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A Novel Soluble Peptide with pH-Responsive Membrane Insertion
252a
Monday, February 29, 2016
1252-Pos Board B229 Use of Transmembrane Peptides to Understand Ionization Properties of Histidine Residues in Lipid Bilayers Ashley N. Martfeld, Denise V. Greathouse, Roger E. Koeppe II. Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR, USA. With a pKa near 6, the imidazole side chain of histidine (His; H) can be positively charged or neutral under physiological conditions. Many membrane proteins are likely to contain functionally important His residues within their transmembrane domains; therefore it is critical to understand the ionization properties of His residues at various locations within the lipid bilayer. To address this problem experimentally, we have employed GWALP23 (acetylGGALW5LALALAL12AL14ALALW19LAGA-amide), a designed transmembrane peptide with interfacial tryptophan anchors. Within the GWALP23 sequence, we have substituted either His12 or His14 near the center of the helix, and incorporated specific 2H-labeled alanine residues for detection by means of solid-state 2H NMR. Based upon the quadrupolar splittings of the labeled Ala residues in each peptide, we observe that the behavior of these peptide isomers strongly depends on the location and ionization state of the His residue. Above pH 4, we infer that H12 is neutral because GWALP23-H12 adopts a stable transmembrane orientation in bilayers of DLPC, DMPC, or DOPC, nearly identical to that of GWALP23 itself. However, in DOPC bilayers, when the pH is lowered from 4 to 2, H12 becomes positively charged, and the spectral changes indicate multi-state behavior, similar to previous observations for charged K12 and R12 (see PNAS 110, 1692). The neutral and charged forms of H14 confer different transmembrane orientations for the GWALP23-H14 helix in DOPC, similar to previous observations for neutral and charged K14. From these experiments, we deduce a pKa of below 3 for His12 and between 3 and 5 for His 14 in DOPC bilayers. 1253-Pos Board B230 Varied Approaches to the Ionization Behavior of Specific Glu Residues that Face the Lipids in Transmembrane Helices Venkatesan Rajagopalan, Denise V. Greathouse, Roger E. Koeppe. Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR, USA. GWALP23 (acetyl-GGALW5LALALAL12AL14AL16ALW19LAGA-amide) is a useful low-dynamic model peptide for investigations of single-residue influence on protein-lipid interactions and the properties of membranespanning helices (J. Biol. Chem. 285, 31723). To investigate the pH dependence, ionization behavior and orientational constraints when potentially negatively charged glutamic acid side chains are present, we have substituted a single Leu residue with Glu at different positions and have incorporated specific 2H-alanines in the core helix or near the ends (Ala-3 and Ala-21) of GWALP23. Solid-state NMR experiments show that the 2H-Ala quadrupolar splittings of core labels change very little over the pH range of 4 to 11.5 when E12, E14 or E16 is present in GWALP23, but show modest shifts above pH 12. The spectra from deuterium labels on alanines 3 and 21 show changes in the unwinding of helix terminals in all three mutants between pH 4.0 and 13. The combined results suggest that these Glu residues may titrate with a pKa near 12 in DLPC and DOPC lipid bilayers. The rather modest shifts in the core alanine 2H quadrupolar splittings, nevertheless, suggest that the orientation of the transmembrane helix actually may change rather little at high pH. Rather, the unwinding of helix ends may be a key indicator of Glu residue titration. Our results are consistent with the expectation that the buried negative charges will aggressively hold their waters of hydration. It is conceivable that the close proximity of E16 to the indole ring of W19 could influence the results. 1254-Pos Board B231 Effects of Lipid Membrane Curvatures on Binding, Secondary Structure, and Aggregation of Amyloid-Beta Protein Yuuki Sugiura, Keisuke Ikeda, Minoru Nakano. Graduate School of Pharmaceutical Sciences, University of Toyama, Toyama, Japan. Conversion of amyloid-beta (Abeta) protein from a non-toxic monomer into the toxic aggregates is the possible pathogenic pathways in Alzheimer’s disease. Recent studies have suggested that lipid membranes play key roles in protein aggregation. It has been demonstrated that Abeta accumulated and formed amyloid fibrils in specific intra- or extracellular membrane structures such as intraluminal vesicles of multivesicular bodies (MVBs) and exosomes with a diameter that ranges from 30 to 80 nm. These studies raise the possibility that Abeta preferentially binds to small-sized intra- or extracellular vesicles with a high membrane curvature, although the presence of specific lipid
components might also influence the interaction. Indeed, it has been reported that small lipid vesicles (%50 nm) composed of PC accelerated Abeta fibrillation. However, the binding modes and the mechanisms of Abeta aggregation on lipid vesicles of high curvature are not fully understood. Here, we observed that a high positive curvature of lipid vesicles with diameters of ~30 nm enhanced the binding of Abeta with anionic PG membranes in the liquid crystalline phase and with zwitterionic PC membranes in the gel phase. The binding modes of Abeta to these membranes differ in terms of the depth of the protein in the membrane and of the protein secondary structure. Amyloid fibril formation of Abeta was accelerated in the presence of the vesicles and at high protein-to-lipid ratios. Under these conditions, the protein highly accumulated on the surfaces, as demonstrated by high (~10^7 M^-1) binding constants. Our findings suggest that packing defects on membranes with high curvatures might result in the accumulation of toxic protein aggregates. 1255-Pos Board B232 Lactoferricin Peptides: The Importance of Methyl-Tryptophan and Glutamine for Structure and Activity Alexandrea H. Kim, Denise V. Greathouse. Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR, USA. The rise in antibiotic-resistant strains of bacteria has led to an active search for more potent antimicrobial drugs. The hexapeptide (LfB6: RRWQWRNH2) from the N-terminal of the protein lactoferricin was shown to be the antimicrobial ‘active core’ by Tomita (Acta Paediatr Jpn, 1994, 36:585). A heptapeptide synthesized in our lab, which contains 4 positively charged arginines and 2 methylated tryptophans (LfB7 MeTrp3,5: RRMeWQMeWRR-NH2), exhibits enhanced activity when compared to LfB6 against gram positive and gram negative bacteria. To determine if Trp-methylation is required for the increased efficacy, a control peptide without methylated Trp (LfB7: RRWQWRR-NH2) has been synthesized and characterized. In addition, glutamine was replaced with alanine in both peptides to examine the importance of H-bonding at the central position (LfB7 MeTrp3,5 Ala4: RRMeWAMeWRR-NH2and LfB7 Ala4: RRWAWRR-NH2). Replacement of the central Gln with Ala reduced activity 5-fold against E. coli. LfB7 MeTrp3,5 exhibits the highest activity, with a 4- and 6-fold increase compared to LfB7, against E. coli and S. aureus, respectively. The Ala, Trp and MeTrp residues were labeled with deuterium, and the peptide was studied by solid-state deuterium NMR in bacterial- and mammalian-like membranes. The 2H-quadrupolar splittings, whether from labeled Ala, MeTrp or Trp, are larger when MeTrp is present in the sequence, indicating a change in peptide conformation and/or dynamics upon Trp-methylation. Tryptophan emission fluorescence spectra reveal that the Trp and MeTrp residues are more deeply buried in anionic compared to neutral lipids, and circular dichroism spectra suggest that anionic lipids promote a conformational change that results in Trp-Trp (and MeTrp-MeTrp) interactions. These results emphasize the importance of Gln at the central position and of Trp-methylation for structure and activity of lactoferricin. 1256-Pos Board B233 A Novel Soluble Peptide with pH-Responsive Membrane Insertion Vanessa P. Nguyen, Daiane S. Alves, Haden L. Scott, Forrest L. Davis, Francisco N. Barrera. BCMB, University of Tennessee - Knoxville, Knoxville, TN, USA. Several diseases, such as cancer, are characterized by acidification of the extracellular environment. Acidosis can be employed as a target to specifically direct therapies to the diseased tissue. We have used first principles to design an acidity-triggered rational membrane (ATRAM) peptide with high solubility in solution that is able to interact with lipid membranes in a pH dependent fashion. Biophysical studies show that the ATRAM peptide binds to the surface of lipid membranes at pH 8.0. However, acidification leads to the peptide inserting into the lipid bilayer as a transmembrane a-helix. The insertion of ATRAM into membranes occurs at a moderately acidic pH (with a pK of 6.5), similar to the extracellular pH found in solid tumors. Studies with human cell lines showed a highly efficient pH-dependent membrane targeting, without causing toxicity. Here we show that it is possible to rationally design a soluble peptide that selectively targets cell membranes in acidic environments. 1257-Pos Board B234 Lipid Environment of Aquaporin Z Victoria Schmidt1, Marlon Sidore1, Frede´ric Carrie`re2, Jean-Pierre Duneau1, James N. Sturgis1. 1 IMM, Marseille, France, 2EIPL, Marseille, France. Aquaporin Z is a membrane protein of E. coli that is responsible for water transfer across the membrane. The protein normally forms a tetramer in the
Monday, February 29, 2016
1252-Pos Board B229 Use of Transmembrane Peptides to Understand Ionization Properties of Histidine Residues in Lipid Bilayers Ashley N. Martfeld, Denise V. Greathouse, Roger E. Koeppe II. Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR, USA. With a pKa near 6, the imidazole side chain of histidine (His; H) can be positively charged or neutral under physiological conditions. Many membrane proteins are likely to contain functionally important His residues within their transmembrane domains; therefore it is critical to understand the ionization properties of His residues at various locations within the lipid bilayer. To address this problem experimentally, we have employed GWALP23 (acetylGGALW5LALALAL12AL14ALALW19LAGA-amide), a designed transmembrane peptide with interfacial tryptophan anchors. Within the GWALP23 sequence, we have substituted either His12 or His14 near the center of the helix, and incorporated specific 2H-labeled alanine residues for detection by means of solid-state 2H NMR. Based upon the quadrupolar splittings of the labeled Ala residues in each peptide, we observe that the behavior of these peptide isomers strongly depends on the location and ionization state of the His residue. Above pH 4, we infer that H12 is neutral because GWALP23-H12 adopts a stable transmembrane orientation in bilayers of DLPC, DMPC, or DOPC, nearly identical to that of GWALP23 itself. However, in DOPC bilayers, when the pH is lowered from 4 to 2, H12 becomes positively charged, and the spectral changes indicate multi-state behavior, similar to previous observations for charged K12 and R12 (see PNAS 110, 1692). The neutral and charged forms of H14 confer different transmembrane orientations for the GWALP23-H14 helix in DOPC, similar to previous observations for neutral and charged K14. From these experiments, we deduce a pKa of below 3 for His12 and between 3 and 5 for His 14 in DOPC bilayers. 1253-Pos Board B230 Varied Approaches to the Ionization Behavior of Specific Glu Residues that Face the Lipids in Transmembrane Helices Venkatesan Rajagopalan, Denise V. Greathouse, Roger E. Koeppe. Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR, USA. GWALP23 (acetyl-GGALW5LALALAL12AL14AL16ALW19LAGA-amide) is a useful low-dynamic model peptide for investigations of single-residue influence on protein-lipid interactions and the properties of membranespanning helices (J. Biol. Chem. 285, 31723). To investigate the pH dependence, ionization behavior and orientational constraints when potentially negatively charged glutamic acid side chains are present, we have substituted a single Leu residue with Glu at different positions and have incorporated specific 2H-alanines in the core helix or near the ends (Ala-3 and Ala-21) of GWALP23. Solid-state NMR experiments show that the 2H-Ala quadrupolar splittings of core labels change very little over the pH range of 4 to 11.5 when E12, E14 or E16 is present in GWALP23, but show modest shifts above pH 12. The spectra from deuterium labels on alanines 3 and 21 show changes in the unwinding of helix terminals in all three mutants between pH 4.0 and 13. The combined results suggest that these Glu residues may titrate with a pKa near 12 in DLPC and DOPC lipid bilayers. The rather modest shifts in the core alanine 2H quadrupolar splittings, nevertheless, suggest that the orientation of the transmembrane helix actually may change rather little at high pH. Rather, the unwinding of helix ends may be a key indicator of Glu residue titration. Our results are consistent with the expectation that the buried negative charges will aggressively hold their waters of hydration. It is conceivable that the close proximity of E16 to the indole ring of W19 could influence the results. 1254-Pos Board B231 Effects of Lipid Membrane Curvatures on Binding, Secondary Structure, and Aggregation of Amyloid-Beta Protein Yuuki Sugiura, Keisuke Ikeda, Minoru Nakano. Graduate School of Pharmaceutical Sciences, University of Toyama, Toyama, Japan. Conversion of amyloid-beta (Abeta) protein from a non-toxic monomer into the toxic aggregates is the possible pathogenic pathways in Alzheimer’s disease. Recent studies have suggested that lipid membranes play key roles in protein aggregation. It has been demonstrated that Abeta accumulated and formed amyloid fibrils in specific intra- or extracellular membrane structures such as intraluminal vesicles of multivesicular bodies (MVBs) and exosomes with a diameter that ranges from 30 to 80 nm. These studies raise the possibility that Abeta preferentially binds to small-sized intra- or extracellular vesicles with a high membrane curvature, although the presence of specific lipid
components might also influence the interaction. Indeed, it has been reported that small lipid vesicles (%50 nm) composed of PC accelerated Abeta fibrillation. However, the binding modes and the mechanisms of Abeta aggregation on lipid vesicles of high curvature are not fully understood. Here, we observed that a high positive curvature of lipid vesicles with diameters of ~30 nm enhanced the binding of Abeta with anionic PG membranes in the liquid crystalline phase and with zwitterionic PC membranes in the gel phase. The binding modes of Abeta to these membranes differ in terms of the depth of the protein in the membrane and of the protein secondary structure. Amyloid fibril formation of Abeta was accelerated in the presence of the vesicles and at high protein-to-lipid ratios. Under these conditions, the protein highly accumulated on the surfaces, as demonstrated by high (~10^7 M^-1) binding constants. Our findings suggest that packing defects on membranes with high curvatures might result in the accumulation of toxic protein aggregates. 1255-Pos Board B232 Lactoferricin Peptides: The Importance of Methyl-Tryptophan and Glutamine for Structure and Activity Alexandrea H. Kim, Denise V. Greathouse. Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR, USA. The rise in antibiotic-resistant strains of bacteria has led to an active search for more potent antimicrobial drugs. The hexapeptide (LfB6: RRWQWRNH2) from the N-terminal of the protein lactoferricin was shown to be the antimicrobial ‘active core’ by Tomita (Acta Paediatr Jpn, 1994, 36:585). A heptapeptide synthesized in our lab, which contains 4 positively charged arginines and 2 methylated tryptophans (LfB7 MeTrp3,5: RRMeWQMeWRR-NH2), exhibits enhanced activity when compared to LfB6 against gram positive and gram negative bacteria. To determine if Trp-methylation is required for the increased efficacy, a control peptide without methylated Trp (LfB7: RRWQWRR-NH2) has been synthesized and characterized. In addition, glutamine was replaced with alanine in both peptides to examine the importance of H-bonding at the central position (LfB7 MeTrp3,5 Ala4: RRMeWAMeWRR-NH2and LfB7 Ala4: RRWAWRR-NH2). Replacement of the central Gln with Ala reduced activity 5-fold against E. coli. LfB7 MeTrp3,5 exhibits the highest activity, with a 4- and 6-fold increase compared to LfB7, against E. coli and S. aureus, respectively. The Ala, Trp and MeTrp residues were labeled with deuterium, and the peptide was studied by solid-state deuterium NMR in bacterial- and mammalian-like membranes. The 2H-quadrupolar splittings, whether from labeled Ala, MeTrp or Trp, are larger when MeTrp is present in the sequence, indicating a change in peptide conformation and/or dynamics upon Trp-methylation. Tryptophan emission fluorescence spectra reveal that the Trp and MeTrp residues are more deeply buried in anionic compared to neutral lipids, and circular dichroism spectra suggest that anionic lipids promote a conformational change that results in Trp-Trp (and MeTrp-MeTrp) interactions. These results emphasize the importance of Gln at the central position and of Trp-methylation for structure and activity of lactoferricin. 1256-Pos Board B233 A Novel Soluble Peptide with pH-Responsive Membrane Insertion Vanessa P. Nguyen, Daiane S. Alves, Haden L. Scott, Forrest L. Davis, Francisco N. Barrera. BCMB, University of Tennessee - Knoxville, Knoxville, TN, USA. Several diseases, such as cancer, are characterized by acidification of the extracellular environment. Acidosis can be employed as a target to specifically direct therapies to the diseased tissue. We have used first principles to design an acidity-triggered rational membrane (ATRAM) peptide with high solubility in solution that is able to interact with lipid membranes in a pH dependent fashion. Biophysical studies show that the ATRAM peptide binds to the surface of lipid membranes at pH 8.0. However, acidification leads to the peptide inserting into the lipid bilayer as a transmembrane a-helix. The insertion of ATRAM into membranes occurs at a moderately acidic pH (with a pK of 6.5), similar to the extracellular pH found in solid tumors. Studies with human cell lines showed a highly efficient pH-dependent membrane targeting, without causing toxicity. Here we show that it is possible to rationally design a soluble peptide that selectively targets cell membranes in acidic environments. 1257-Pos Board B234 Lipid Environment of Aquaporin Z Victoria Schmidt1, Marlon Sidore1, Frede´ric Carrie`re2, Jean-Pierre Duneau1, James N. Sturgis1. 1 IMM, Marseille, France, 2EIPL, Marseille, France. Aquaporin Z is a membrane protein of E. coli that is responsible for water transfer across the membrane. The protein normally forms a tetramer in the