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Sequence Permutation of Positive Charges in a Model Peptide Antibiotic Produces Differing Enthalpic and Entropic Contributions to the Lipid-Peptide Binding Affinity
422a
Tuesday, March 1, 2016
cardiolipin binding. We utilize optimized titration techniques that generate reproducible quantitative data that can then be fit to extrapolate the binding contribution to cardiolipin for each amino acid investigated. Monitoring the binding of Cytc variants to cardiolipin vesicles allows us to investigate the role of individual amino acids in the protein-lipid interaction and elucidate the contribution of different sites on cytochrome c to cardiolipin binding. 2091-Pos Board B235 Exploring Oxidation State Dependent Conformational Changes of Cytochrome C on Cardiolipin Containing Liposomes Bridget Milorey, Lee Serpas, Leah Pandiscia, Reinhard Schweitzer-Stenner. Drexel University, Philadelphia, PA, USA. :A combination of circular dichroism, absorption and fluorescence spectroscopies were used to study the binding of oxidized and reduced cytochrome c to cardiolipin (CL) containing liposomes with different CL content as a function of cardiolipin concentration. Recently, our group provided evidence for a two-step binding process of the oxidized (ferricytochrome) state to CL containing liposomes. The second step of binding converts a native-like to a more unfolded structure in which M80 is replaced either by a lysine of a histidine as axial ligand. The conversion into this more unfolded conformation was found to be partially inhibited in the presence of NaCl. Currently we are investigating whether the initial binding and/or the equilibrium between the two membrane bound conformers depends on pH. Our work with the reduced (ferrocytochrome) state has revealed a dependence on NaCl for stability. In the absence of NaCl and at aerobic environment, the protein becomes oxidized upon binding to CL containing liposomes. When NaCl is added to the solution, it stabilizes the reduced state on the liposome surface and inhibits the auto-oxidation. At anaerobic conditions and in the absence of NaCl, the protein undergoes a notable conformational change, but clearly maintains its reduced state. This shows that the obtained oxidation of the protein involves the reduction of molecular oxygen. We are in the process of exploring the pH dependence of the underlying change of the protein’s redox potential. 2092-Pos Board B236 Sequence Permutation of Positive Charges in a Model Peptide Antibiotic Produces Differing Enthalpic and Entropic Contributions to the LipidPeptide Binding Affinity Brianna Haight, Ellen R. Arndt, Adrienne P. Loh. Chemistry & Biochemistry, University of Wisconsin - La Crosse, La Crosse, WI, USA. With rising disease rates and decreasing effectiveness of conventional antibiotics, there is an immediate need for new antibiotics. One promising solution is through cationic antimicrobial peptides, which act by perturbing bacterial membranes. The interaction of model peptides with membranes can provide valuable information about the role of helical structure and peptide charge distribution on peptide-membrane interactions. We are investigating model peptide antibiotics composed primarily of the hydrophobic dialkylated amino acid Aib (a-aminoisobutyric acid), which imparts a strong 310-helical bias due to steric hindrance at the a-carbon. Cationic lysine residues are placed in adjacent locations in the center of the helix (KK45) or one full turn apart (KK36). Large unilamellar vesicles (LUVs) composed of DMPC or DMPG were used as zwitterionic or anionic membrane models, respectively. NMR data indicates that in a membrane mimicing solvent (DMSO), KK45 adopts a kinked helical stucture, while KK36 adopts a canonical helical structure. We have previously seen that KK45 binding to anionic micells is more favorable than binding of KK36 to anionic micelles. However, unlike binding to micelles, NMR data suggests that binding of peptides to DMPG vesicles is more favorable for KK36 than for KK45. Here we present thermodynamic data characterizing the peptide-LUV interactions using isothermal titration calorimetry (ITC). Preliminary data suggests that binding to DMPG vesicles is exothermic, while binding to DMPC vesicles is endothermic. Complete characterization of the enthalpy of binding, entropy of binding, and thermodynamic binding constant will allow us to elucidate the relationship between peptide structure and the favorability of membrane binding. 2093-Pos Board B237 Inducing a Helical Kink in a Model Peptide Antibiotic Reduces PeptideMembrane Interaction Favorability in Vesicles Riley Larson, Ellen R. Arndt, Adrienne P. Loh. Chemistry & Biochemistry, U. Wisconsin - La Crosse, La Crosse, WI, USA. Peptide antibiotics offer promise as robust alternatives to conventional small molecule antibiotics. While the mechanism of function of peptide antibiotics remains an active area of research, it is generally understood that these peptide interact with and disrupt cell membranes. We are studying the interactions of
model peptide antibiotics with large unilamellar vesicles (LUVs) in order to understand how helical shape influences the peptide-membrane interaction. Our model antibiotic peptides are octamers composed primarily of the hydrophobic dialkylated amino acid Aib (a -aminoisobutyric acid), which imparts a strong 310-helical bias due to steric hindrance at the a-carbon. Positively charged lysine residues are placed in either adjacent positions (KK45) or separated by one helical turn (KK36) in the sequence. Using NMR spectroscopy, we are measuring the binding constant for these two peptides with zwitterionic (DMPC) and anionic (DMPG) LUVs by measuring the disappearance of the amide 1H NMR signal as LUVs are added to peptide solution. The large size of the peptide-LUV complex results in a long rotational correlation time, and thus dramatic broadening (disappearance) of the peptide signal. Preliminary experiments indicate that neither peptide binds appreciably to DMPC LUVs, while KK36 has a greater affinity for DMPG LUVs than KK45 (KD, KK36=0.34 þ/- 0.09 mM versus KD,KK45=1.1 þ/- 0.1 mM). Circular dichroism experiments indicate that KK45 remains largely 310-helical in water and when bound to DMPG LUVs, while KK36 undergoes a transition from largely [a]-helical in water to largely 310-helical when bound to DMPG LUVs. These results together suggest that both charge and structure are important factors in peptide-lipid association. 2094-Pos Board B238 Does Lipid Composition Regulate Anthrax Toxin Uptake? Nnanya U. Kalu1, Clare Kenney2, Ekaterina Nestorovich1. 1 Biology, The Catholic University of America, NE, DC, USA, 2Chemistry, The Catholic University of America, NE, DC, USA. Biological roles of lipids are not limited toproviding structural stability to bilayer membranes. Lipids have numerous functions, e.g. acting in cellular signaling and protein regulation. Cells adjust bilayer biophysical parameters by controlling lipid content and gradient of the plasma and intracellular organelle membranes. Thus, membrane composition changes along the endocytic pathway, with early endosomal membranes resembling plasma membranes, and late endosomal membranes showing dramatic increase in anionic lysobisphosphatidic acid level. Here we investigate the role of the lipid environmentin the anthrax toxin uptake. The anthrax toxin, formed by three proteins (binding protective antigen, PA, and enzymatic lethal factor, LF and edema factor, EF), follows a multistep internalization process which involves PA binding to its cellular receptors, proteolytic cleavage to PA63, oligomerization to form prepores, and LF and EF binding followed by endocytosis. The acidic endosomal environment causes conformational changes in PA63 leading to its insertion into the early endosomal membrane and ion channel formation. The delivery of LF to cytoplasm was suggested to take place later in the endocytic pathway via the back-fusion of intraluminal vesicles of late endosomes. Given the variations in membrane lipid composition of the early endosome, the intraluminal vesicles and the late endosome, it becomes important to understand if these changes influence the PA63 insertion and structural dynamics, LF and EF binding and translocation. Using bilayer lipid membrane technique, we mimic the endosomal membrane composition probing its properties in-vitro. We show that at pH 5.5, PA63 insertion rate increases dramatically in PS compared with PC bilayers, whereas LF binds ten times less effectively to PA63 in PS compared to PC. When the solution pH is raised from 5 to 6.75, this difference is diminished. 2095-Pos Board B239 Investigation of the Physiochemical Properties of the Phospholipid Cardiolipin: Implications for Oxphos Regulation and Barth Syndrome Murugappan Sathappa, Matthew Greenwood, Nathan Alder. University of Connecticut, Storrs, CT, USA. Cardiolipin (CL) is the signature phospholipid of the mitochondrial inner membrane, which houses respiratory complexes that generate electrochemical ion gradients to drive processes such as ATP synthesis by oxidative phosphorylation. Nascent CL, which is enriched in saturated acyl chains of variable lengths, is subject to a two-stage remodeling process: a deacylase first removes an acyl chain to generate the monolysocardiolpin (MLCL) intermediate, and the transacylase tafazzin regenerates the four-chain lipid with highly unsaturated acyl chains with a high degree of symmetry. Mutations in the TAZ gene that encodes tafazzin underpin Barth syndrome, a multi-system mitochondrial disorder characterized by gross abnormalities in lipid profiles, cardiac and skeletal myopathy, neutropenia, growth retardation and mortality in early childhood. First, we analyze the role of CL in activating OXPHOS complexes using nanoscale lipid bilayers containing headgroup and acyl chain variants of CL. To this end, purified cytochrome c oxidase was reconstituted into nanodiscs of defined lipid composition and assayed for CL-dependent redox activity. Second, we have analyzed the binding characteristics of cytochrome c to bilayers containing
Tuesday, March 1, 2016
cardiolipin binding. We utilize optimized titration techniques that generate reproducible quantitative data that can then be fit to extrapolate the binding contribution to cardiolipin for each amino acid investigated. Monitoring the binding of Cytc variants to cardiolipin vesicles allows us to investigate the role of individual amino acids in the protein-lipid interaction and elucidate the contribution of different sites on cytochrome c to cardiolipin binding. 2091-Pos Board B235 Exploring Oxidation State Dependent Conformational Changes of Cytochrome C on Cardiolipin Containing Liposomes Bridget Milorey, Lee Serpas, Leah Pandiscia, Reinhard Schweitzer-Stenner. Drexel University, Philadelphia, PA, USA. :A combination of circular dichroism, absorption and fluorescence spectroscopies were used to study the binding of oxidized and reduced cytochrome c to cardiolipin (CL) containing liposomes with different CL content as a function of cardiolipin concentration. Recently, our group provided evidence for a two-step binding process of the oxidized (ferricytochrome) state to CL containing liposomes. The second step of binding converts a native-like to a more unfolded structure in which M80 is replaced either by a lysine of a histidine as axial ligand. The conversion into this more unfolded conformation was found to be partially inhibited in the presence of NaCl. Currently we are investigating whether the initial binding and/or the equilibrium between the two membrane bound conformers depends on pH. Our work with the reduced (ferrocytochrome) state has revealed a dependence on NaCl for stability. In the absence of NaCl and at aerobic environment, the protein becomes oxidized upon binding to CL containing liposomes. When NaCl is added to the solution, it stabilizes the reduced state on the liposome surface and inhibits the auto-oxidation. At anaerobic conditions and in the absence of NaCl, the protein undergoes a notable conformational change, but clearly maintains its reduced state. This shows that the obtained oxidation of the protein involves the reduction of molecular oxygen. We are in the process of exploring the pH dependence of the underlying change of the protein’s redox potential. 2092-Pos Board B236 Sequence Permutation of Positive Charges in a Model Peptide Antibiotic Produces Differing Enthalpic and Entropic Contributions to the LipidPeptide Binding Affinity Brianna Haight, Ellen R. Arndt, Adrienne P. Loh. Chemistry & Biochemistry, University of Wisconsin - La Crosse, La Crosse, WI, USA. With rising disease rates and decreasing effectiveness of conventional antibiotics, there is an immediate need for new antibiotics. One promising solution is through cationic antimicrobial peptides, which act by perturbing bacterial membranes. The interaction of model peptides with membranes can provide valuable information about the role of helical structure and peptide charge distribution on peptide-membrane interactions. We are investigating model peptide antibiotics composed primarily of the hydrophobic dialkylated amino acid Aib (a-aminoisobutyric acid), which imparts a strong 310-helical bias due to steric hindrance at the a-carbon. Cationic lysine residues are placed in adjacent locations in the center of the helix (KK45) or one full turn apart (KK36). Large unilamellar vesicles (LUVs) composed of DMPC or DMPG were used as zwitterionic or anionic membrane models, respectively. NMR data indicates that in a membrane mimicing solvent (DMSO), KK45 adopts a kinked helical stucture, while KK36 adopts a canonical helical structure. We have previously seen that KK45 binding to anionic micells is more favorable than binding of KK36 to anionic micelles. However, unlike binding to micelles, NMR data suggests that binding of peptides to DMPG vesicles is more favorable for KK36 than for KK45. Here we present thermodynamic data characterizing the peptide-LUV interactions using isothermal titration calorimetry (ITC). Preliminary data suggests that binding to DMPG vesicles is exothermic, while binding to DMPC vesicles is endothermic. Complete characterization of the enthalpy of binding, entropy of binding, and thermodynamic binding constant will allow us to elucidate the relationship between peptide structure and the favorability of membrane binding. 2093-Pos Board B237 Inducing a Helical Kink in a Model Peptide Antibiotic Reduces PeptideMembrane Interaction Favorability in Vesicles Riley Larson, Ellen R. Arndt, Adrienne P. Loh. Chemistry & Biochemistry, U. Wisconsin - La Crosse, La Crosse, WI, USA. Peptide antibiotics offer promise as robust alternatives to conventional small molecule antibiotics. While the mechanism of function of peptide antibiotics remains an active area of research, it is generally understood that these peptide interact with and disrupt cell membranes. We are studying the interactions of
model peptide antibiotics with large unilamellar vesicles (LUVs) in order to understand how helical shape influences the peptide-membrane interaction. Our model antibiotic peptides are octamers composed primarily of the hydrophobic dialkylated amino acid Aib (a -aminoisobutyric acid), which imparts a strong 310-helical bias due to steric hindrance at the a-carbon. Positively charged lysine residues are placed in either adjacent positions (KK45) or separated by one helical turn (KK36) in the sequence. Using NMR spectroscopy, we are measuring the binding constant for these two peptides with zwitterionic (DMPC) and anionic (DMPG) LUVs by measuring the disappearance of the amide 1H NMR signal as LUVs are added to peptide solution. The large size of the peptide-LUV complex results in a long rotational correlation time, and thus dramatic broadening (disappearance) of the peptide signal. Preliminary experiments indicate that neither peptide binds appreciably to DMPC LUVs, while KK36 has a greater affinity for DMPG LUVs than KK45 (KD, KK36=0.34 þ/- 0.09 mM versus KD,KK45=1.1 þ/- 0.1 mM). Circular dichroism experiments indicate that KK45 remains largely 310-helical in water and when bound to DMPG LUVs, while KK36 undergoes a transition from largely [a]-helical in water to largely 310-helical when bound to DMPG LUVs. These results together suggest that both charge and structure are important factors in peptide-lipid association. 2094-Pos Board B238 Does Lipid Composition Regulate Anthrax Toxin Uptake? Nnanya U. Kalu1, Clare Kenney2, Ekaterina Nestorovich1. 1 Biology, The Catholic University of America, NE, DC, USA, 2Chemistry, The Catholic University of America, NE, DC, USA. Biological roles of lipids are not limited toproviding structural stability to bilayer membranes. Lipids have numerous functions, e.g. acting in cellular signaling and protein regulation. Cells adjust bilayer biophysical parameters by controlling lipid content and gradient of the plasma and intracellular organelle membranes. Thus, membrane composition changes along the endocytic pathway, with early endosomal membranes resembling plasma membranes, and late endosomal membranes showing dramatic increase in anionic lysobisphosphatidic acid level. Here we investigate the role of the lipid environmentin the anthrax toxin uptake. The anthrax toxin, formed by three proteins (binding protective antigen, PA, and enzymatic lethal factor, LF and edema factor, EF), follows a multistep internalization process which involves PA binding to its cellular receptors, proteolytic cleavage to PA63, oligomerization to form prepores, and LF and EF binding followed by endocytosis. The acidic endosomal environment causes conformational changes in PA63 leading to its insertion into the early endosomal membrane and ion channel formation. The delivery of LF to cytoplasm was suggested to take place later in the endocytic pathway via the back-fusion of intraluminal vesicles of late endosomes. Given the variations in membrane lipid composition of the early endosome, the intraluminal vesicles and the late endosome, it becomes important to understand if these changes influence the PA63 insertion and structural dynamics, LF and EF binding and translocation. Using bilayer lipid membrane technique, we mimic the endosomal membrane composition probing its properties in-vitro. We show that at pH 5.5, PA63 insertion rate increases dramatically in PS compared with PC bilayers, whereas LF binds ten times less effectively to PA63 in PS compared to PC. When the solution pH is raised from 5 to 6.75, this difference is diminished. 2095-Pos Board B239 Investigation of the Physiochemical Properties of the Phospholipid Cardiolipin: Implications for Oxphos Regulation and Barth Syndrome Murugappan Sathappa, Matthew Greenwood, Nathan Alder. University of Connecticut, Storrs, CT, USA. Cardiolipin (CL) is the signature phospholipid of the mitochondrial inner membrane, which houses respiratory complexes that generate electrochemical ion gradients to drive processes such as ATP synthesis by oxidative phosphorylation. Nascent CL, which is enriched in saturated acyl chains of variable lengths, is subject to a two-stage remodeling process: a deacylase first removes an acyl chain to generate the monolysocardiolpin (MLCL) intermediate, and the transacylase tafazzin regenerates the four-chain lipid with highly unsaturated acyl chains with a high degree of symmetry. Mutations in the TAZ gene that encodes tafazzin underpin Barth syndrome, a multi-system mitochondrial disorder characterized by gross abnormalities in lipid profiles, cardiac and skeletal myopathy, neutropenia, growth retardation and mortality in early childhood. First, we analyze the role of CL in activating OXPHOS complexes using nanoscale lipid bilayers containing headgroup and acyl chain variants of CL. To this end, purified cytochrome c oxidase was reconstituted into nanodiscs of defined lipid composition and assayed for CL-dependent redox activity. Second, we have analyzed the binding characteristics of cytochrome c to bilayers containing