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Olaf S. Andersen - One of the best experts on this subject based on the ideXlab platform.
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all atom simulation and continuum elastic theory of Gramicidin a in binary component lipid bilayers
Biophysical Journal, 2014Co-Authors: Andrew H Beaven, Denise V. Greathouse, Olaf S. Andersen, Roger E. Koeppe, Alexander J Sodt, Richard W Pastor, Wonpil ImAbstract:The linear Gramicidins are a group of peptides with alternating L and D chirality that fold into β-helices. The prototypical Gramicidin is [Val1] Gramicidin A (gA), which has been extensively studied using electrophysiology, spectroscopy, and molecular dynamics simulations. gA channels form by transmembrane dimerization and have been used to examine the interactions between membrane proteins and their host bilayer. This study focuses on gA channels in lipid bilayers composed of two phosphatidylcholines with different acyl chain lengths. The bilayers were formed from equimolar mixtures of DC16:1PC+DC24:1PC or DC18:1PC+DC22:1PC mixture, as well as pure DC20:1PC, all of which have the same average tail length. These gA-bilayer systems were simulated for 3.5 μs to explore the characteristics and energetics of lateral lipid redistribution around a protein. The simulations indicate: i) the overall bilayer thickness profile adjacent to the channel is similar in the three systems tested; ii) in the DC16:1PC+DC24:1PC mixture, the shorter DC16:1PC is enriched by nearly a factor of two in the first lipid shell around the channel; iii) thickness matching is dominant, even when the disparity between lengths is large; and iv) the acyl chains adopt non-native conformations in order to match achieve hydrophobic matching between the gA dimer and the bilayer core. In contrast to the results in the DC16:1PC+DC24:1PC mixture, enrichment in the DC18:1PC+DC22:1PC mixture is statistically insignificant. The preference for the better matching lipid (DC16:1PC) near the channel in the DC16:1PC+DC24:1PC mixture can be explained by a continuum model that accounts for the energetic penalty associated with compressing the longer lipid (DC24:1PC) to match the channel's hydrophobic length.
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Gramicidin Channels as Cation Nanotubes
Molecular- and Nano-Tubes, 2011Co-Authors: Roger E. Koeppe, Sigrid E. Schmutzer, Olaf S. AndersenAbstract:The linear Gramicidins constitute a family of peptide antibiotics produced by the soil bacterium Bacillus brevis. The first antibiotics to be used in clinical practice, the linear Gramicidins exert their antibacterial activity by forming bilayer-spanning channels that increase the monovalent cation permeability of target bacterial plasma membranes. Gramicidin channels are synthesized by non-ribosomal peptide synthesis on large protein complexes and contain both D- and L-amino acid residues; they were the first channels of known chemical composition to be studied. The channels effectively serve as cation-selective organic nanotubes that span lipid bilayer membranes and provide a basis for examining many aspects of ion-channel function and channel-lipid bilayer interactions. The nanotube properties can be tuned by means of mutations or chemical changes to the subunit architecture, as well as by altering the channels’ bilayer environment (e.g., the bilayer thickness). Indeed, many analogue sequences within the extended peptide family have been prepared by semi-synthesis or total synthesis. Diverse applications of Gramicidin channels have enhanced our understanding of the microphysics of ion permeation, lipid-protein interactions and membrane protein function.
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screening for small molecules bilayer modifying potential using a Gramicidin based fluorescence assay
Assay and Drug Development Technologies, 2010Co-Authors: Helgi I Ingolfsson, Olaf S. AndersenAbstract:Abstract Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter lipid bilayer properties. This is important because membrane proteins are energetically coupled to their host bilayer by hydrophobic interactions. Changes in bilayer properties thus alter membrane protein function, which provides a possible mechanism for “off-target” drug effects. We have previously shown that channels formed by the linear Gramicidins are suitable probes for changes in lipid bilayer properties, as experienced by bilayer-spanning proteins. We now report a Gramicidin-based fluorescence assay for changes in bilayer properties. The assay is based on measuring the time course of fluorescence quenching in fluorophore-loaded large unilamellar vesicles, due to entry of a Gramicidin channel-permeable quencher. The method is scalable and suitable for both mechanistic studies and high-throughput screening for bilayer-perturbing, potential ...
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Design and characterization of Gramicidin channels.
Methods in Enzymology, 2004Co-Authors: Denise V. Greathouse, Lyndon L. Providence, Roger E. Koeppe, S. Shobana, Olaf S. AndersenAbstract:Publisher Summary Gramicidin channels are miniproteins that are comprised of two tryptophan- rich transmembrane subunits. Gramicidin channels are useful models for understanding the principles that govern the structure and function of proteins (and lipids) in biological membranes. Through modifications of the parent Gramicidin A (gA) sequence, Gramicidins have been designed for specific purposes. This chapter describes methods for the solid-phase synthesis of Gramicidins and acylGramicidins, for the purification of the resulting peptides and acylpeptides, and for the characterization of the channels that are formed by these molecules in phospholipid membranes, using spectroscopic, chromatographic, computational, and electrophysiologic approaches.
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Gramicidin channel controversy the structure in a lipid environment
Nature Structural & Molecular Biology, 1999Co-Authors: Olaf S. Andersen, Roger E. Koeppe, Ernst Bamberg, Fred J. Sigworth, Hansjurgen Apell, David D Busath, Gabor Szabo, Dan W Urry, Andrew G WoolleyAbstract:Membrane-spanning Gramicidin channels remain unique because of their small size, well-characterized function and well-defined structure. In organic solvents, the Gramicidins are conformationally polymorphic; but a large body of work (summarized in Refs 1,2) shows conclusively that the predominant channel form is a head-to-head dimer of two single-stranded (SS) -helices confirming an early prediction3. We therefore take issue with the suggestion in a recent editorial in Nature Structural Biology4 that the major conformer responsible for ion movement across membranes is a double-stranded (DS) dimer. The DS Gramicidin crystal structures determined by Duax and collaborators5, which were presented at the conference described in the editorial, constitute an important advance, but they do not relate to the active channel structure.
Roger E. Koeppe - One of the best experts on this subject based on the ideXlab platform.
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all atom simulation and continuum elastic theory of Gramicidin a in binary component lipid bilayers
Biophysical Journal, 2014Co-Authors: Andrew H Beaven, Denise V. Greathouse, Olaf S. Andersen, Roger E. Koeppe, Alexander J Sodt, Richard W Pastor, Wonpil ImAbstract:The linear Gramicidins are a group of peptides with alternating L and D chirality that fold into β-helices. The prototypical Gramicidin is [Val1] Gramicidin A (gA), which has been extensively studied using electrophysiology, spectroscopy, and molecular dynamics simulations. gA channels form by transmembrane dimerization and have been used to examine the interactions between membrane proteins and their host bilayer. This study focuses on gA channels in lipid bilayers composed of two phosphatidylcholines with different acyl chain lengths. The bilayers were formed from equimolar mixtures of DC16:1PC+DC24:1PC or DC18:1PC+DC22:1PC mixture, as well as pure DC20:1PC, all of which have the same average tail length. These gA-bilayer systems were simulated for 3.5 μs to explore the characteristics and energetics of lateral lipid redistribution around a protein. The simulations indicate: i) the overall bilayer thickness profile adjacent to the channel is similar in the three systems tested; ii) in the DC16:1PC+DC24:1PC mixture, the shorter DC16:1PC is enriched by nearly a factor of two in the first lipid shell around the channel; iii) thickness matching is dominant, even when the disparity between lengths is large; and iv) the acyl chains adopt non-native conformations in order to match achieve hydrophobic matching between the gA dimer and the bilayer core. In contrast to the results in the DC16:1PC+DC24:1PC mixture, enrichment in the DC18:1PC+DC22:1PC mixture is statistically insignificant. The preference for the better matching lipid (DC16:1PC) near the channel in the DC16:1PC+DC24:1PC mixture can be explained by a continuum model that accounts for the energetic penalty associated with compressing the longer lipid (DC24:1PC) to match the channel's hydrophobic length.
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Gramicidin Channels as Cation Nanotubes
Molecular- and Nano-Tubes, 2011Co-Authors: Roger E. Koeppe, Sigrid E. Schmutzer, Olaf S. AndersenAbstract:The linear Gramicidins constitute a family of peptide antibiotics produced by the soil bacterium Bacillus brevis. The first antibiotics to be used in clinical practice, the linear Gramicidins exert their antibacterial activity by forming bilayer-spanning channels that increase the monovalent cation permeability of target bacterial plasma membranes. Gramicidin channels are synthesized by non-ribosomal peptide synthesis on large protein complexes and contain both D- and L-amino acid residues; they were the first channels of known chemical composition to be studied. The channels effectively serve as cation-selective organic nanotubes that span lipid bilayer membranes and provide a basis for examining many aspects of ion-channel function and channel-lipid bilayer interactions. The nanotube properties can be tuned by means of mutations or chemical changes to the subunit architecture, as well as by altering the channels’ bilayer environment (e.g., the bilayer thickness). Indeed, many analogue sequences within the extended peptide family have been prepared by semi-synthesis or total synthesis. Diverse applications of Gramicidin channels have enhanced our understanding of the microphysics of ion permeation, lipid-protein interactions and membrane protein function.
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Semisynthesis of linear Gramicidins using diphenyl phosphorazidate (DPPA).
International Journal of Peptide and Protein Research, 2009Co-Authors: Lawrence B. Weiss, Roger E. KoeppeAbstract:: Sequential HPLC analysis has been used to optimize a synthetic scheme for the preparation of semisynthetic position 1 analogues of the channel-forming pentadecapeptide, Gramicidin A. Diphenyl phosphorazidate (DPPA) was more efficient than dicyclohexylcarbodiimide (DCC) for the coupling of N-formyl amino acids to des(formyl-valyl) Gramicidin A. The DPPA coupling proceeds rapidly, and with high yield, at 0 degree. The absence of significant (less than 0.1%) racemization is demonstrated by the lack of electrophysiologically active formyl-L-valine Gramicidin A in preparations of formyl-D-valine Gramicidin A. This scheme has proved useful for the preparation of microgram to gram amounts of position-1-substituted Gramicidin analogues suitable for crystallography and electrophysiology. Details of the techniques for the preparation of these highly purified analogues are discussed.
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Interaction of Gramicidin Derivatives with Phospholipid Monolayers
Langmuir, 2004Co-Authors: Conor Whitehouse, Roger E. Koeppe, David Gidalevitz, Marjolaine Cahuzac, Andrew NelsonAbstract:A study of the interaction of Gramicidin A (gA), tert-butyloxycarbonyl-Gramicidin (g-BOC), and desformyl Gramicidin (g-des) with dioleoyl phosphatidylcholine (DOPC) and DOPC/phosphatidylserine (PS) mixed monolayers on a mercury electrode is reported in this paper. Experiments were carried out in electrolytes KCl (0.1 mol dm-3) and Mg(NO3)2 (0.05 mol dm-3). The channel-forming properties of the Gramicidins were studied by following the reduction of Tl(I) to Tl(Hg). The frequency dependence of the complex impedance of coated electrode surfaces in the presence and absence of the Gramicidins was estimated between 65 000 and 0.1 Hz at potentials of −0.4 V versus Ag/AgCl with 3.5 mol dm-3 KCl. Epifluorescence microscopy was used to qualitatively correlate the interaction of the Gramicidin peptides with dipalmitoyl phosphatidylcholine (DPPC) and dipalmitoyl phosphatidylglycerol (DPPG) at the air−water interface. gA was shown to form Tl+ conducting channels in a DOPC monolayer, while g-BOC and g-des did not. In D...
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Interaction of Gramicidin Derivatives with Phospholipid Monolayers
Langmuir : the ACS journal of surfaces and colloids, 2004Co-Authors: Conor Whitehouse, Roger E. Koeppe, David Gidalevitz, Marjolaine Cahuzac, Andrew NelsonAbstract:A study of the interaction of Gramicidin A (gA), tert-butyloxycarbonyl-Gramicidin (g-BOC), and desformyl Gramicidin (g-des) with dioleoyl phosphatidylcholine (DOPC) and DOPC/phosphatidylserine (PS) mixed monolayers on a mercury electrode is reported in this paper. Experiments were carried out in electrolytes KCl (0.1 mol dm(-3)) and Mg(NO3)2 (0.05 mol dm(-3)). The channel-forming properties of the Gramicidins were studied by following the reduction of Tl(I) to Tl(Hg). The frequency dependence of the complex impedance of coated electrode surfaces in the presence and absence of the Gramicidins was estimated between 65,000 and 0.1 Hz at potentials of -0.4 V versus Ag/AgCl with 3.5 mol dm(-3) KCl. Epifluorescence microscopy was used to qualitatively correlate the interaction of the Gramicidin peptides with dipalmitoyl phosphatidylcholine (DPPC) and dipalmitoyl phosphatidylglycerol (DPPG) at the air-water interface. gA was shown to form Tl+ conducting channels in a DOPC monolayer, while g-BOC and g-des did not. In DOPC-30% PS (DOPC-0.3PS) layers, there is a marked increase in channel activity of all three Gramicidin derivatives. None of the peptides facilitate the permeability of the DOPC-0.3PS layer to Cd2+. All three peptides interact with the layer as shown by capacitance-potential curves and impedance spectroscopy indicated by penetration of the peptide into the dielectric, an increase in surface "roughness", and an increased significance of low-frequency relaxations. The order of interaction is gA > g-des > g-BOC. The epifluorescence study of DPPC and DPPG layers at the air-water interface shows a selective action of the different Gramicidins.
Denise V. Greathouse - One of the best experts on this subject based on the ideXlab platform.
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all atom simulation and continuum elastic theory of Gramicidin a in binary component lipid bilayers
Biophysical Journal, 2014Co-Authors: Andrew H Beaven, Denise V. Greathouse, Olaf S. Andersen, Roger E. Koeppe, Alexander J Sodt, Richard W Pastor, Wonpil ImAbstract:The linear Gramicidins are a group of peptides with alternating L and D chirality that fold into β-helices. The prototypical Gramicidin is [Val1] Gramicidin A (gA), which has been extensively studied using electrophysiology, spectroscopy, and molecular dynamics simulations. gA channels form by transmembrane dimerization and have been used to examine the interactions between membrane proteins and their host bilayer. This study focuses on gA channels in lipid bilayers composed of two phosphatidylcholines with different acyl chain lengths. The bilayers were formed from equimolar mixtures of DC16:1PC+DC24:1PC or DC18:1PC+DC22:1PC mixture, as well as pure DC20:1PC, all of which have the same average tail length. These gA-bilayer systems were simulated for 3.5 μs to explore the characteristics and energetics of lateral lipid redistribution around a protein. The simulations indicate: i) the overall bilayer thickness profile adjacent to the channel is similar in the three systems tested; ii) in the DC16:1PC+DC24:1PC mixture, the shorter DC16:1PC is enriched by nearly a factor of two in the first lipid shell around the channel; iii) thickness matching is dominant, even when the disparity between lengths is large; and iv) the acyl chains adopt non-native conformations in order to match achieve hydrophobic matching between the gA dimer and the bilayer core. In contrast to the results in the DC16:1PC+DC24:1PC mixture, enrichment in the DC18:1PC+DC22:1PC mixture is statistically insignificant. The preference for the better matching lipid (DC16:1PC) near the channel in the DC16:1PC+DC24:1PC mixture can be explained by a continuum model that accounts for the energetic penalty associated with compressing the longer lipid (DC24:1PC) to match the channel's hydrophobic length.
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Design and characterization of Gramicidin channels.
Methods in Enzymology, 2004Co-Authors: Denise V. Greathouse, Lyndon L. Providence, Roger E. Koeppe, S. Shobana, Olaf S. AndersenAbstract:Publisher Summary Gramicidin channels are miniproteins that are comprised of two tryptophan- rich transmembrane subunits. Gramicidin channels are useful models for understanding the principles that govern the structure and function of proteins (and lipids) in biological membranes. Through modifications of the parent Gramicidin A (gA) sequence, Gramicidins have been designed for specific purposes. This chapter describes methods for the solid-phase synthesis of Gramicidins and acylGramicidins, for the purification of the resulting peptides and acylpeptides, and for the characterization of the channels that are formed by these molecules in phospholipid membranes, using spectroscopic, chromatographic, computational, and electrophysiologic approaches.
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Gramicidin channels--a solvable membrane "protein" folding problem.
Indian journal of biochemistry & biophysics, 1996Co-Authors: Olaf S. Andersen, Denise V. Greathouse, Gayatri Saberwal, Roger E. KoeppeAbstract:The linear Gramicidins are peptide antibiotics that form cation-selective channels in lipid bilayers. Gramicidin channels have very well-defined functional characteristics, and the structure of membrane-spanning Gramicidin A channels is known at atomic resolution. These features make the Gramicidins well suited to study how the amino acid sequence encodes the structure and function of a membrane-spanning channel. We show how one can use electrophysiological measurements to obtain structural information about conducting channels and to quantify the conformational preferences of sequence-substituted Gramicidin mutants.
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Gramicidin channel function does not depend on phospholipid chirality.
Biochemistry, 1995Co-Authors: Lyndon L. Providence, Denise V. Greathouse, Olaf S. Andersen, Roger E. Koeppe, Robert BittmanAbstract:: Chiral interactions are often important determinants for molecular recognition in chemistry and biochemistry. In order to determine whether the phospholipid backbone could be important for the conformational preference of membrane-spanning channels, we made use of the linear pentadecapeptide antibiotic Gramicidin A (gA+) and a Trp-->Phe-substituted gA+ analogue, Gramicidin M+ (gM+), as well as their enantiomers [Gramicidin A- (gA-) and Gramicidin M- (gM-), respectively]. All four analogues form conducting channels in planar bilayers formed from the dialkylphospholipids (R)- or (S)- dioleylphosphatidylcholine or from the diacylphospholipid (R)-dioleoylphosphatidylcholine. The characteristics of channels formed by the two Gramicidin A enantiomers, or the two Gramicidin M enantiomers, in membranes formed by either of the dioleylphosphatidylcholine enantiomers are indistinguishable. Similarly, channels formed by either pair of Gramicidin enantiomers in dioleoylphosphatidylcholine bilayers are indistinguishable. We conclude that chiral interactions between Gramicidin channels and the lipids in the host bilayer cannot be important determinants of Gramicidin channel structure or function. The membrane/solution interface, therefore, seems to organize the channel structure because of the general characteristics of the nonpolar/polar transition at the interface rather than because of specific chemical interactions.
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On the helix sense of Gramicidin A single channels.
Proteins, 1992Co-Authors: Roger E. Koeppe, Y Trudelle, Frederic Heitz, Denise V. Greathouse, Neil Purdie, Lyndon L. Providence, Olaf S. AndersenAbstract:: In order to resolve whether Gramicidin A channels are formed by right- or left-handed beta-helices, we synthesized an optically reversed (or mirror image) analogue of Gramicidin A, called Gramicidin A-, to test whether it forms channels that have the same handedness as channels formed by Gramicidin M- (F. Heitz et al., Biophys. J. 40:87-89, 1982). In Gramicidin M- the four tryptophan residues have been replaced with phenylalanine, and the circular dichroism (CD) spectrum therefore reflects almost exclusively contributions from the polypeptide backbone. The CD spectrum of Gramicidin M- in dimyristoylphosphatidylcholine vesicles is consistent with a left-handed helical backbone folding motif (F. Heitz et al., Biophys. Chem. 24:149-160, 1986), and the CD spectra of Gramicidins A and A- are essentially mirror images of each other. Based on hybrid channel experiments, Gramicidin A- and M- channels are structurally equivalent, while Gramicidin A and A- channels are nonequivalent, being of opposite helix sense. Gramicidin A- channels are therefore left-handed, and natural Gramicidin A channels in phospholipid bilayers are right-handed beta 6.3-helical dimers.
Lyndon L. Providence - One of the best experts on this subject based on the ideXlab platform.
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Design and characterization of Gramicidin channels.
Methods in Enzymology, 2004Co-Authors: Denise V. Greathouse, Lyndon L. Providence, Roger E. Koeppe, S. Shobana, Olaf S. AndersenAbstract:Publisher Summary Gramicidin channels are miniproteins that are comprised of two tryptophan- rich transmembrane subunits. Gramicidin channels are useful models for understanding the principles that govern the structure and function of proteins (and lipids) in biological membranes. Through modifications of the parent Gramicidin A (gA) sequence, Gramicidins have been designed for specific purposes. This chapter describes methods for the solid-phase synthesis of Gramicidins and acylGramicidins, for the purification of the resulting peptides and acylpeptides, and for the characterization of the channels that are formed by these molecules in phospholipid membranes, using spectroscopic, chromatographic, computational, and electrophysiologic approaches.
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Gramicidin channel function does not depend on phospholipid chirality.
Biochemistry, 1995Co-Authors: Lyndon L. Providence, Denise V. Greathouse, Olaf S. Andersen, Roger E. Koeppe, Robert BittmanAbstract:: Chiral interactions are often important determinants for molecular recognition in chemistry and biochemistry. In order to determine whether the phospholipid backbone could be important for the conformational preference of membrane-spanning channels, we made use of the linear pentadecapeptide antibiotic Gramicidin A (gA+) and a Trp-->Phe-substituted gA+ analogue, Gramicidin M+ (gM+), as well as their enantiomers [Gramicidin A- (gA-) and Gramicidin M- (gM-), respectively]. All four analogues form conducting channels in planar bilayers formed from the dialkylphospholipids (R)- or (S)- dioleylphosphatidylcholine or from the diacylphospholipid (R)-dioleoylphosphatidylcholine. The characteristics of channels formed by the two Gramicidin A enantiomers, or the two Gramicidin M enantiomers, in membranes formed by either of the dioleylphosphatidylcholine enantiomers are indistinguishable. Similarly, channels formed by either pair of Gramicidin enantiomers in dioleoylphosphatidylcholine bilayers are indistinguishable. We conclude that chiral interactions between Gramicidin channels and the lipids in the host bilayer cannot be important determinants of Gramicidin channel structure or function. The membrane/solution interface, therefore, seems to organize the channel structure because of the general characteristics of the nonpolar/polar transition at the interface rather than because of specific chemical interactions.
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On the helix sense of Gramicidin A single channels.
Proteins, 1992Co-Authors: Roger E. Koeppe, Y Trudelle, Frederic Heitz, Denise V. Greathouse, Neil Purdie, Lyndon L. Providence, Olaf S. AndersenAbstract:: In order to resolve whether Gramicidin A channels are formed by right- or left-handed beta-helices, we synthesized an optically reversed (or mirror image) analogue of Gramicidin A, called Gramicidin A-, to test whether it forms channels that have the same handedness as channels formed by Gramicidin M- (F. Heitz et al., Biophys. J. 40:87-89, 1982). In Gramicidin M- the four tryptophan residues have been replaced with phenylalanine, and the circular dichroism (CD) spectrum therefore reflects almost exclusively contributions from the polypeptide backbone. The CD spectrum of Gramicidin M- in dimyristoylphosphatidylcholine vesicles is consistent with a left-handed helical backbone folding motif (F. Heitz et al., Biophys. Chem. 24:149-160, 1986), and the CD spectra of Gramicidins A and A- are essentially mirror images of each other. Based on hybrid channel experiments, Gramicidin A- and M- channels are structurally equivalent, while Gramicidin A and A- channels are nonequivalent, being of opposite helix sense. Gramicidin A- channels are therefore left-handed, and natural Gramicidin A channels in phospholipid bilayers are right-handed beta 6.3-helical dimers.
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Formation of non-beta 6.3-helical Gramicidin channels between sequence-substituted Gramicidin analogues.
Biophysical Journal, 1992Co-Authors: John T. Durkin, Lyndon L. Providence, R. E. Koeppe, Olaf S. AndersenAbstract:Using the linear Gramicidins as an example, we have previously shown how the statistical properties of heterodimeric (hybrid) channels (formed between the parent [Val1]Gramicidin A (gA) and a sequence-altered analogue) can be used to assess whether the analogue forms channels that are structurally equivalent to the parent channels (Durkin, J. T., R. E. Koeppe II, and O. S. Andersen. 1990. J. Mol. Biol. 211:221–234). Generally, the Gramicidins are tolerant of amino acid sequence alterations. We report here an exception. The optically reversed analogue, Gramicidin M- (gM-) (Heitz, F., G. Spach, and Y. Trudelle. 1982. Biophys. J. 40:87–89), forms channels that are the mirror-image of [Val1]gA channels; gM- should thus form no hybrid channels with analogues having the same helix sense as [Val1]gA. Surprisingly, however, gM- forms hybrid channels with the shortened analogues des-Val1-[Ala2]gA and des-Val1-gC, but these channels differ fundamentally from the parent channels: (a) the appearance rate of these heterodimers is only approximately 1/10 of that predicted from the random assortment of monomers into conducting dimers, indicating the existence of an energy barrier to their formation (e.g., monomer refolding into a new channel-forming conformation); and (b), once formed, the hybrid channels are stabilized approximately 1,000-fold relative to the parent channels. The increased stability suggests a structure that is joined by many hydrogen bonds, such as one of the double-stranded helical dimers shown to be adopted by Gramicidins in organic solvents (Veatch, W. R., E. T. Fossel, and E. R. Blout. 1974. Biochemistry. 13:5249–5256).
Frederic Heitz - One of the best experts on this subject based on the ideXlab platform.
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synthesis and characterization of retro Gramicidin a dala Gramicidin a a 31 residue long Gramicidin analogue
International Journal of Peptide and Protein Research, 2009Co-Authors: Dominique Lelièvre, Y Trudelle, Frederic Heitz, Gerard SpachAbstract:A 31-residue peptide, retro Gramicidin A-dAla-Gramicidin A, mimicking a Gramicidin A dimer was prepared by solid phase method using a 4-(oxymethyl)-Pam resin and BOC as α amino protecting group. To avoid any possible formation of Gramicidin A, couplings of fragments were used together with stepwise growing of the peptide chain. After purification by silica gel chromatography, this peptide was shown to form long living channels in lipid bilayer. Preliminary physicochemical investigations suggest that the peptide does not adopt the expected π6.3DL helical conformation.
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Synthesis and characterization of retro Gramicidin A‐dAla‐Gramicidin A, a 31‐residue‐long Gramicidin analogue
International Journal of Peptide and Protein Research, 2009Co-Authors: Dominique Lelièvre, Y Trudelle, Frederic Heitz, Gerard SpachAbstract:A 31-residue peptide, retro Gramicidin A-dAla-Gramicidin A, mimicking a Gramicidin A dimer was prepared by solid phase method using a 4-(oxymethyl)-Pam resin and BOC as α amino protecting group. To avoid any possible formation of Gramicidin A, couplings of fragments were used together with stepwise growing of the peptide chain. After purification by silica gel chromatography, this peptide was shown to form long living channels in lipid bilayer. Preliminary physicochemical investigations suggest that the peptide does not adopt the expected π6.3DL helical conformation.
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On the helix sense of Gramicidin A single channels.
Proteins, 1992Co-Authors: Roger E. Koeppe, Y Trudelle, Frederic Heitz, Denise V. Greathouse, Neil Purdie, Lyndon L. Providence, Olaf S. AndersenAbstract:: In order to resolve whether Gramicidin A channels are formed by right- or left-handed beta-helices, we synthesized an optically reversed (or mirror image) analogue of Gramicidin A, called Gramicidin A-, to test whether it forms channels that have the same handedness as channels formed by Gramicidin M- (F. Heitz et al., Biophys. J. 40:87-89, 1982). In Gramicidin M- the four tryptophan residues have been replaced with phenylalanine, and the circular dichroism (CD) spectrum therefore reflects almost exclusively contributions from the polypeptide backbone. The CD spectrum of Gramicidin M- in dimyristoylphosphatidylcholine vesicles is consistent with a left-handed helical backbone folding motif (F. Heitz et al., Biophys. Chem. 24:149-160, 1986), and the CD spectra of Gramicidins A and A- are essentially mirror images of each other. Based on hybrid channel experiments, Gramicidin A- and M- channels are structurally equivalent, while Gramicidin A and A- channels are nonequivalent, being of opposite helix sense. Gramicidin A- channels are therefore left-handed, and natural Gramicidin A channels in phospholipid bilayers are right-handed beta 6.3-helical dimers.
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Gramicidin channels that have no tryptophan residues.
Biochemistry, 1992Co-Authors: V. Fonseca, Y Trudelle, Frederic Heitz, P. Daumas, L. Ranjalahy-rasoloarijao, R. Lazaro, Olaf S. AndersenAbstract:: In order to understand how aromatic residues modulate the function of membrane-spanning proteins, we examined the role of the four tryptophans in Gramicidin A (gA) in determining the average duration and permeability characteristics of membrane-spanning Gramicidin channels; the tryptophan residues were replaced by tyrosine (Gramicidin T, gT), tyrosine O-benzyl ether [Gramicidin T(Bzl), gT(Bzl)], naphthylalanine (Gramicidin N, gN), and phenylalanine (Gramicidin M enantiomer, gM-). These analogues form channels with durations and conductances that differ some 10- and 16-fold, respectively. The single-channel conductance was invariably decreased by the Trp----Yyy replacement, and the relative conductance alterations were similar in phosphatidylcholine (DPhPC) and monoglyceride (GMO) bilayers. The duration variations exhibited a more complex pattern, which was quite different in the two membrane environments: in DPhPC bilayers, gN channels have an average duration that is approximately 2-fold longer than that of gA channels; in GMO bilayers, the average duration of gN channels is about one-tenth that of gA channels. The sequence-dependent alterations in channel function do not result from alterations in the channels' peptide backbone structure, because heterodimers can form between the different analogues and Gramicidine A, and there is no energetic cost associated with heterodimer formation [cf. Durkin, J. T., Koeppe, R. E., II, & Andersen, O. S. (1990) J. Mol. Biol. 211, 221]. The alterations in permeability properties are consistent with the notion that Trp residues alter the energy profile for ion permeation through long-range electrostatic interactions.
