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1.
Nat Commun ; 15(1): 7281, 2024 Aug 23.
Article in English | MEDLINE | ID: mdl-39179607

ABSTRACT

Membrane active peptides are known to porate lipid bilayers, but their exact permeabilization mechanism and the structure of the nanoaggregates they form in membranes have often been difficult to determine experimentally. For many sequences at lower peptide concentrations, transient leakage is observed in experiments, suggesting the existence of transient pores. For two well-know peptides, alamethicin and melittin, we show here that molecular mechanics simulations i) can directly distinguish equilibrium poration and non-equilibrium transient leakage processes, and ii) can be used to observe the detailed pore structures and mechanism of permeabilization in both cases. Our results are in very high agreement with numerous experimental evidence for these two peptides. This suggests that molecular simulations can capture key membrane poration phenomena directly and in the future may develop to be a useful tool that can assist experimental peptide design.


Subject(s)
Lipid Bilayers , Melitten , Molecular Dynamics Simulation , Melitten/chemistry , Melitten/metabolism , Lipid Bilayers/metabolism , Lipid Bilayers/chemistry , Alamethicin/chemistry , Alamethicin/metabolism , Cell Membrane Permeability , Permeability
2.
Nanoscale ; 16(34): 15984-15994, 2024 Aug 29.
Article in English | MEDLINE | ID: mdl-39141323

ABSTRACT

The presence of non-essential metals in the environment as contaminants is prone to cause hazardous health problems following accumulation in the human body and the ensuing toxic effects. This calls for continuous discovery and innovation in the realm of developing easy-to-operate, cheap and sensitive sensors. Herein, we describe the proof of concept approach for designing a molecular receptor-like, chimeric sensor based on the pore-forming peptide alamethicin (Alm), tethered via a linker with an ultrashort peptide nucleic acid (PNA) moiety, capable of generating functional ion channel oligomers in planar lipid membranes. The working principle of the sensor exploits the ability of Hg2+ ions to complex mismatching thymine-thymine sequences between the PNA receptor moiety on Alm oligomers and free, thymine-based, single-stranded DNAs (ssDNAs) in solution, thus creating a stable base pair at the oligomer entrance. This generates a transducing mechanism which converts the metal ion complexation into a specific electrical signature of the self-assembled Alm oligomers, enabling selective Hg2+ ion detection. The platform is programmable, whereby the simple exchange of the PNA sequence and its ssDNA counterpart in solution rendered the system selective for Cu2+ ion detection. With further optimization, the presented solution has the potential to translate into miniaturized, cost-effective biosensors suitable for the real-time, label-free and continuous detection of metal ions or other biomolecules.


Subject(s)
Copper , DNA, Single-Stranded , Mercury , Peptide Nucleic Acids , Mercury/analysis , Mercury/chemistry , Peptide Nucleic Acids/chemistry , Copper/chemistry , DNA, Single-Stranded/chemistry , Alamethicin/chemistry , Ion Channels/metabolism , Ion Channels/chemistry , Biosensing Techniques , Antimicrobial Peptides/chemistry , Antimicrobial Peptides/pharmacology , Ions/chemistry
3.
Eur Biophys J ; 53(5-6): 267-276, 2024 Aug.
Article in English | MEDLINE | ID: mdl-38849514

ABSTRACT

Alamethicin, a peptide consisted of 20 amino acid residues, has been known to function as an antibiotic. The peptides self-associate in biological membranes, form an ion channel, and then induce cell death by leaking intracellular contents through a transmembrane pore of an ion channel. We investigated conformation and its thermal stability of alamethicin-A6 and -U6 in ethanol using proton nuclear magnetic resonance (NMR) spectroscopy; alamethicin-A6 and -U6 have the amino acid sequences of UPUAUAQUVUGLUPVUUQQO and UPUAUUQUVUGLUPVUUQQO, respectively, where U and O represent α-aminoisobutyric acid and phenylalaninol, respectively. As indicated by the under bars in the sequences, only the residue 6 differs between the alamethicins. We show that the alamethicins in ethanol form helix conformation in the region of the residues 2-11 and a non-regular conformation in the regions of the N- and C-termini, and that the helices are maintained up to 66 °C at least. Conformations in the region of the residues 12-18 of the alamethicins, however, are not well identified due to the lack of NMR data. In addition, we demonstrate that the amide proton chemical shift temperature coefficients' method, which is known as an indicator for intramolecular hydrogen bonds in peptides and proteins in aqueous solutions, can be also applied to the alamethicins in ethanol. Further, we show that the conformation around the C-terminus of alamethicin-A6 is restrained by intramolecular hydrogen bonds, whereas that of alamethicin-U6 is either restrained or unrestrained by intramolecular hydrogen bonds; the alamethicin-U6 molecules having the restrained and unrestrained conformations coexist in ethanol. We discuss the two types of conformations using a model chain consisting of particles linked by rigid bonds called as the free jointed chain.


Subject(s)
Alamethicin , Ethanol , Magnetic Resonance Spectroscopy , Alamethicin/chemistry , Ethanol/chemistry , Protein Conformation , Amino Acid Sequence , Temperature
4.
Biophys J ; 122(12): 2531-2543, 2023 06 20.
Article in English | MEDLINE | ID: mdl-37161094

ABSTRACT

Transmembrane assemblies of the peptaibol alamethicin (ALM) are among the most extensively studied ion channels not only because of their antimicrobial activity but also as models for channel structure and aggregation. In this study, several oligomeric states of ALM are investigated with molecular dynamics simulations to establish properties of the channel and obtain free energy profiles for ion transport and the corresponding values of conductance. The hexamer, heptamer, and octamer of ALM in phospholipid membrane are found to be stable but highly dynamic in barrel-stave structures, with calculated conductance equal to 18, 195, and 1270 pS, respectively, in 1 M KCl ion solution. The corresponding free energy profiles, reported for the first time, are reconstructed from simulations at applied voltage of 200 mV with the aid of the electrodiffusion model both with and without the knowledge of diffusivity. The calculated free energy barriers are equal to 2.5, 1.5, and 0.5 kcal/mol for K+ and 4.0, 2.2, and 1.5 kcal/mol for Cl-, for hexamer, heptamer, and octamer, respectively. The calculated conductance and the ratio between conductance in consecutive states are in good agreement with those measured experimentally. This suggests that the hexamer is the lowest conducting state, with measured conductance equal to 19 pS. The selectivity of K+ over Cl- is calculated as 1.5 and 2.3 for the octameric and heptameric channels, close to the selectivity measured for high-conductance states. Selectivity increases to 13 in the hexameric channel in which the narrowest Gln7 site has a pore radius of only ∼1.6 Å, again in accord with experiment. A good agreement found between calculated and measured conductance through a hexamer templated on cyclodextrin lands additional support for the results of our simulations, and the comparison with ALM reveals the dependence of conductance on the nature of phospholipid membrane.


Subject(s)
Alamethicin , Ion Channels , Alamethicin/chemistry , Molecular Dynamics Simulation , Peptaibols , Phospholipids
5.
J Pept Sci ; 29(8): e3479, 2023 Aug.
Article in English | MEDLINE | ID: mdl-36652104

ABSTRACT

Peptaibols are naturally occurring, antimicrobial peptides endowed with well-defined helical conformations and resistance to proteolysis. Both features stem from the presence in their sequence of several, Cα -tetrasubstituted, α-aminoisobutyric acid (Aib) residues. Peptaibols interact with biological membranes, usually causing their leakage. All of the peptaibol-membrane interaction mechanisms proposed so far begin with peptide aggregation or accumulation. The long-length alamethicin, the most studied peptaibol, acts by forming pores in the membranes. Conversely, the carpet mechanism has been claimed for short-length peptaibols, such as trichogin. The mechanism of medium-length peptaibols is far less studied, and this is partly due to the difficulties of their synthesis. They are believed to perturb membrane permeability in different ways, depending on the membrane properties. The present work focuses on pentadecaibin, a recently discovered, medium-length peptaibol. In contrast to the majority of its family members, its sequence does not comprise hydroxyprolines or prolines, and its helix is not kinked. A reliable and effective synthesis procedure is described that allowed us to produce also two shorter analogs. By a combination of techniques, we were able to establish a 3D-structure-activity relationship. In particular, the membrane activity of pentadecaibin heavily depends on the presence of three consecutive Aib residues that are responsible for the clear, albeit modest, amphiphilic character of its helix. The shortest analog, devoid of two of these three Aib residues, preserves a well-defined helical conformation, but not its amphipathicity, and loses almost completely the ability to cause membrane leakage. We conclude that pentadecaibin amphiphilicity is probably needed for the peptide ability to perturb model membranes.


Subject(s)
Alamethicin , Peptaibols , Peptaibols/analysis , Peptaibols/chemistry , Peptaibols/metabolism , Alamethicin/analysis , Alamethicin/chemistry , Alamethicin/metabolism , Cell Membrane/chemistry , Molecular Conformation , Biological Transport , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemistry
6.
Phys Chem Chem Phys ; 25(5): 3752-3757, 2023 Feb 01.
Article in English | MEDLINE | ID: mdl-36644888

ABSTRACT

The linear 15-mer peptide gramicidin A (gA) produced by Bacillus brevis is known to form the simplest natural ion channel in lipid membranes representing a head-to-head transmembrane dimer. Its incorporation into a planar lipid bilayer manifests itself in regular electrical current transitions. If two gA subunits are tightly connected by a water-soluble, flexible linker of a certain length, the current transitions become heterogeneous: in a part of them, the amplitude is almost twofold higher than that of a single channel, thereby demonstrating the synchronous opening of two single channels. The lifetime, i.e. the open-state duration, of this dual channel is by several orders of magnitude longer than that of the single channel. Here, we used the ideas of the theory of excitons to hypothesize about the mechanism of synchronous opening and closing of two adjacent channels. Two independent (uncoupled) single channels can be described by two independent conformational coordinates q1 and q2, while two closely located channels can exhibit collective behavior, if the coupling between them produces mixing of the individual states (q1,0) and (0,q2). We suppose that a similar phenomenon can occur not only with synthetic derivatives of gA, but also with such natural channel-forming peptide antibiotics and toxins as alamethicin and syringomycin. In particular, channel clustering observed with these peptides may be also associated with formation of collective conductance states, resulting from mixing of their monomeric states, which allows us to explain the fact that clusters of these channels transmit ions and nonelectrolytes of the same size as the original single channels.


Subject(s)
Gramicidin , Ion Channels , Gramicidin/chemistry , Ion Channels/chemistry , Alamethicin/metabolism , Molecular Conformation , Lipid Bilayers/chemistry
7.
J Phys Chem B ; 126(50): 10712-10720, 2022 12 22.
Article in English | MEDLINE | ID: mdl-36440848

ABSTRACT

We report total internal reflection (TIR)-Raman spectroscopy to study intermolecular interactions between membrane-binding peptides and lipid bilayer membranes. The method was applied to alamethicin (ALM), a model peptide for channel proteins, interacting with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayer membranes at a silica/water interface. After a dimethyl sulfoxide (DMSO) solution of ALM was added into the water subphase of the DPPC/DPPC bilayer, Raman signals in the CH stretching region increased in intensity reflecting the appearance of the Raman bands due to ALM and DMSO. To identify ALM-dependent spectral changes, we removed DPPC and DMSO contributions from the Raman spectra. We first subtracted the spectrum of the DPPC bilayer from those after the addition of the ALM solution. The contribution of DMSO was then removed by subtracting a DMSO spectrum from the resultant spectra. The DMSO spectrum was obtained in a similar way from a control experiment where DMSO alone was added into the subphase. With the use of this double difference approach, the ALM-dependent changes were successfully obtained. Experiments with DPPC bilayers with deuterated acyl chains revealed that most of the spectral change observed after the addition of ALM was due to the vibrational bands of ALM, not originated from ALM-induced conformational changes of the lipid bilayers.


Subject(s)
Lipid Bilayers , Water , Lipid Bilayers/chemistry , Water/chemistry , Dimethyl Sulfoxide , Peptides , Peptaibols , 1,2-Dipalmitoylphosphatidylcholine/chemistry , Alamethicin
8.
Biochim Biophys Acta Biomembr ; 1864(10): 184001, 2022 10 01.
Article in English | MEDLINE | ID: mdl-35817122

ABSTRACT

Natural liquid crystalline membranes are made up of many different lipids carrying a mixture of saturated and unsaturated fatty acyl chains. Whereas in the past considerable attention has been paid to cholesterol content, the phospholipid head groups and the membrane surface charge the detailed fatty acyl composition was often considered less important. However, recent investigations indicate that the detailed fatty acyl chain composition has pronounced effects on the oligomerization of the transmembrane helical anchoring domains of the MHC II receptor or the membrane alignment of the cationic antimicrobial peptide PGLa. In contrast the antimicrobial peptides magainin 2 and alamethicin are less susceptible to lipid saturation. Using histidine-rich LAH4 designer peptides the high energetic contributions of lipid saturation in stabilizing transmembrane helical alignments are quantitatively evaluated. These observations can have important implications for the biological regulation of membrane proteins and should be taken into considerations during biophysical or structural experiments.


Subject(s)
Alamethicin , Lipid Bilayers , Biophysics , Lipid Bilayers/chemistry , Magainins/chemistry , Phospholipids
9.
Biochim Biophys Acta Biomembr ; 1864(10): 183997, 2022 10 01.
Article in English | MEDLINE | ID: mdl-35718208

ABSTRACT

Hybrid membranes built from phospholipids and amphiphilic block copolymers seek to capitalize on the benefits of both constituents for constructing biomimetic interfaces with improved performance. However, hybrid membranes have not been formed or studied using the droplet interface bilayer (DIB) method, an approach that offers advantages for revealing nanoscale changes in membrane structure and mechanics and offers a path toward assembling higher-order tissues. We report on hybrid droplet interface bilayers (hDIBs) formed in hexadecane from binary mixtures of synthetic diphytanoyl phosphatidylcholine (DPhPC) lipids and low molecular weight 1,2 polybutadiene-b-polyethylene oxide (PBPEO) amphiphilic block copolymers and use electrophysiology measurements and imaging to assess the effects of PBPEO in the membrane. This work reveals that hDIBs containing up to 15 mol% PBPEO plus DPhPC are homogeneously mixtures of lipids and polymers, remain highly resistive to ion transport, and are stable-including under applied voltage. Moreover, they exhibit hydrophobic thicknesses similar to DPhPC-only bilayers, but also have significantly lower values of membrane tension. These characteristics coincide with reduced energy of adhesion between droplets and the formation of alamethicin ion channels at significantly lower threshold voltages, demonstrating that even moderate amounts of amphiphilic block copolymers in a lipid bilayer provide a route for tuning the physical properties of a biomimetic membrane.


Subject(s)
Phosphatidylcholines , Phospholipids , Alamethicin , Lipid Bilayers/chemistry , Phosphatidylcholines/chemistry , Phospholipids/chemistry
10.
Langmuir ; 38(27): 8398-8406, 2022 07 12.
Article in English | MEDLINE | ID: mdl-35749587

ABSTRACT

The inhibition effect of amiloride on alamethicin ion channels was studied in a model zwitterionic floating bilayer lipid membrane (fBLM). The EIS studies indicated that amiloride prevents the transport of ions through the alamethicin channels leading to an overall increase in membrane resistance. The PM-IRRAS data demonstrated that amiloride has no influence on the secondary structure of alamethicin but restricts the insertion of the peptides into the bilayer and blocks ion transport through preformed alamethicin channels. The effect of amiloride on ion channel formation in the floating bilayer formed by a zwitterionic lipid was compared to those of previous studies involving negatively charged fBLMs and tethered zwitterionic lipid bilayers. The findings from these studies show that the effects of amiloride on ion channel formation strongly depend on the mobility and charge of the membrane lipids.


Subject(s)
Alamethicin , Amiloride , Alamethicin/chemistry , Alamethicin/pharmacology , Amiloride/pharmacology , Ion Channels/chemistry , Ions , Lipid Bilayers/chemistry , Phospholipids
11.
Xenobiotica ; 52(12): 1011-1019, 2022 Dec.
Article in English | MEDLINE | ID: mdl-36594659

ABSTRACT

Uridine diphosphate glucuronosyltransferase (UGT) enzymes conjugate many lipophilic chemicals, such as drugs, environmental contaminants, and endogenous compounds, promoting their excretion. The complexity of UGT kinetics, and the location of enzyme active site in endoplasmic reticulum lumen, requires an accurate optimisation of enzyme assays.In the present study, we characterised UGT activity in liver microsomes of green turtles (Chelonia mydas), an endangered species. The conditions for measuring UGT activity were standardised through spectrofluorimetric methods, using the substrates 4-methylumbelliferone (4-MU) and uridine diphosphate glucuronic acid (UDPGA) at 30 °C and pH 7.4.The green turtles showed UGT activity at the saturating concentrations of substrates of 250 µM to 4-MU and 7 mM to UDPGA. The alamethicin, Brij®58, bovine serum albumin (BSA), and magnesium increased UGT activity. The assay using alamethicin (22 µg per mg of protein), magnesium (1 mM), and BSA (0.25%) reached the highest Vmax (1203 pmol·min-1mg·protein-1). Lithocholic acid and diclofenac inhibited UGT activity in green turtles.This study is the first report of UGT activity in the liver of green turtles and provides a base for future studies to understand the mechanisms of toxicity by exposure to contaminants in this charismatic species.


Subject(s)
Turtles , Uridine Diphosphate Glucuronic Acid , Animals , Uridine Diphosphate Glucuronic Acid/metabolism , Turtles/metabolism , Magnesium , Uridine Diphosphate , Glucuronosyltransferase/metabolism , Microsomes, Liver/metabolism , Alamethicin/pharmacology
12.
Methods Mol Biol ; 2363: 77-84, 2022.
Article in English | MEDLINE | ID: mdl-34545487

ABSTRACT

We here describe measurements of respiratory enzymes in situ, which can be done on very small cell samples and make mitochondrial isolation unnecessary. The method is based on the ability of the fungal peptide alamethicin to permeate biological membranes from the net positively charged side, and form nonspecific ion channels. These channels allow rapid transport of substrates and products across the plasma membrane, the inner mitochondrial membrane, and the inner plastid envelope. In this way, mitochondrial enzyme activities can be studied without disrupting the cells. The enzymes can be investigated in their natural proteinaceous environment and the activity of enzymes, also those sensitive to detergents or to dilution, can be quantified on a whole cell basis. We here present protocols for in situ measurement of two mitochondrial enzymatic activities: malate oxidation measured as oxygen consumption by the electron transport chain, which is sensitive to detergents, and NAD+-isocitrate dehydrogenase, a tricarboxylic acid cycle enzyme that dissociates upon dilution.


Subject(s)
Alamethicin/metabolism , Alamethicin/pharmacology , Detergents/metabolism , Mitochondria/metabolism , Mitochondrial Membranes , Oxidation-Reduction , Oxygen Consumption
13.
Xenobiotica ; 51(11): 1247-1254, 2021 Nov.
Article in English | MEDLINE | ID: mdl-34727004

ABSTRACT

Adenine-related compounds are allosteric inhibitors of UDP-glucuronosyltransferase (UGT) in rat liver microsomes (RLM) and human UGT isoforms treated with detergent or pore-forming peptide, alamethicin.To clarify whether the same is true beyond species, the effects of adenine-related compounds on 4-methylumbelliferone (4-MU) glucuronidation were examined using detergent-treated mouse liver microsomes (MLM).Brij-58 treatment of MLM increased the Vmax and the Michaelis constant, Km, of 4-MU. This study was performed using Brij-58-treated MLM as an enzyme source. ATP- and ADP-inhibited 4-MU glucuronidation. In contrast, AMP caused a 1.5-fold increase in glucuronidation. Oxidised forms, NAD+ and NADP+, potently inhibited 4-MU glucuronidation, whereas the reduced forms, NADH and NADPH, did not. Furthermore, the IC50 values of ATP, ADP, NAD+, and NADP+ were approximately 15 µM.In our previous study, ATP was the strongest inhibitor of UGT activity in RLM. However, in this study, the above-mentioned compounds inhibited 4-MU UGT in a comparable and non-competitive manner. Furthermore, AMP antagonised the inhibitory effects of ATP and ADP.These results suggest that ATP, ADP, NAD+, and NADP+ are common endogenous inhibitors of UGT beyond species.


Subject(s)
Adenine , Microsomes, Liver , Adenine/pharmacology , Alamethicin , Animals , Glucuronides , Glucuronosyltransferase , Mice , Microsomes , Rats , Uridine Diphosphate
14.
Soft Matter ; 17(23): 5671-5681, 2021 Jun 16.
Article in English | MEDLINE | ID: mdl-33942045

ABSTRACT

Biological membranes are composed of complex mixtures of lipids and proteins that influence each other's structure and function. The biological activities of many channel-forming peptides and proteins are known to depend on the material properties of the surrounding lipid bilayer. However, less is known about how membrane-spanning channels affect the lipid bilayer properties, and in particular, their collective fluctuation dynamics. Here we use neutron spin echo spectroscopy (NSE) to measure the collective bending and thickness fluctuation dynamics in dimyristoylphosphatidylcholine (di 14 : 0 PC, DMPC) lipid membranes containing two different antimicrobial peptides, alamethicin (Ala) and gramicidin (gD). Ala and gD are both well-studied antimicrobial peptides that form oligomeric membrane-spanning channels with different structures. At low concentrations, the peptides did not have a measurable effect on the average bilayer structure, yet significantly changed the collective membrane dynamics. Despite both peptides forming transmembrane channels, they had opposite effects on the relaxation time of the collective bending fluctuations and associated effective bending modulus, where gD addition stiffened the membrane while Ala addition softened the membrane. Meanwhile, the lowest gD concentrations enhanced the collective thickness fluctuation dynamics, while the higher gD concentrations and all studied Ala concentrations dampened these dynamics. The results highlight the synergy between lipids and proteins in determining the collective membrane dynamics and that not all peptides can be universally treated as rigid bodies when considering their effects on the lipid bilayer fluctuations.


Subject(s)
Alamethicin , Dimyristoylphosphatidylcholine , Cell Membrane , Gramicidin , Lipid Bilayers
15.
Acc Chem Res ; 54(8): 1878-1890, 2021 04 20.
Article in English | MEDLINE | ID: mdl-33750106

ABSTRACT

The rise of multidrug resistant bacteria has significantly compromised our supply of antibiotics and poses an alarming medical and economic threat to society. To combat this problem, it is imperative that new antibiotics and treatment modalities be developed, especially those toward which bacteria are less capable of developing resistance. Peptide natural products stand as promising candidates to meet this need as bacterial resistance is typically slow in response to their unique modes of action. They also have additional benefits including favorable modulation of host immune responses and often possess broad-spectrum activity against notoriously treatment resistant bacterial biofilms. Moreover, nature has provided a wealth of peptide-based natural products from a range of sources, including bacteria and fungi, which can be hijacked in order to combat more dangerous clinically relevant infections.This Account highlights recent advances in the total synthesis and development of a range of peptide-based natural product antibiotics and details the medicinal chemistry approaches used to optimize their activity.In the context of antibiotics with potential to treat Gram-positive bacterial infections, this Account covers the synthesis and optimization of the natural products daptomycin, glycocin F, and alamethicin. In particular, the reported synthesis of daptomycin highlights the utility of on-resin ozonolysis for accessing a key kynurenine residue from the canonical amino acid tryptophan. Furthermore, the investigation into glycocin F analogues uncovered a potent lead compound against Lactobacillus plantarum that bears a non-native thioacetal linkage to a N-acetyl-d-glucosamine (GlcNAc) sugar, which is otherwise O-linked in its native form.For mycobacterial infections, this Account covers the synthesis and optimization of teixobactin, callyaerin A, lassomycin, and trichoderin A. The synthesis of callyaerin A, in particular, highlighted the importance of a (Z)-2,3-diaminoacrylamide motif for antimicrobial activity against Mycobacterium tuberculosis, while the synthesis of trichoderin A highlighted the importance of (R)-stereoconfiguration in a key 2-amino-6-hydroxy-4-methyl-8-oxodecanoic acid (AHMOD) residue.Lastly, this Account covers lipopeptide antibiotics bearing activity toward Gram-negative bacterial infections, namely, battacin and paenipeptin C. In both cases, optimization of the N-terminal lipid tails led to the identification of analogues with potent activity toward Escherichia coli and Pseudomonas aeruginosa.


Subject(s)
Anti-Bacterial Agents/chemical synthesis , Peptides/chemical synthesis , Alamethicin/chemical synthesis , Alamethicin/pharmacology , Amino Acid Sequence , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Bacteriocins/chemical synthesis , Bacteriocins/pharmacology , Daptomycin/chemical synthesis , Daptomycin/pharmacology , Drug Evaluation, Preclinical , Drug Resistance, Multiple, Bacterial/drug effects , Gram-Positive Bacteria/drug effects , Lipopeptides/chemical synthesis , Lipopeptides/pharmacology , Microbial Sensitivity Tests , Ozone/chemistry , Peptides/chemistry , Peptides/pharmacology , Structure-Activity Relationship
16.
J Phys Chem Lett ; 12(7): 1786-1792, 2021 Feb 25.
Article in English | MEDLINE | ID: mdl-33576633

ABSTRACT

The structures of many membrane-bound proteins and polypeptides depend on the membrane potential. However, spectroscopically studying their structures under an applied field is challenging, because a potential is difficult to generate across more than a few bilayers. We study the voltage-dependent structures of the membrane-bound polypeptide, alamethicin, using a spectroelectrochemical cell coated with a rough, gold film to create surface plasmons. The plasmons sufficiently enhance the 2D IR signal to measure a single bilayer. The film is also thick enough to conduct current and thereby apply a potential. The 2D IR spectra resolve features from both 310- and α-helical structures and cross-peaks connecting the two. We observe changes in the peak intensity, not their frequencies, upon applying a voltage. A similar change occurs with pH, which is known to alter the angle of alamethicin relative to the surface normal. The spectra are modeled using a vibrational exciton Hamiltonian, and the voltage-dependent spectra are consistent with a change in angle of the 310- and α-helices in the membrane from 55 to 44°and from 31 to 60°, respectively. The 310- and α-helices are coupled by approximately 10 cm-1. These experiments provide new structural information about alamethicin under a potential difference and demonstrate a technique that might be applied to voltage-gated membrane proteins and compared to molecular dynamics structures.


Subject(s)
Alamethicin/chemistry , Biomedical Enhancement/methods , Membrane Proteins/chemistry , Amino Acid Sequence , Lipid Bilayers/chemistry , Membrane Potentials , Molecular Dynamics Simulation , Protein Conformation , Spectrophotometry, Infrared , Surface Properties , Vibration
17.
J Phys Chem B ; 124(25): 5095-5102, 2020 06 25.
Article in English | MEDLINE | ID: mdl-32428410

ABSTRACT

Macromolecular crowding is known to modulate chemical equilibria, reaction rates, and molecular binding events, both in aqueous solutions and at lipid bilayer membranes, natural barriers that enclose the crowded environments of cells and their subcellular compartments. Previous studies on the effects of macromolecular crowding in aqueous compartments on conduction through membranes have focused on single-channel ionic conduction through previously formed pores at thermodynamic equilibrium. Here, the effects of macromolecular crowding on the mechanism of pore formation itself were studied using the droplet interface bilayer (DIB) technique with the voltage-dependent pore-forming peptide alamethicin (alm). Macromolecular crowding was varied using 8 kDa molecular weight polyethylene glycol (PEG8k) or 500 kDa dextran (DEX500k) in two aqueous droplets on both sides of the bilayer membrane. In general, voltage thresholds for pore formation in the presence of crowders in the droplets decreased compared to their values in the absence of crowders, due to excluded volume effects, water binding by PEG, and changes in the ordering of water molecules and hydrogen-bonding interactions involving the polar lipid headgroups. In addition, asymmetric crowder loading (e.g., PEG8k-DEX500k on either side of the membrane) resulted in transmembrane osmotic pressure gradients that either enhanced or degraded the ionic conduction through the pores.


Subject(s)
Alamethicin , Lipid Bilayers , Hydrogen Bonding , Macromolecular Substances , Membranes
18.
Plant Sci ; 294: 110459, 2020 May.
Article in English | MEDLINE | ID: mdl-32234218

ABSTRACT

Glutathione reductase (GR; EC 1.6.4.2) is a key NADPH-dependent flavo-protein oxidoreductase which can catalyze the oxidized glutathione (GSSG) to reduced glutathione (GSH) to protect plant cells from oxidative damage induced by Reactive oxygen species (ROS) burst. To investigate the biochemical characteristics and functional divergence of Populus GR family, three GR genes (PtGR1.1/1.2/2) were cloned from Populus trichocarpa and their biochemical characteristics were analyzed in this study. All the three genes were expressed in root, stem, leaf and bud, and the expression of PtGR genes were general upregulated under salicylic acid and alamethicin treatment. PtGR1.1 and PtGR1.2 were localized in cytoplasm, while PtGR2 was in chloroplast. The three PtGR proteins showed different enzymatic activities, apparent kinetic characteristic and thermal stability profiles. However, they have similar bivalent metal ions (Cu2+, Cd2+, Zn2+ and Pb2+) sensitivity and optimum pH profiles. Our study sheds light on a comprehensive information of glutathione reductase family in P. trichocarpa, and proved PtGR genes play critical roles when suffering different stresses.


Subject(s)
Glutathione Reductase/metabolism , Plant Proteins/metabolism , Populus/enzymology , Alamethicin/metabolism , Cadmium/metabolism , Copper/metabolism , Gene Expression Regulation, Plant/genetics , Gene Expression Regulation, Plant/physiology , Glutathione Reductase/genetics , Kinetics , Lead/metabolism , Plant Proteins/genetics , Plant Roots/genetics , Plant Roots/metabolism , Plant Stems/genetics , Plant Stems/metabolism , Populus/genetics , Populus/metabolism , Reactive Oxygen Species/metabolism , Salicylic Acid/metabolism , Zinc/metabolism
19.
Eur Biophys J ; 49(1): 113-124, 2020 Jan.
Article in English | MEDLINE | ID: mdl-31912177

ABSTRACT

Alamethicin is an antibiotic peptide comprising 20 amino acid residues and functions as an ion channel in biological membranes. Natural alamethicins have a variety of amino acid sequences. Two of them, used as a mixed sample in this study, are: UPUAUAQUVUGLUPVUUQQO and UPUAUUQUVUGLUPVUUQQO, where U and O represent α-aminoisobutyric acid and phenylalaninol, respectively. As indicated, only the amino acid at position six differs, and the two alamethicins are referred to as alamethicin-A6 and -U6, respectively. The conformation and thermal stability of alamethicin-A6 and -U6 in methanol were examined using proton nuclear magnetic resonance (NMR) spectroscopy. Both alamethicins form an α-helix between the 2nd and 11th residues. The N-terminal, 19th and C-terminal residues take a non-helical conformation. The structure between the 12th and 18th residues has not been well determined due to the absence of cross peaks in the two-dimensional NMR data. The α-helices are maintained up to 54 °C at least. In contrast to these similarities, it has been found that the length of the α-helix of alamethicin-U6 is somewhat shorter than that of alamethicin-A6, the intra-molecular hydrogen bonds formed by the amide proton of the seventh residue is much more thermally stable for alamethicin-U6 than for alamethicin-A6, and the C-terminal residue of alamethicin-U6 has higher mobility than that of alamethicin-A6. The mobility of the N- and C-terminal residues is discussed on the basis of a model chain which consists of particles connected by rigid links, and the physiological significance of the mobility is emphasized.


Subject(s)
Alamethicin/chemistry , Molecular Dynamics Simulation , Hydrogen Bonding , Methanol/chemistry , Protein Conformation , Protein Stability , Proton Magnetic Resonance Spectroscopy
20.
Anal Chim Acta ; 1099: 111-118, 2020 Feb 22.
Article in English | MEDLINE | ID: mdl-31986267

ABSTRACT

The pharmaceutical industry's focus has expanded to include peptide and protein-based therapeutics; however, some analytical challenges have arisen along the way, including the urgent need for fast and robust measurement of the membrane permeability of peptides and small proteins. In this study, a simple and efficient approach that utilizes MALDI-TOF-MS to study peptide and protein permeability through an artificial liposome membrane in conjunction with a differential hydrogen-deuterium exchange (HDX) methodology is described. A non-aqueous (aprotic) matrix was evaluated for use with MALDI sample preparation in order to eliminate undesirable hydrogen-deuterium back-exchange. Peptides and proteins were incubated with liposomes and their penetration into the liposome membrane over time was measured by MALDI-MS. A differential HDX approach was used to distinguish the peptides outside of the liposome from those inside. In this regard, the peptides on the outside of the liposomes were labeled using short exposure to deuterium oxide, while the peptides inside of the liposomes were protected from labeling. Subsequently, the unlabeled versus labeled peak area ratios for peptide and protein samples were compared using MALDI-TOF-MS. In this proof-of-concept study, we developed the Liposome Artificial Membrane Permeability Assay (LAMPA) workflow to study three well-known membrane-active model peptides (melittin, alamethicin, and gramicidin) and two model proteins (aprotinin and ubiquitin). The permeability results obtained from this were corroborated by previously reported data for studied peptides and proteins. The proposed LAMPA by MALDI-HDX-MS can be applied in an ultra-high-throughput manner for studying and rank-ordering membrane permeability of peptides and small proteins.


Subject(s)
Alamethicin/analysis , Aprotinin/analysis , Gramicidin/analysis , Melitten/analysis , Ubiquitin/analysis , Hydrogen Deuterium Exchange-Mass Spectrometry , Liposomes/chemistry , Membranes, Artificial , Permeability
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