to 50 amino acids. Of particular interest are a-helical AMPs that kill bacteria through either membrane lysis or translocation and binding to internal targets. Due to their mechanism of action, the ability of a-helical AMPs to interact with biological membranes selectively is critical to generating AMPs with a wide therapeutic window. Of the many biophysical factors to consider when studying this membrane selectivity, secondary structure has been shown to play a critical role. To elucidate the role of helicity in determining AMP membrane selectivity and potency, we have created a library of AMPs with varying stabilized a-helical structures and determined the degree of peptide folding in the presence of liposomes modeled after bacterial and mammalian membranes using circular dichroism (CD) spectroscopy. By correlating our measurements with data from antimicrobial and hemolytic assays, we found that a threshold point existed above which increasing a-helicity diminishes potency and membrane selectivity. The results from this study will aid us in building a biophysical model that could be used for the computational design of new AMPs that have a better chance of being translated into viable clinical treatments. 2060-Pos Board B204 Adaptation of Escherichia Coli Spheroplasts to the Characterization of Antimicrobial Peptides Lei Wei1, Donald E. Elmore2. 1 Biochemistry Program, Wellesley College, Wellesley, MA, USA, 2 Department of Chemistry and Biochemistry Program, Wellesley College, Wellesley, MA, USA. With activity against a broad range of microorganisms and low susceptibility to the resistance mechanisms developed by multidrug resistant organisms, naturally-occurring antimicrobial peptides (AMPs) have the potential to serve as effective antimicrobial agents to combat the ever-increasing number of antibiotic-resistant illnesses. In addition to various spectroscopic measurements, antimicrobial activity assays, and molecular modeling, the mechanisms of action of novel AMPs can be further characterized using confocal microscopy. This imaging technique utilizes fluorescently-labeled peptides or fluorophore-containing peptide conjugates to examine the localization of antimicrobial peptides in bacterial cells, providing insight into a peptide’s mechanism of action. However, the small size and the rod shape of bacteria make it difficult to acquire clear and conclusive images to determine the localization of the peptide in the bacterial cell. The present study focuses on the adaptation of Escherichia coli spheroplasts, which are spherical and at least four times larger than normal bacteria, as a model for the characterization of the mechanisms of action of AMPs. The larger size of the spheroplasts increases the ease of focusing on the target and allows clearer images with increased resolution and more convincing evidence of peptide localization. The spherical shape of the spheroplasts allows for equivalent images to be taken from any angle of the sample. During the optimization process for this approach, we found that the presence of the stabilizing agent, divalent magnesium cation, affects the translocation of cell-penetrating AMPs. Ongoing work is focused on finding a suitable replacement for magnesium in spheroplast stabilization. The successful adaptation of E. coli spheroplasts protocols with confocal microscopy will allow the system to be used for the further characterization of a wide variety of molecules, including AMPs and other cell-penetrating, non-antimicrobial peptides. 2061-Pos Board B205 The Role of Arginine and Lysine in Histone Derived Antimicrobial Peptides Dania Figueroa1, Carla Perez2, Donald E. Elmore3. 1 Biochemistry Program, Wellesley College, Wellesley, MA, USA, 2 Department of Chemistry, Wellesley College, Wellesley, MA, USA, 3 Department of Chemistry and Biochemistry Program, Wellesley College, Wellesley, MA, USA. To combat the rapid increase of antibiotic resistant bacteria scientists have turned to antimicrobial peptides. Antimicrobial peptides (AMPs) are small proteins within the innate immune system with the ability to kill bacteria, fungi and viruses. Previous research has shown that a greater number of arginine amino acids enhance the antimicrobial activity of some AMPs. This study investigates the role of basic amino acids in three histone derived antimicrobial peptides (HDAPs): buforin II, parasin and DesHDAP1. Variants of all three were made by mutating all lysine residues to arginine or all arginine residues to lysine. We were interested in characterizing the mutant AMPs antimicrobial activity and method of action against a variety of gram positive and gram negative bacterial strains. Using a radial diffusion assay (RDA) we observed increased levels of antimicrobial activity in the arginine mutants and decreased activity in the lysine mutants compared to the wild type AMPs. A propidium iodide assay
(PI) for membrane permeabilization illustrated a similar trend, as permeabilization activity increased in the arginine mutants and decreased in the lysine mutants compared to wild type AMPs. RDA and PI assays also point to potential differences in the activity of mutant AMPs against gram positive and gram negative bacterial membranes. Together, these observations will help promote the rational design of AMPs with enhanced activity. 2062-Pos Board B206 Using Molecular Dynamics Simulations to Characterize the Role Played by Basic Residues in Interactions of HDAPs and Bacterial Lipid Membranes Sung Hyun Lee1, Donald E. Elmore2. 1 Department of Chemistry, Wellesley College, Wellesley, MA, USA, 2 Department of Chemistry and Biochemistry Program, Wellesley College, Wellesley, MA, USA. Antimicrobial peptides are small, cationic peptides that induce bacterial cell death via cell lysis or interactions with intracellular components. Currently, with growing bacterial resistance to existing antimicrobial drugs there is an urgent need to study the potentcy of antimicrobial peptides and find ways to improve their activity. Most AMPs include positively charged amino acids that allow binding to negatively-charged bacterial membranes. Previous studies have shown that an increased composition of arginine versus lysine enhances the activity of two histone-derived antimicrobial peptides (HDAPs), buforin II (BF2) and DesHDAP1. Variants of these peptides with all basic residues modified to arginine had increased antibacterial activity and membrane translocation but unchanged membrane permeabilization. In this study, we use molecular dynamics simulations to study how the arginine-modified peptides interact with model bacterial lipid membranes (1:3 POPG:POPE) differently than wild-type and lysine-modified peptides. Simulations were performed with one and four peptides for wild type BF2 (BF2wt), argininemodified BF2 (BF2R), and lysine-modified BF2 (BF2K). The results of 100ns simulations of each system show that BF2R has increased interactions with the lipid membrane and structural stability compared to the other peptides. The analysis of hydrogen bonding per lipids supports that anionic POPG lipids interact more with the peptides than zwitterionic POPE lipids, further emphasizing the role of the basic residues. Analogous simulations considering the lipid interactions of DesHDAP1 and its arginine and lysine mutants are ongoing. Our findings elucidate mechanistic details of peptidelipid interactions that will become helpful in the development of AMPs with improved activity. 2063-Pos Board B207 Mimicking and Understanding the Agglutination Effect of the Antimicrobial Peptide Thanatin using Model Phospholipid Vesicles E´mile Robert, Thierry Lefe`vre, Matthieu Fillion, Benjamin Martial, Justine Dionne, Miche`le Auger. Chemistry, Universite Laval, Quebec, QC, Canada. Thanatin is a cationic 21-residue antimicrobial and antifongical peptide found in the spined soldier bug Podisus maculiventris. It is believed that it does not permeabilize membranes, but rather induces the agglutination of bacteria and inhibits cellular respiration. To clarify its mode of action, lipid vesicle organization and aggregation propensity as well as peptide secondary structure have been studied using different membrane models. Dynamic light scattering and turbidimetry results show that specific mixtures of negatively charged and zwitterionic phospholipid vesicles are able to mimic the agglutination effect of thanatin observed on G- and Gþ bacterial cells, while mono-constituent (‘‘conventional’’) models cannot reproduce this phenomenon. The model of eukaryotic cell reveals no particular interaction with thanatin, consistently with the literature. Infrared spectroscopy shows that in the conditions where vesicle agglutination occurs, thanatin exhibits a particular spectral pattern in the amide I’ region and in the region associated with Arg side-chains. The data suggest that thanatin mainly retains its hairpin structure, Arg residues being involved in strong interactions with anionic groups of phospholipids. In the absence of vesicle agglutination, the peptide conformation and Arg side-chain environment are similar to those observed in solution. The data show that a negatively charged membrane is required for thanatin to be active, but this condition is insufficient. The activity of thanatin seems to be modulated by charge surface density of membranes and thanatin concentration. 2064-Pos Board B208 2H NMR Studies of Living Bacteria Interacting with Antimicrobial Peptides Nury P. Santisteban1, Michael R. Morrow1, Valerie Booth2. 1 Physics and Physical Oceanography, Memorial University of Newfoundland, St. John’s, NL, Canada, 2Biochemistry, Memorial University of Newfoundland, St. John’s, NL, Canada.