conformation-dependent atomic proximity, the substitutions D121C and R972C were introduced (indiv... more conformation-dependent atomic proximity, the substitutions D121C and R972C were introduced (individually and concurrently) into the ouabain-insensitive C104Y-Xenopus-a1. Each mutant was coexpressed with Xenopus-b3 in Xenopus oocytes and their function tested in Na þ -loaded oocytes under twoelectrode voltage clamp. K þ o -activated Na/K-pump currents were observed in oocytes injected with D121C or R972C, but not in those injected with D121C-R972C, unless the eggs were exposed to TCEP (10-50 mM,~20 min), consistent with the presence of a pump-inhibiting disulfide. To identify the conformation locked by the disulfide, we used palytoxin to transform Na/K-pumps into channels. Palytoxin-induced currents (I PTX ) in outside-out patches from oocytes expressing D121C-R972C, bathed in Na þ solutions, were insensitive to MTSET þ application. Patch exposure to DTT restored MTSET þ -sensitivity (~65% I PTX reduction) without affecting I PTX amplitude. Palytoxin stabilizes an E2P-like structure; thus, the lack of DTT effect on I PTX suggest that cross-linking between D121C and R972C occurs in E2P, with the external cation pathway open. Moreover, pump inhibition by spontaneous disulfide formation indicates that conformational mobility between these residues is required for the E2 to E1 transition. The slow component of ouabain-sensitive transient charge movement in 125 mM Na o þ was measured in Na þ -loaded oocytes expressing these mutants. The center of the equilibrium distribution of charge (voltage of equal occupancy of E1 and E2) for R972C (V 1/2 ¼ -3458 mV) was identical to that of the C104Y-a1 template (V 1/2 ¼ -3553 mV). Therefore, it appears that a D121-R972 salt bridge is not necessary for E2P stabilization among Na þ -occupied states. Antimicrobial peptides (AMPs) are a promising new class of antibiotics that are believed to kill pathogens by permealizing their cell membranes. We present a model for the transient transport that takes place in a bacterial cell as a result of exposure to high concentrations of protegrin, a particularly potent AMP found in porcine leukocytes. In particular, we focus on the efflux of potassium, the decay of the transmembrane potential, and the volume changes associated with osmotic flow across the membrane, all of which are coupled phenomena. The model that we employ is based on the classic nonequilibrium thermodynamics approach for transport of solutes across permeable membranes, commonly referred to as Kedem-Katchalsky formalism. In our model, the cellular interior and the exterior bath are assumed to be well-mixed compartments, separated by a thin homogeneous membrane region. Overall mass balances on each diffusing species and an overall volume balance yield a tractable set of initial-value, ordinary differential equations; some complexities arise in the modeling of the electrostatic potential and the hydrostatic pressure differences across the membrane. The model parameters that relate to membrane properties appear as parameters in the flux expressions. This work allows us to investigate the timeline of events that follow protegrin treatment leading to cell death, as well as assessing the role of osmotic lysis as a mechanism of action for antimicrobial peptides.
One of the great challenges in pharmacokinetics is to find a means to optimize the transport acro... more One of the great challenges in pharmacokinetics is to find a means to optimize the transport across cell barriers. In this work, permeation across a cell monolayer, such as the tight endothelia in the blood-brain barrier, was modeled using a homologous series of amphipatic molecules, 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-labeled alkyl chain amphiphiles (NBD-Cn, n = 2 to 16), to obtain rules that relate permeant structure to permeability. The amphiphile enters the system from the serum, equilibrated with serum albumin and lipoproteins, and its sequestration by serum components, interaction with the endothelium, and accumulation in the tissue is followed over time. The dependence of the permeability coefficient on the number of carbons of the amphiphile's alkyl chain has a parabolic-like shape. After a threshold value, an increase in the hydrophobicity of the amphiphile, along the homologous series, results in a decrease in the characteristic rate of permeation to the tissue. A sensitivity analysis was performed, and the rate limiting steps for permeation of each amphiphile were identified. Sequestration in the serum and rate of interaction with the endothelium, particularly the rate of desorption, were found to be the determinant processes for some amphiphiles, while for others translocation was the rate limiting step. Additionally, for some amphiphiles a single rate limiting step could not be identified, with several steps contributing significantly to the overall permeation. Finally, we derived analytical equations that adequately describe the rate of amphiphile accumulation in the tissue for the cases where permeation is controlled by a single rate limiting step.
The rate of noncatalyzed transfer of cholesterol (Chol) among lipoproteins and cells in the blood... more The rate of noncatalyzed transfer of cholesterol (Chol) among lipoproteins and cells in the blood is of fundamental importance as a baseline to assess the role of active transport mechanisms, but remains unknown. Here we address this gap by characterizing the associa-tion of the Chol analog, ergosta-5,7,9(11),22-tetraen-3β-ol (DHE), with the lipoproteins VLDL, LDL, HDL2, and HDL3. Combining these results with data for the association of DHE with liposomes, we elaborated a kinetic model for the noncatalyzed exchange of free Chol among blood compartments. The computational results are in good agreement with experimental values. The small deviations are explained by the nonequilibrium distribution of unesterified Chol in vivo, due to esterification and entry of new unesterified Chol, and eventual effects introduced by incubations at low temperatures. The kinetic profile of the homeostasis of unesterified Chol in the blood predicted by the model developed in this work is in good agreeme...
One of the great challenges in membrane biophysics is to find a means to foster the transport of ... more One of the great challenges in membrane biophysics is to find a means to foster the transport of drugs across complex membrane structures. In this spirit, we elucidate methodological challenges associated with free energy computations of complex chainlike molecules across lipid membranes. As an appropriate standard molecule to this end, we consider 7-nitrobenz-2-oxa-1,3-diazol-4-yl-labeled fatty amine, NBD-Cn, which is here dealt with as a homologous series with varying chain lengths. We found the membrane-water interface region to be highly sensitive to details in free energy computations. Despite considerable simulation times, we observed substantial hysteresis, the cause being the small frequency of insertion/desorption events of the amphiphile's alkyl chain in the membrane interface. The hysteresis was most pronounced when the amphiphile was pulled from water to the membrane and compromised the data that were not in line with experiments. The subtleties in umbrella sampling for computing distance along the transition path were also observed to be potential causes of artifacts. With the PGD (pull geometry distance) scheme, in which the distance from the molecule was computed to a reference plane determined by an average over all lipids in the membrane, we found marked deformations in membrane structure when the amphiphile was close to the membrane. The deformations were weaker with the PGC (pull geometry cylinder) method, where the reference plane is chosen based on lipids that are within a cylinder of radius 1.7 nm from the amphiphile. Importantly, the free energy results given by PGC were found to be qualitatively consistent with experimental data, while the PGD results were not. We conclude that with long amphiphiles there is reason for concern with regard to computations of their free energy profiles. The membrane-water interface is the region where the greatest care is warranted.
The lipidic α-amino acid with 11 carbons in the alkyl lateral chain (α-aminotridecanoic acid) was... more The lipidic α-amino acid with 11 carbons in the alkyl lateral chain (α-aminotridecanoic acid) was synthesized via multicomponent hydroformylation/Strecker reaction, which is a greener synthetic approach to promote this transformation relative to previously described methods. Its solubility and aggregation behavior in aqueous solutions was characterized, as well as the interaction with lipid bilayers. Lipidic amino acids are very promising molecules in the development of prodrugs with increased bioavailability due to the presence of the two polar functional groups and nonpolar alkyl chain. They are also biocompatible surfactants that may be used in the food and pharmaceutical industry. In this work we have conjugated the lipidic amino acid with a fluorescent polar group (7-nitrobenz-2-oxa-1,3-diazol-4-yl), to mimic drug conjugates, and its association with serum proteins and lipid bilayers was characterized. The results obtained indicate that conjugates of polar molecules with lipidic α-amino acid, via covalent attachment to the amine group, have a relatively high solubility in aqueous solutions due to their negative global charge. They bind to serum albumin with intermediate affinity and show a very high partition coefficient into lipid bilayers in the liquid-disordered state. The attachment of the polar group to the lipidic amino acid increased strongly the aqueous solubility of the amphiphile, although the partition coefficient into lipid membranes was not significantly reduced. Conjugation of polar drugs with lipidic amino acids is therefore an efficient approach to increase their affinity for biomembranes.
The role transmembrane helices (TMH) play in biological systems includes creating ion transport p... more The role transmembrane helices (TMH) play in biological systems includes creating ion transport pathways, cell signaling and facilitating photon absorption in photosynthetic complexes. The packing and spatial organization of those helices was found to be important for their functional properties. It was shown (Walters & DeGrado, PNAS (2006); 13, 37) that despite the large number of available conformations, experimentally observed helix-helix interactions can be classified into very few interaction clusters. This suggests that a basic, universal set of interactions might govern the helix packing. Using a coarsegrained model we investigate the interaction of helical peptides in a lipid bilayer using the dissipative particle dynamics (DPD) simulation technique. We incorporate in our model basic hydrophobic-hydrophilic interactions without referencing a specific TMH, thereby studying the common motifs in lipid mediated protein interactions. Our model successfully reproduces the effect of hydrophobic mismatch on peptides in a lipid bilayer (de Meyer, Venturoli & Smit, Biophys. J (2008); 95, 4) and predicts a selective aggregation pattern. A more detailed representation of a helix further reveals the characteristics of the helix-helix interactions
The role transmembrane helices (TMH) play in biological systems includes creating ion transport p... more The role transmembrane helices (TMH) play in biological systems includes creating ion transport pathways, cell signaling and facilitating photon absorption in photosynthetic complexes. The packing and spatial organization of those helices was found to be important for their functional properties. It was shown (Walters & DeGrado, PNAS (2006); 13, 37) that despite the large number of available conformations, experimentally observed helix-helix interactions can be classified into very few interaction clusters. This suggests that a basic, universal set of interactions might govern the helix packing. Using a coarsegrained model we investigate the interaction of helical peptides in a lipid bilayer using the dissipative particle dynamics (DPD) simulation technique. We incorporate in our model basic hydrophobic-hydrophilic interactions without referencing a specific TMH, thereby studying the common motifs in lipid mediated protein interactions. Our model successfully reproduces the effect of hydrophobic mismatch on peptides in a lipid bilayer (de Meyer, Venturoli & Smit, Biophys. J (2008); 95, 4) and predicts a selective aggregation pattern. A more detailed representation of a helix further reveals the characteristics of the helix-helix interactions
conformation-dependent atomic proximity, the substitutions D121C and R972C were introduced (indiv... more conformation-dependent atomic proximity, the substitutions D121C and R972C were introduced (individually and concurrently) into the ouabain-insensitive C104Y-Xenopus-a1. Each mutant was coexpressed with Xenopus-b3 in Xenopus oocytes and their function tested in Na þ -loaded oocytes under twoelectrode voltage clamp. K þ o -activated Na/K-pump currents were observed in oocytes injected with D121C or R972C, but not in those injected with D121C-R972C, unless the eggs were exposed to TCEP (10-50 mM,~20 min), consistent with the presence of a pump-inhibiting disulfide. To identify the conformation locked by the disulfide, we used palytoxin to transform Na/K-pumps into channels. Palytoxin-induced currents (I PTX ) in outside-out patches from oocytes expressing D121C-R972C, bathed in Na þ solutions, were insensitive to MTSET þ application. Patch exposure to DTT restored MTSET þ -sensitivity (~65% I PTX reduction) without affecting I PTX amplitude. Palytoxin stabilizes an E2P-like structure; thus, the lack of DTT effect on I PTX suggest that cross-linking between D121C and R972C occurs in E2P, with the external cation pathway open. Moreover, pump inhibition by spontaneous disulfide formation indicates that conformational mobility between these residues is required for the E2 to E1 transition. The slow component of ouabain-sensitive transient charge movement in 125 mM Na o þ was measured in Na þ -loaded oocytes expressing these mutants. The center of the equilibrium distribution of charge (voltage of equal occupancy of E1 and E2) for R972C (V 1/2 ¼ -3458 mV) was identical to that of the C104Y-a1 template (V 1/2 ¼ -3553 mV). Therefore, it appears that a D121-R972 salt bridge is not necessary for E2P stabilization among Na þ -occupied states. Antimicrobial peptides (AMPs) are a promising new class of antibiotics that are believed to kill pathogens by permealizing their cell membranes. We present a model for the transient transport that takes place in a bacterial cell as a result of exposure to high concentrations of protegrin, a particularly potent AMP found in porcine leukocytes. In particular, we focus on the efflux of potassium, the decay of the transmembrane potential, and the volume changes associated with osmotic flow across the membrane, all of which are coupled phenomena. The model that we employ is based on the classic nonequilibrium thermodynamics approach for transport of solutes across permeable membranes, commonly referred to as Kedem-Katchalsky formalism. In our model, the cellular interior and the exterior bath are assumed to be well-mixed compartments, separated by a thin homogeneous membrane region. Overall mass balances on each diffusing species and an overall volume balance yield a tractable set of initial-value, ordinary differential equations; some complexities arise in the modeling of the electrostatic potential and the hydrostatic pressure differences across the membrane. The model parameters that relate to membrane properties appear as parameters in the flux expressions. This work allows us to investigate the timeline of events that follow protegrin treatment leading to cell death, as well as assessing the role of osmotic lysis as a mechanism of action for antimicrobial peptides.
conformation-dependent atomic proximity, the substitutions D121C and R972C were introduced (indiv... more conformation-dependent atomic proximity, the substitutions D121C and R972C were introduced (individually and concurrently) into the ouabain-insensitive C104Y-Xenopus-a1. Each mutant was coexpressed with Xenopus-b3 in Xenopus oocytes and their function tested in Na þ -loaded oocytes under twoelectrode voltage clamp. K þ o -activated Na/K-pump currents were observed in oocytes injected with D121C or R972C, but not in those injected with D121C-R972C, unless the eggs were exposed to TCEP (10-50 mM,~20 min), consistent with the presence of a pump-inhibiting disulfide. To identify the conformation locked by the disulfide, we used palytoxin to transform Na/K-pumps into channels. Palytoxin-induced currents (I PTX ) in outside-out patches from oocytes expressing D121C-R972C, bathed in Na þ solutions, were insensitive to MTSET þ application. Patch exposure to DTT restored MTSET þ -sensitivity (~65% I PTX reduction) without affecting I PTX amplitude. Palytoxin stabilizes an E2P-like structure; thus, the lack of DTT effect on I PTX suggest that cross-linking between D121C and R972C occurs in E2P, with the external cation pathway open. Moreover, pump inhibition by spontaneous disulfide formation indicates that conformational mobility between these residues is required for the E2 to E1 transition. The slow component of ouabain-sensitive transient charge movement in 125 mM Na o þ was measured in Na þ -loaded oocytes expressing these mutants. The center of the equilibrium distribution of charge (voltage of equal occupancy of E1 and E2) for R972C (V 1/2 ¼ -3458 mV) was identical to that of the C104Y-a1 template (V 1/2 ¼ -3553 mV). Therefore, it appears that a D121-R972 salt bridge is not necessary for E2P stabilization among Na þ -occupied states. Antimicrobial peptides (AMPs) are a promising new class of antibiotics that are believed to kill pathogens by permealizing their cell membranes. We present a model for the transient transport that takes place in a bacterial cell as a result of exposure to high concentrations of protegrin, a particularly potent AMP found in porcine leukocytes. In particular, we focus on the efflux of potassium, the decay of the transmembrane potential, and the volume changes associated with osmotic flow across the membrane, all of which are coupled phenomena. The model that we employ is based on the classic nonequilibrium thermodynamics approach for transport of solutes across permeable membranes, commonly referred to as Kedem-Katchalsky formalism. In our model, the cellular interior and the exterior bath are assumed to be well-mixed compartments, separated by a thin homogeneous membrane region. Overall mass balances on each diffusing species and an overall volume balance yield a tractable set of initial-value, ordinary differential equations; some complexities arise in the modeling of the electrostatic potential and the hydrostatic pressure differences across the membrane. The model parameters that relate to membrane properties appear as parameters in the flux expressions. This work allows us to investigate the timeline of events that follow protegrin treatment leading to cell death, as well as assessing the role of osmotic lysis as a mechanism of action for antimicrobial peptides.
One of the great challenges in pharmacokinetics is to find a means to optimize the transport acro... more One of the great challenges in pharmacokinetics is to find a means to optimize the transport across cell barriers. In this work, permeation across a cell monolayer, such as the tight endothelia in the blood-brain barrier, was modeled using a homologous series of amphipatic molecules, 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-labeled alkyl chain amphiphiles (NBD-Cn, n = 2 to 16), to obtain rules that relate permeant structure to permeability. The amphiphile enters the system from the serum, equilibrated with serum albumin and lipoproteins, and its sequestration by serum components, interaction with the endothelium, and accumulation in the tissue is followed over time. The dependence of the permeability coefficient on the number of carbons of the amphiphile's alkyl chain has a parabolic-like shape. After a threshold value, an increase in the hydrophobicity of the amphiphile, along the homologous series, results in a decrease in the characteristic rate of permeation to the tissue. A sensitivity analysis was performed, and the rate limiting steps for permeation of each amphiphile were identified. Sequestration in the serum and rate of interaction with the endothelium, particularly the rate of desorption, were found to be the determinant processes for some amphiphiles, while for others translocation was the rate limiting step. Additionally, for some amphiphiles a single rate limiting step could not be identified, with several steps contributing significantly to the overall permeation. Finally, we derived analytical equations that adequately describe the rate of amphiphile accumulation in the tissue for the cases where permeation is controlled by a single rate limiting step.
The rate of noncatalyzed transfer of cholesterol (Chol) among lipoproteins and cells in the blood... more The rate of noncatalyzed transfer of cholesterol (Chol) among lipoproteins and cells in the blood is of fundamental importance as a baseline to assess the role of active transport mechanisms, but remains unknown. Here we address this gap by characterizing the associa-tion of the Chol analog, ergosta-5,7,9(11),22-tetraen-3β-ol (DHE), with the lipoproteins VLDL, LDL, HDL2, and HDL3. Combining these results with data for the association of DHE with liposomes, we elaborated a kinetic model for the noncatalyzed exchange of free Chol among blood compartments. The computational results are in good agreement with experimental values. The small deviations are explained by the nonequilibrium distribution of unesterified Chol in vivo, due to esterification and entry of new unesterified Chol, and eventual effects introduced by incubations at low temperatures. The kinetic profile of the homeostasis of unesterified Chol in the blood predicted by the model developed in this work is in good agreeme...
One of the great challenges in membrane biophysics is to find a means to foster the transport of ... more One of the great challenges in membrane biophysics is to find a means to foster the transport of drugs across complex membrane structures. In this spirit, we elucidate methodological challenges associated with free energy computations of complex chainlike molecules across lipid membranes. As an appropriate standard molecule to this end, we consider 7-nitrobenz-2-oxa-1,3-diazol-4-yl-labeled fatty amine, NBD-Cn, which is here dealt with as a homologous series with varying chain lengths. We found the membrane-water interface region to be highly sensitive to details in free energy computations. Despite considerable simulation times, we observed substantial hysteresis, the cause being the small frequency of insertion/desorption events of the amphiphile's alkyl chain in the membrane interface. The hysteresis was most pronounced when the amphiphile was pulled from water to the membrane and compromised the data that were not in line with experiments. The subtleties in umbrella sampling for computing distance along the transition path were also observed to be potential causes of artifacts. With the PGD (pull geometry distance) scheme, in which the distance from the molecule was computed to a reference plane determined by an average over all lipids in the membrane, we found marked deformations in membrane structure when the amphiphile was close to the membrane. The deformations were weaker with the PGC (pull geometry cylinder) method, where the reference plane is chosen based on lipids that are within a cylinder of radius 1.7 nm from the amphiphile. Importantly, the free energy results given by PGC were found to be qualitatively consistent with experimental data, while the PGD results were not. We conclude that with long amphiphiles there is reason for concern with regard to computations of their free energy profiles. The membrane-water interface is the region where the greatest care is warranted.
The lipidic α-amino acid with 11 carbons in the alkyl lateral chain (α-aminotridecanoic acid) was... more The lipidic α-amino acid with 11 carbons in the alkyl lateral chain (α-aminotridecanoic acid) was synthesized via multicomponent hydroformylation/Strecker reaction, which is a greener synthetic approach to promote this transformation relative to previously described methods. Its solubility and aggregation behavior in aqueous solutions was characterized, as well as the interaction with lipid bilayers. Lipidic amino acids are very promising molecules in the development of prodrugs with increased bioavailability due to the presence of the two polar functional groups and nonpolar alkyl chain. They are also biocompatible surfactants that may be used in the food and pharmaceutical industry. In this work we have conjugated the lipidic amino acid with a fluorescent polar group (7-nitrobenz-2-oxa-1,3-diazol-4-yl), to mimic drug conjugates, and its association with serum proteins and lipid bilayers was characterized. The results obtained indicate that conjugates of polar molecules with lipidic α-amino acid, via covalent attachment to the amine group, have a relatively high solubility in aqueous solutions due to their negative global charge. They bind to serum albumin with intermediate affinity and show a very high partition coefficient into lipid bilayers in the liquid-disordered state. The attachment of the polar group to the lipidic amino acid increased strongly the aqueous solubility of the amphiphile, although the partition coefficient into lipid membranes was not significantly reduced. Conjugation of polar drugs with lipidic amino acids is therefore an efficient approach to increase their affinity for biomembranes.
The role transmembrane helices (TMH) play in biological systems includes creating ion transport p... more The role transmembrane helices (TMH) play in biological systems includes creating ion transport pathways, cell signaling and facilitating photon absorption in photosynthetic complexes. The packing and spatial organization of those helices was found to be important for their functional properties. It was shown (Walters & DeGrado, PNAS (2006); 13, 37) that despite the large number of available conformations, experimentally observed helix-helix interactions can be classified into very few interaction clusters. This suggests that a basic, universal set of interactions might govern the helix packing. Using a coarsegrained model we investigate the interaction of helical peptides in a lipid bilayer using the dissipative particle dynamics (DPD) simulation technique. We incorporate in our model basic hydrophobic-hydrophilic interactions without referencing a specific TMH, thereby studying the common motifs in lipid mediated protein interactions. Our model successfully reproduces the effect of hydrophobic mismatch on peptides in a lipid bilayer (de Meyer, Venturoli & Smit, Biophys. J (2008); 95, 4) and predicts a selective aggregation pattern. A more detailed representation of a helix further reveals the characteristics of the helix-helix interactions
The role transmembrane helices (TMH) play in biological systems includes creating ion transport p... more The role transmembrane helices (TMH) play in biological systems includes creating ion transport pathways, cell signaling and facilitating photon absorption in photosynthetic complexes. The packing and spatial organization of those helices was found to be important for their functional properties. It was shown (Walters & DeGrado, PNAS (2006); 13, 37) that despite the large number of available conformations, experimentally observed helix-helix interactions can be classified into very few interaction clusters. This suggests that a basic, universal set of interactions might govern the helix packing. Using a coarsegrained model we investigate the interaction of helical peptides in a lipid bilayer using the dissipative particle dynamics (DPD) simulation technique. We incorporate in our model basic hydrophobic-hydrophilic interactions without referencing a specific TMH, thereby studying the common motifs in lipid mediated protein interactions. Our model successfully reproduces the effect of hydrophobic mismatch on peptides in a lipid bilayer (de Meyer, Venturoli & Smit, Biophys. J (2008); 95, 4) and predicts a selective aggregation pattern. A more detailed representation of a helix further reveals the characteristics of the helix-helix interactions
conformation-dependent atomic proximity, the substitutions D121C and R972C were introduced (indiv... more conformation-dependent atomic proximity, the substitutions D121C and R972C were introduced (individually and concurrently) into the ouabain-insensitive C104Y-Xenopus-a1. Each mutant was coexpressed with Xenopus-b3 in Xenopus oocytes and their function tested in Na þ -loaded oocytes under twoelectrode voltage clamp. K þ o -activated Na/K-pump currents were observed in oocytes injected with D121C or R972C, but not in those injected with D121C-R972C, unless the eggs were exposed to TCEP (10-50 mM,~20 min), consistent with the presence of a pump-inhibiting disulfide. To identify the conformation locked by the disulfide, we used palytoxin to transform Na/K-pumps into channels. Palytoxin-induced currents (I PTX ) in outside-out patches from oocytes expressing D121C-R972C, bathed in Na þ solutions, were insensitive to MTSET þ application. Patch exposure to DTT restored MTSET þ -sensitivity (~65% I PTX reduction) without affecting I PTX amplitude. Palytoxin stabilizes an E2P-like structure; thus, the lack of DTT effect on I PTX suggest that cross-linking between D121C and R972C occurs in E2P, with the external cation pathway open. Moreover, pump inhibition by spontaneous disulfide formation indicates that conformational mobility between these residues is required for the E2 to E1 transition. The slow component of ouabain-sensitive transient charge movement in 125 mM Na o þ was measured in Na þ -loaded oocytes expressing these mutants. The center of the equilibrium distribution of charge (voltage of equal occupancy of E1 and E2) for R972C (V 1/2 ¼ -3458 mV) was identical to that of the C104Y-a1 template (V 1/2 ¼ -3553 mV). Therefore, it appears that a D121-R972 salt bridge is not necessary for E2P stabilization among Na þ -occupied states. Antimicrobial peptides (AMPs) are a promising new class of antibiotics that are believed to kill pathogens by permealizing their cell membranes. We present a model for the transient transport that takes place in a bacterial cell as a result of exposure to high concentrations of protegrin, a particularly potent AMP found in porcine leukocytes. In particular, we focus on the efflux of potassium, the decay of the transmembrane potential, and the volume changes associated with osmotic flow across the membrane, all of which are coupled phenomena. The model that we employ is based on the classic nonequilibrium thermodynamics approach for transport of solutes across permeable membranes, commonly referred to as Kedem-Katchalsky formalism. In our model, the cellular interior and the exterior bath are assumed to be well-mixed compartments, separated by a thin homogeneous membrane region. Overall mass balances on each diffusing species and an overall volume balance yield a tractable set of initial-value, ordinary differential equations; some complexities arise in the modeling of the electrostatic potential and the hydrostatic pressure differences across the membrane. The model parameters that relate to membrane properties appear as parameters in the flux expressions. This work allows us to investigate the timeline of events that follow protegrin treatment leading to cell death, as well as assessing the role of osmotic lysis as a mechanism of action for antimicrobial peptides.
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Papers by Hugo Filipe