Extent of Cysteine Modification of SNAP-25 In Vitro Alex McGregor DaBell Department of Physiology... more Extent of Cysteine Modification of SNAP-25 In Vitro Alex McGregor DaBell Department of Physiology and Developmental Biology, BYU Master of Science Exocytosis, the fusion of a vesicle to a cellular membrane, involves a protein named SNAP-25. This protein, containing two alpha helices connected with a linker region, is localized to the cell membrane via palmitic acids attached to the cysteine residues of its linker region in a process called palmitoylation. Are cysteine residues of the SNAP-25 linker region palmitoylated in an ordered manner and to a particular extent? The answer to this question may give insight into the regulated nature of exocytosis. While it is generally accepted that SNAP-25 must be palmitoylated in order to perform its exocytotic functions, the details surrounding this process are still being discovered, defined, and understood. In these studies we replicate the oxidation, reduction, and palmitoylation of SNAP-25 in vitro. Palmitoylating SNAP-25 in vitro, a proc...
ABSTRACT Some results of our simulation studies of selectivity and conductance of calcium ion cha... more ABSTRACT Some results of our simulation studies of selectivity and conductance of calcium ion channels in membranes are reported. Ion channels regulate many important physiological functions. To simulate the system we use periodic boundary conditions. We have noted previously that the use of such boundary conditions does not affect our results in any significant manner. The structure of the filter of a calcium channel is reasonably well understood. An important structural element is the set of four glutamate residues that are attached to the wall of the channel. Since the glutamates are long and flexible, Nonner and Eisenberg et al. have suggested that these glutamates can be modeled by eight half-charged oxygen ions that represent the carboxylates at the end of each residue. These oxygen ions are confined to the filter but otherwise are mobile. We have used this model in simulations of the selectivity and conductance of this model calcium channel filter. Our studies confirm that this model behaves as a calcium filter; calcium ions will drive sodium ions from the filter. In many of our simulations, explicit water molecules are used with water molecules in both the filter and bath.
The M2 protein from influenza A is a proton channel as a tetramer, with a single transmembrane he... more The M2 protein from influenza A is a proton channel as a tetramer, with a single transmembrane helix from each monomer lining the pore. Val27 and Trp41 form gates at either end of the pore and His37 mediates the shuttling of protons across a central barrier between the N-terminal and C-terminal aqueous pore regions. Numerous structures of this transmembrane domain and of a longer construct that includes an amphipathic helix are now in the Protein Data Bank. Many structural differences are apparent from samples obtained in a variety of membrane mimetic environments. High-resolution structural results in lipid bilayers have provided novel insights into the functional mechanism of the unique HxxxW cluster in the M2 proton channel.
The mechanism of action of volatile general anesthetics has not yet been resolved. In order to id... more The mechanism of action of volatile general anesthetics has not yet been resolved. In order to identify the effects of isoflurane on the membrane, we measured the steady-state anisotropy of two fluorescent probes that reside at different depths. Incorporation of anesthetic was confirmed by shifting of the main phase transition temperature. In liquid crystalline dipalmitoylphosphatidylcholine liposomes, isoflurane (7-25 mM in the bath) increases trimethylammonium-diphenylhexatriene fluorescence anisotropy by ~0.02 units and decreases diphenylhexatriene anisotropy by the same amount. The anisotropy data suggest that isoflurane decreases non-axial dye mobility in the headgroup region, while increasing it in the tail region. We propose that these results reflect changes in the lateral pressure profile of the membrane.
International Journal of Peptide and Protein Research, 2009
ABSTRACT The 13C-D-Leu12,14 gramicidin A was synthesized by the solid phase method incorporating ... more ABSTRACT The 13C-D-Leu12,14 gramicidin A was synthesized by the solid phase method incorporating 13C-D-leucine in positions 12 and 14 with about 25 and 50% enrichment, respectively. The pentadecapeptide was removed from the resin by ethanolamine treatment, with the N-protecting group (Boc) still on. After removal of the protecting group, the peptide was formylated and purified by preparative t.l.c. to obtain 13C-D-Leu12,14 gramicidin A in a very pure state in an overall yield of about 12.5%. The peptide was then thoroughly characterized by HPLC which gave one single peak with the same retention time as that of Val1-gramicidin A of the natural gramicidin mixture. The CD spectra of the synthetic and the HPLC purified natural Val1 -GA were obtained and found to be identical, indicating the optical purity of the sample. The synthetic GA was characterized by 13C n.m.r. spectrum and compared with that of natural GA. Single channel conductance parameters of the synthetic GA were determined and found to be indistinguishable from those of natural Val1-GA in lipid bilayer membranes and the mean channel lifetime was found to be as reported earlier by others.
International Journal of Peptide and Protein Research, 2009
The synthesis of (1-" C)-Phe'-gramicidin (90% enriched) was carried out by the solid phase method... more The synthesis of (1-" C)-Phe'-gramicidin (90% enriched) was carried out by the solid phase method. The peptide was removed from the resin by treatment with ethanolamine, deblocked, formylated and purified by preparative t.1.c. to obtain the gramicidin analog in an overall yield of 24%. The peptide was verified and characterized by high pressure liquid chromatography, carbon-13 nuclear magnetic resonance, circular dichroism and single channel currents. Single channel conductances were found to be similar to those of (l-'3C)-Phe11-GB but significantly lower than that of gramicidin A. When this gramicidin analog was incubated with phospholipid, the characteristic channel spectrum was not obtained and interaction with sodium ion was not observed. A possible explanation for this behavior is discussed.
M2 Out of the Envelope The M2 protein from influenza A virus forms an acid-activated tetrameric p... more M2 Out of the Envelope The M2 protein from influenza A virus forms an acid-activated tetrameric proton channel in the viral envelope and is essential for viral replication. Two manuscripts shed light on the functional mechanism of this channel. Sharma et al. (p. 509 ; see the Perspective by Fiorin et al. ) determined the structure of the conductance domain in a lipid bilayer and propose that a histidine and tryptophan from each monomer form a cluster that guides protons through the channel in a mechanism that involves forming and breaking hydrogen bonds between adjacent pairs of histidines. Hu et al. (p. 505 ; see the Perspective by Fiorin et al. ) focused on the structure and dynamics of the proton-selective histidine at high and low pH, proposing that proton conduction involves histidine deprotonation and reprotonation.
Proteins: Structure, Function, and Bioinformatics, 2009
M 2 transmembrane domain channel (M 2-TMD) permeation properties are studied using molecular dyna... more M 2 transmembrane domain channel (M 2-TMD) permeation properties are studied using molecular dynamics simulations of M 2-TMD (1NYJ) embedded in a lipid bilayer (DMPC) with 1 mol/kg NaCl or KCl saline solution. This study allows examination of spontaneous cation and anion entry into the selectivity filter. Three titration states of the M 2-TMD tetramer are modeled for which the four His37 residues, forming the selectivity filter, are net uncharged, +2 charged, or +3 charged. M 2-TMD structural properties from our simulations are compared with the properties of other models extracted from NMR and X-ray studies. During 10 ns simulations, chloride ions rarely occupy the positively-charged selectivity filter, whereas from umbrella sampling simulations, Cl − has a lower free-energy barrier in the selectivity-filter region than either Na + or , and has a lower free-energy barrier than Na +. For Na + and Cl − , the free-energy barriers are less than 5 kcal/mol, suggesting that the 1NYJ conformation would probably not be exquisitely proton selective. We also point out a rotameric configuration of Trp41 that could fully occlude the channel.
The theory of fluids near surfaces and in pores is reviewed and some simulations of electrolytes ... more The theory of fluids near surfaces and in pores is reviewed and some simulations of electrolytes with a continuum solvent near an electrode that agree qualitatively with experiment but that are inexplicable by current theories are outlined. Recent work with ...
Pfl�gers Archiv European Journal of Physiology, 1986
The capture, transport, and maintenance of live adult Atlantic coast squid (Loligo pealei) are de... more The capture, transport, and maintenance of live adult Atlantic coast squid (Loligo pealei) are described. The objective was to obtain healthy live squid from a coastal region and to maintain them at an inland research facility long enough to provide at least 4 days of electrophysiological research. An inexpensive closed aquarium system is described, which utilizes an ion-exchange resin in the filter, that allows a typical survival time of at least 4.5 days. Similar closed aquarium systems may be of interest to other biophysicists who wish to maintain live squid away from coastal research facilities.
A series of 2-adamantanamines with alkyl adducts of various lengths were examined for efficacy ag... more A series of 2-adamantanamines with alkyl adducts of various lengths were examined for efficacy against strains of influenza A including those having an S31N mutation in M2 proton channel that confer resistance to amantadine and rimantadine. The addition of as little as one CH 2 group to the methyl adduct of the amantadine/rimantadine analogue, 2-methyl-2-aminoadamantane, led to activity in vitro against two M2 S31N viruses A/Calif/07/ 2009 (H1N1) and A/PR/8/34 (H1N1) but not to a third A/WS/33 (H1N1). Solid state NMR of the transmembrane domain (TMD) with a site mutation corresponding to S31N shows evidence of drug binding. But electrophysiology using the full length S31N M2 protein in HEK cells showed no blockade. A wild type strain, A/Hong Kong/1/68 (H3N2) developed resistance to representative drugs within one passage with mutations in M2 TMD, but A/ Calif/07/2009 S31N was slow (>8 passages) to develop resistance in vitro, and the resistant virus had no mutations in M2 TMD. The results indicate that 2alkyl-2-aminoadamantane derivatives with sufficient adducts can persistently block p2009 influenza A in vitro through an alternative mechanism. The observations of an HA1 mutation, N160D, near the sialic acid binding site in both 6-resistant A/ Calif/07/2009(H1N1) and the broadly resistant A/WS/33(H1N1) and of an HA1 mutation, I325S, in the 6-resistant virus at a cell-culture stable site suggest that the drugs tested here may block infection by direct binding near these critical sites for virus entry to the host cell.
Membrane proteins are structurally characterized in membrane mimetic environments, environments t... more Membrane proteins are structurally characterized in membrane mimetic environments, environments that may or may not reflect the properties of native membrane proteins. This is critically important since the structure of membrane proteins is influenced by the protein's environment. Here we will show structural data from magic angle spinning solid state NMR of the full length M2 protein from Influenza A observed in E. coli membranes. This protein has never been exposed to the denaturing influence of detergents nor to the length isolation, purification and reconstitution protocols that typify membrane protein sample preparation for structural studies. The data will be compared to the purified full length protein in synthetic liposomes and to spectra of smaller constructs for which there is a high resolution structure in lipid bilayers.
Empirical energy function calculations were used to evaluate the effects of minimization on the s... more Empirical energy function calculations were used to evaluate the effects of minimization on the structure of a gramicidin A channel and to analyze the energies of interaction between three cations (guanidinium, acetamidinium, formamidinium) and the channel as a function of position along the channel axis. The energy minimized model of the gramicidin channel, which was based on the results of Venkatachalam and Urry (1983), has a constriction at the channel entrance. If the channel is not allowed to relax in the presence of the ions (rigid model), there is a large potential energy barrier for all three cations. The barrier varies with cation size and is due to high van der Waals and ion deformation energies. If the channel is minimized in the presence of the ions, the potential energy barrier to formamidinium entry is almost eliminated, but a residual barrier remains for guanidinium and acetamidinium. The residual barrier is primarily due, not to the expansion of the helix, but, to the disruption of hydrogen bonds between the terminal ethanoloamine and the next turn of the helix which occurs when the carbonyls of the outer turn of the helix librate inward toward the ion as it enters the channel. The residual potential energy barriers could be a possible explanation for the measured selectivity of gramicidin for formamidinium over guanidinium. The results of this full-atomic model address the applicability of the size-exclusion concept for the selectivity of the gramicidin channel.
Compared to the N-formyl gramicidin A (GA), the N-acetyl gramicidin A (NAG) channel has unchanged... more Compared to the N-formyl gramicidin A (GA), the N-acetyl gramicidin A (NAG) channel has unchanged conductance in 1 M NH+ (YNN/YGG = 1, conductance ratio) but reduced conductance in 1 M K+ (YNN/YGG = 0.6) methylammonium (YNN/YGG = 0.3), and formamidinium (YNN/YGG = 0.1) solutions. Except with formamidinium, '"licker blocks" are evident even at low cutoff frequencies. For all cations studied, channel lifetimes of N-acetyl homodimers (NN) are-50-fold shorter than those of the GA homodimer (GG). The novel properties of GA channels in formamidinium solution (supralinear current-voltage relations and dimer stabilization (Seoh and Busath, 1993)) also appear in NN channels. The average single channel lifetime in 1 M formamidinium solution at 100 mV is 6-7-fold longer than in K+ and methylammonium solutions and, like in the GA channel, significantly decreases with increasing membrane potential. Experiments with mixtures of the two peptides, GA and NAG, showed three main conductance peaks. Oriented hybrids were formed utilizing the principle that monomers remain in one leaflet of the bilayer (O'Connell et al., 1990). With GA at the polarized side and NAG at the grounded side, at positive potentials (in which case hybrids were designated GN) and at negative potentials (in which case hybrids were designated NG), channels had the same conductances and channel properties at all potentials studied. Flicker blocks were not evident in the hybrid channels, which suggests that both N-acetyl methyl groups at the junction of the dimer are required to cause flickers. Channel lifetimes in hybrids are only-threefold shorter than those of the GG channels, and channel conductances are similar to those of GG rather than NN channels. We suggest that acetyl-acetyl crowding at the dimeric junction in NN channels causes dimer destabilization, flickers, and increased selectivity in N-acetyl gramicidin channels.
Guanidinium and acetamidinium, when added to the bathing solution in concentrations of 0.1 M, cau... more Guanidinium and acetamidinium, when added to the bathing solution in concentrations of 0.1 M, cause brief blocks in the single channel potassium currents from channels formed in planar lipid bilayers by gramicidin A. Single channel lifetimes are not affected indicating that the channel structure is not modified by the blockers. Guanidinium block durations and interblock times are approximately exponential in distribution. Block frequencies increase with guanidinium concentration whereas block durations are unaffected. Increases in membrane potential cause an increase in block frequency as expected for a positively charged blocker but a decrease in block duration suggesting that the block is relieved when the blocker passes through the channel. At low pH, urea, formamide, and acetamide cause similar blocks suggesting that the protonated species of these molecules also block. Arginine and several amines do not block. This indicates that only iminium ions which are small enough to enter the channel can cause blocks in gramicidin channels.
The conductance properties of organic cations in single gramicidin A channels were studied using ... more The conductance properties of organic cations in single gramicidin A channels were studied using planar lipid bilayers. From measurements at 10 mM and at 27 mV the overall selectivity sequence was found to be NH4+> K+ > hydrazinium > formamidinium > Na+ > methylammonium, which corresponds to Eisenman polyatomic cation sequence X'. Methylammonium and formamidinium exhibit self block, suggesting multiple occupancy and single filing. Formamidinium has an apparent dissociation constant (which is similar to those of alkali metal cations) for the first ion being 22 mM from the Eadie-Hofstee plot (Go vs. Go/C), 12 mM from the rate constants of a three-step kinetic model. The rate-limiting step for formamidinium is translocation judging from supralinear I-V relations at low concentrations. 1 M formamidinium solutions yields exceptionally long single channel lifetimes, 20-fold longer than methylammonium, which yields lifetimes similar to those found with alkali metal cations. The average lifetime in formamidinium solution significantly decreases with increasing voltage up to 100 mV but is relatively voltage independent between 100 and 200 mV. At lower voltages (.100 mV), the temperature and concentration dependences of the average lifetime of formamidinium were steep. At very low salt concentrations (0.01 M, 100 mV), there was no significant difference in average lifetime from that formed with 0.01 M methylammonium or hydrazinium. We conclude that formamidinium very effectively stabilizes the dimeric channel while inside the channel and speculate that it does so by affecting tryptophan-reorientation or tryptophan-lipid interactions at binding sites.
Extent of Cysteine Modification of SNAP-25 In Vitro Alex McGregor DaBell Department of Physiology... more Extent of Cysteine Modification of SNAP-25 In Vitro Alex McGregor DaBell Department of Physiology and Developmental Biology, BYU Master of Science Exocytosis, the fusion of a vesicle to a cellular membrane, involves a protein named SNAP-25. This protein, containing two alpha helices connected with a linker region, is localized to the cell membrane via palmitic acids attached to the cysteine residues of its linker region in a process called palmitoylation. Are cysteine residues of the SNAP-25 linker region palmitoylated in an ordered manner and to a particular extent? The answer to this question may give insight into the regulated nature of exocytosis. While it is generally accepted that SNAP-25 must be palmitoylated in order to perform its exocytotic functions, the details surrounding this process are still being discovered, defined, and understood. In these studies we replicate the oxidation, reduction, and palmitoylation of SNAP-25 in vitro. Palmitoylating SNAP-25 in vitro, a proc...
ABSTRACT Some results of our simulation studies of selectivity and conductance of calcium ion cha... more ABSTRACT Some results of our simulation studies of selectivity and conductance of calcium ion channels in membranes are reported. Ion channels regulate many important physiological functions. To simulate the system we use periodic boundary conditions. We have noted previously that the use of such boundary conditions does not affect our results in any significant manner. The structure of the filter of a calcium channel is reasonably well understood. An important structural element is the set of four glutamate residues that are attached to the wall of the channel. Since the glutamates are long and flexible, Nonner and Eisenberg et al. have suggested that these glutamates can be modeled by eight half-charged oxygen ions that represent the carboxylates at the end of each residue. These oxygen ions are confined to the filter but otherwise are mobile. We have used this model in simulations of the selectivity and conductance of this model calcium channel filter. Our studies confirm that this model behaves as a calcium filter; calcium ions will drive sodium ions from the filter. In many of our simulations, explicit water molecules are used with water molecules in both the filter and bath.
The M2 protein from influenza A is a proton channel as a tetramer, with a single transmembrane he... more The M2 protein from influenza A is a proton channel as a tetramer, with a single transmembrane helix from each monomer lining the pore. Val27 and Trp41 form gates at either end of the pore and His37 mediates the shuttling of protons across a central barrier between the N-terminal and C-terminal aqueous pore regions. Numerous structures of this transmembrane domain and of a longer construct that includes an amphipathic helix are now in the Protein Data Bank. Many structural differences are apparent from samples obtained in a variety of membrane mimetic environments. High-resolution structural results in lipid bilayers have provided novel insights into the functional mechanism of the unique HxxxW cluster in the M2 proton channel.
The mechanism of action of volatile general anesthetics has not yet been resolved. In order to id... more The mechanism of action of volatile general anesthetics has not yet been resolved. In order to identify the effects of isoflurane on the membrane, we measured the steady-state anisotropy of two fluorescent probes that reside at different depths. Incorporation of anesthetic was confirmed by shifting of the main phase transition temperature. In liquid crystalline dipalmitoylphosphatidylcholine liposomes, isoflurane (7-25 mM in the bath) increases trimethylammonium-diphenylhexatriene fluorescence anisotropy by ~0.02 units and decreases diphenylhexatriene anisotropy by the same amount. The anisotropy data suggest that isoflurane decreases non-axial dye mobility in the headgroup region, while increasing it in the tail region. We propose that these results reflect changes in the lateral pressure profile of the membrane.
International Journal of Peptide and Protein Research, 2009
ABSTRACT The 13C-D-Leu12,14 gramicidin A was synthesized by the solid phase method incorporating ... more ABSTRACT The 13C-D-Leu12,14 gramicidin A was synthesized by the solid phase method incorporating 13C-D-leucine in positions 12 and 14 with about 25 and 50% enrichment, respectively. The pentadecapeptide was removed from the resin by ethanolamine treatment, with the N-protecting group (Boc) still on. After removal of the protecting group, the peptide was formylated and purified by preparative t.l.c. to obtain 13C-D-Leu12,14 gramicidin A in a very pure state in an overall yield of about 12.5%. The peptide was then thoroughly characterized by HPLC which gave one single peak with the same retention time as that of Val1-gramicidin A of the natural gramicidin mixture. The CD spectra of the synthetic and the HPLC purified natural Val1 -GA were obtained and found to be identical, indicating the optical purity of the sample. The synthetic GA was characterized by 13C n.m.r. spectrum and compared with that of natural GA. Single channel conductance parameters of the synthetic GA were determined and found to be indistinguishable from those of natural Val1-GA in lipid bilayer membranes and the mean channel lifetime was found to be as reported earlier by others.
International Journal of Peptide and Protein Research, 2009
The synthesis of (1-" C)-Phe'-gramicidin (90% enriched) was carried out by the solid phase method... more The synthesis of (1-" C)-Phe'-gramicidin (90% enriched) was carried out by the solid phase method. The peptide was removed from the resin by treatment with ethanolamine, deblocked, formylated and purified by preparative t.1.c. to obtain the gramicidin analog in an overall yield of 24%. The peptide was verified and characterized by high pressure liquid chromatography, carbon-13 nuclear magnetic resonance, circular dichroism and single channel currents. Single channel conductances were found to be similar to those of (l-'3C)-Phe11-GB but significantly lower than that of gramicidin A. When this gramicidin analog was incubated with phospholipid, the characteristic channel spectrum was not obtained and interaction with sodium ion was not observed. A possible explanation for this behavior is discussed.
M2 Out of the Envelope The M2 protein from influenza A virus forms an acid-activated tetrameric p... more M2 Out of the Envelope The M2 protein from influenza A virus forms an acid-activated tetrameric proton channel in the viral envelope and is essential for viral replication. Two manuscripts shed light on the functional mechanism of this channel. Sharma et al. (p. 509 ; see the Perspective by Fiorin et al. ) determined the structure of the conductance domain in a lipid bilayer and propose that a histidine and tryptophan from each monomer form a cluster that guides protons through the channel in a mechanism that involves forming and breaking hydrogen bonds between adjacent pairs of histidines. Hu et al. (p. 505 ; see the Perspective by Fiorin et al. ) focused on the structure and dynamics of the proton-selective histidine at high and low pH, proposing that proton conduction involves histidine deprotonation and reprotonation.
Proteins: Structure, Function, and Bioinformatics, 2009
M 2 transmembrane domain channel (M 2-TMD) permeation properties are studied using molecular dyna... more M 2 transmembrane domain channel (M 2-TMD) permeation properties are studied using molecular dynamics simulations of M 2-TMD (1NYJ) embedded in a lipid bilayer (DMPC) with 1 mol/kg NaCl or KCl saline solution. This study allows examination of spontaneous cation and anion entry into the selectivity filter. Three titration states of the M 2-TMD tetramer are modeled for which the four His37 residues, forming the selectivity filter, are net uncharged, +2 charged, or +3 charged. M 2-TMD structural properties from our simulations are compared with the properties of other models extracted from NMR and X-ray studies. During 10 ns simulations, chloride ions rarely occupy the positively-charged selectivity filter, whereas from umbrella sampling simulations, Cl − has a lower free-energy barrier in the selectivity-filter region than either Na + or , and has a lower free-energy barrier than Na +. For Na + and Cl − , the free-energy barriers are less than 5 kcal/mol, suggesting that the 1NYJ conformation would probably not be exquisitely proton selective. We also point out a rotameric configuration of Trp41 that could fully occlude the channel.
The theory of fluids near surfaces and in pores is reviewed and some simulations of electrolytes ... more The theory of fluids near surfaces and in pores is reviewed and some simulations of electrolytes with a continuum solvent near an electrode that agree qualitatively with experiment but that are inexplicable by current theories are outlined. Recent work with ...
Pfl�gers Archiv European Journal of Physiology, 1986
The capture, transport, and maintenance of live adult Atlantic coast squid (Loligo pealei) are de... more The capture, transport, and maintenance of live adult Atlantic coast squid (Loligo pealei) are described. The objective was to obtain healthy live squid from a coastal region and to maintain them at an inland research facility long enough to provide at least 4 days of electrophysiological research. An inexpensive closed aquarium system is described, which utilizes an ion-exchange resin in the filter, that allows a typical survival time of at least 4.5 days. Similar closed aquarium systems may be of interest to other biophysicists who wish to maintain live squid away from coastal research facilities.
A series of 2-adamantanamines with alkyl adducts of various lengths were examined for efficacy ag... more A series of 2-adamantanamines with alkyl adducts of various lengths were examined for efficacy against strains of influenza A including those having an S31N mutation in M2 proton channel that confer resistance to amantadine and rimantadine. The addition of as little as one CH 2 group to the methyl adduct of the amantadine/rimantadine analogue, 2-methyl-2-aminoadamantane, led to activity in vitro against two M2 S31N viruses A/Calif/07/ 2009 (H1N1) and A/PR/8/34 (H1N1) but not to a third A/WS/33 (H1N1). Solid state NMR of the transmembrane domain (TMD) with a site mutation corresponding to S31N shows evidence of drug binding. But electrophysiology using the full length S31N M2 protein in HEK cells showed no blockade. A wild type strain, A/Hong Kong/1/68 (H3N2) developed resistance to representative drugs within one passage with mutations in M2 TMD, but A/ Calif/07/2009 S31N was slow (>8 passages) to develop resistance in vitro, and the resistant virus had no mutations in M2 TMD. The results indicate that 2alkyl-2-aminoadamantane derivatives with sufficient adducts can persistently block p2009 influenza A in vitro through an alternative mechanism. The observations of an HA1 mutation, N160D, near the sialic acid binding site in both 6-resistant A/ Calif/07/2009(H1N1) and the broadly resistant A/WS/33(H1N1) and of an HA1 mutation, I325S, in the 6-resistant virus at a cell-culture stable site suggest that the drugs tested here may block infection by direct binding near these critical sites for virus entry to the host cell.
Membrane proteins are structurally characterized in membrane mimetic environments, environments t... more Membrane proteins are structurally characterized in membrane mimetic environments, environments that may or may not reflect the properties of native membrane proteins. This is critically important since the structure of membrane proteins is influenced by the protein's environment. Here we will show structural data from magic angle spinning solid state NMR of the full length M2 protein from Influenza A observed in E. coli membranes. This protein has never been exposed to the denaturing influence of detergents nor to the length isolation, purification and reconstitution protocols that typify membrane protein sample preparation for structural studies. The data will be compared to the purified full length protein in synthetic liposomes and to spectra of smaller constructs for which there is a high resolution structure in lipid bilayers.
Empirical energy function calculations were used to evaluate the effects of minimization on the s... more Empirical energy function calculations were used to evaluate the effects of minimization on the structure of a gramicidin A channel and to analyze the energies of interaction between three cations (guanidinium, acetamidinium, formamidinium) and the channel as a function of position along the channel axis. The energy minimized model of the gramicidin channel, which was based on the results of Venkatachalam and Urry (1983), has a constriction at the channel entrance. If the channel is not allowed to relax in the presence of the ions (rigid model), there is a large potential energy barrier for all three cations. The barrier varies with cation size and is due to high van der Waals and ion deformation energies. If the channel is minimized in the presence of the ions, the potential energy barrier to formamidinium entry is almost eliminated, but a residual barrier remains for guanidinium and acetamidinium. The residual barrier is primarily due, not to the expansion of the helix, but, to the disruption of hydrogen bonds between the terminal ethanoloamine and the next turn of the helix which occurs when the carbonyls of the outer turn of the helix librate inward toward the ion as it enters the channel. The residual potential energy barriers could be a possible explanation for the measured selectivity of gramicidin for formamidinium over guanidinium. The results of this full-atomic model address the applicability of the size-exclusion concept for the selectivity of the gramicidin channel.
Compared to the N-formyl gramicidin A (GA), the N-acetyl gramicidin A (NAG) channel has unchanged... more Compared to the N-formyl gramicidin A (GA), the N-acetyl gramicidin A (NAG) channel has unchanged conductance in 1 M NH+ (YNN/YGG = 1, conductance ratio) but reduced conductance in 1 M K+ (YNN/YGG = 0.6) methylammonium (YNN/YGG = 0.3), and formamidinium (YNN/YGG = 0.1) solutions. Except with formamidinium, '"licker blocks" are evident even at low cutoff frequencies. For all cations studied, channel lifetimes of N-acetyl homodimers (NN) are-50-fold shorter than those of the GA homodimer (GG). The novel properties of GA channels in formamidinium solution (supralinear current-voltage relations and dimer stabilization (Seoh and Busath, 1993)) also appear in NN channels. The average single channel lifetime in 1 M formamidinium solution at 100 mV is 6-7-fold longer than in K+ and methylammonium solutions and, like in the GA channel, significantly decreases with increasing membrane potential. Experiments with mixtures of the two peptides, GA and NAG, showed three main conductance peaks. Oriented hybrids were formed utilizing the principle that monomers remain in one leaflet of the bilayer (O'Connell et al., 1990). With GA at the polarized side and NAG at the grounded side, at positive potentials (in which case hybrids were designated GN) and at negative potentials (in which case hybrids were designated NG), channels had the same conductances and channel properties at all potentials studied. Flicker blocks were not evident in the hybrid channels, which suggests that both N-acetyl methyl groups at the junction of the dimer are required to cause flickers. Channel lifetimes in hybrids are only-threefold shorter than those of the GG channels, and channel conductances are similar to those of GG rather than NN channels. We suggest that acetyl-acetyl crowding at the dimeric junction in NN channels causes dimer destabilization, flickers, and increased selectivity in N-acetyl gramicidin channels.
Guanidinium and acetamidinium, when added to the bathing solution in concentrations of 0.1 M, cau... more Guanidinium and acetamidinium, when added to the bathing solution in concentrations of 0.1 M, cause brief blocks in the single channel potassium currents from channels formed in planar lipid bilayers by gramicidin A. Single channel lifetimes are not affected indicating that the channel structure is not modified by the blockers. Guanidinium block durations and interblock times are approximately exponential in distribution. Block frequencies increase with guanidinium concentration whereas block durations are unaffected. Increases in membrane potential cause an increase in block frequency as expected for a positively charged blocker but a decrease in block duration suggesting that the block is relieved when the blocker passes through the channel. At low pH, urea, formamide, and acetamide cause similar blocks suggesting that the protonated species of these molecules also block. Arginine and several amines do not block. This indicates that only iminium ions which are small enough to enter the channel can cause blocks in gramicidin channels.
The conductance properties of organic cations in single gramicidin A channels were studied using ... more The conductance properties of organic cations in single gramicidin A channels were studied using planar lipid bilayers. From measurements at 10 mM and at 27 mV the overall selectivity sequence was found to be NH4+> K+ > hydrazinium > formamidinium > Na+ > methylammonium, which corresponds to Eisenman polyatomic cation sequence X'. Methylammonium and formamidinium exhibit self block, suggesting multiple occupancy and single filing. Formamidinium has an apparent dissociation constant (which is similar to those of alkali metal cations) for the first ion being 22 mM from the Eadie-Hofstee plot (Go vs. Go/C), 12 mM from the rate constants of a three-step kinetic model. The rate-limiting step for formamidinium is translocation judging from supralinear I-V relations at low concentrations. 1 M formamidinium solutions yields exceptionally long single channel lifetimes, 20-fold longer than methylammonium, which yields lifetimes similar to those found with alkali metal cations. The average lifetime in formamidinium solution significantly decreases with increasing voltage up to 100 mV but is relatively voltage independent between 100 and 200 mV. At lower voltages (.100 mV), the temperature and concentration dependences of the average lifetime of formamidinium were steep. At very low salt concentrations (0.01 M, 100 mV), there was no significant difference in average lifetime from that formed with 0.01 M methylammonium or hydrazinium. We conclude that formamidinium very effectively stabilizes the dimeric channel while inside the channel and speculate that it does so by affecting tryptophan-reorientation or tryptophan-lipid interactions at binding sites.
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Papers by David Busath