Interaction of an Amphiphilic Peptide with a Phospholipid Bilayer Surface by Molecular Dynamics Simulation Study

Abstract

Corticotropin-releasing factor (CRF) is the principal neuroregulator of adrenocorticotropic hormone (ACTH) secretion. Previous experiments have demonstrated that CRF binds avidly to the surface of single egg phosphatidylcholine vesicles and its amphiphilic secondary structure might play an important role in the function. In this study, the interaction of the residues 13–41 in human CRF with the surface of a DOPC bilayer was investigated by molecular dynamics (MD) simulation in order to understand the role of the membrane surface in the formation of the amphiphilic α helix as well as to determine the effects of the peptide on the lipid bilayer. The model used included 60 DOPC molecules, 1 helical peptide (CRF_13–41) on the bilayer surface, and explicit waters of solvation in the lipid polar head group regions, together with constant-volume periodic boundary conditions in three dimensions. The MD simulation was carried out for 510 ps. In addition, CRF_13–41, initially in a helical form, was simulated in vacuo as a control. The results indicate that while it was completely unstable in vacuo, the peptide helical form was generally maintained on the bilayer surface, but with distortions near the terminal ends. The peptide was confined to the bilayer headgroup/water region, similar to that reported from neutron diffraction measurement of tripeptides bound to the phosphatidylcholine bilayer surface (Ref 1). The amphiphilicity of the peptide matched that of the bilayer headgroup environment, with the hydrophilic side oriented toward water and the hydrophobic side making contact with the bilayer hydrocarbon core. These results support the hypothesis that the amphiphilic environment of a membrane surface is important in the induction of peptide amphiphilic α-helical secondary structure. Two major effects of the peptide on the lipids were found: the first CH₂ segment in the lipid chains was significantly disordered and the lipid headgroup distribution was broadened towards the water region.     

A Molecular Dynamics Simulation Study of Nanomechanical Properties of Asymmetric Lipid Bilayer

A very important part of the living cells of biological systems is the lipid membrane. The mechanical properties of this membrane play an important role in biophysical studies. Investigation as to how the insertion of additional phospholipids in one leaflet of a bilayer affects the physical properties of the obtained asymmetric lipid membrane is of recent practical interest. In this work a coarse-grained molecular dynamics simulation was carried out in order to compute the pressure tensor, the lateral pressure, the surface tension and the first moment of lateral pressure in each leaflet of such a bilayer. Our simulations indicate that adding more phospholipids into one monolayer results in asymmetrical changes in the lateral pressure of the individual bilayer leaflets. Interestingly, it has been observed that a change in phospholipid density in one leaflet affects the physical properties of unperturbed leaflet as well. The asymmetric behavior of the physical properties of the two leaflets as a result of a change in the contribution of the various intermolecular forces in the presence of additional phospholipids may be expressed formally.     

Molecular Dynamics Simulations of Homo-oligomeric Bundles Embedded Within a Lipid Bilayer

Using molecular dynamics simulations, we studied the structure, interhelix interactions, and dynamics of transmembrane proteins. Specifically, we investigated homooligomeric helical bundle systems consisting of synthetic α-helices with either the sequence Ac-(LSLLLSL)₃-NH₂ (LS2) or Ac-(LSSLLSL)₃-NH₂ (LS3). The LS2 and LS3 helical peptides are designed to have amphipathic characteristics that form ion channels in membrane. We simulated bundles containing one to six peptides that were embedded in palmitoyl-oleoyl-phosphatidylcholine (POPC) lipid bilayer and placed between two lamellae of water. We aim to provide a fundamental understanding of how amphipathic helical peptides interact with each other and their dynamical behaviors in different homooligomeric states. To understand structural properties, we examined the helix lengths, tilt angles of individual helices and the entire bundle, interhelix distances, interhelix cross-angles, helix hydrophobic-to-hydrophilic vector projections, and the average number of interhelix hydrophilic (serine–serine) contacts lining the pore of the transmembrane channel. To analyze dynamical properties, we calculated the rotational autocorrelation function of each helix and the cross-correlation of the rotational velocity between adjacent helices. The observed structural and dynamical characteristics show that higher order bundles containing four to six peptides are composed of multiple lower order bundles of one to three peptides. For example, the LS2 channel was found to be stable in a tetrameric bundle composed of a “dimer of dimers.” In addition, we observed that there is a minimum of two strong hydrophilic contacts between a pair of adjacent helices in the dimer to tetramer systems and only one strong hydrophilic interhelix contact in helix pairs of the pentamer and hexamer systems. We believe these results are general and can be applied to more complex ion channels, providing insight into ion channel stability and assembly.     

Molecular Dynamics Simulations of Homo-oligomeric Bundles Embedded Within a Lipid Bilayer

Using molecular dynamics simulations, we studied the structure, interhelix interactions, and dynamics of transmembrane proteins. Specifically, we investigated homooligomeric helical bundle systems consisting of synthetic α-helices with either the sequence Ac-(LSLLLSL)₃-NH₂ (LS2) or Ac-(LSSLLSL)₃-NH₂ (LS3). The LS2 and LS3 helical peptides are designed to have amphipathic characteristics that form ion channels in membrane. We simulated bundles containing one to six peptides that were embedded in palmitoyl-oleoyl-phosphatidylcholine (POPC) lipid bilayer and placed between two lamellae of water. We aim to provide a fundamental understanding of how amphipathic helical peptides interact with each other and their dynamical behaviors in different homooligomeric states. To understand structural properties, we examined the helix lengths, tilt angles of individual helices and the entire bundle, interhelix distances, interhelix cross-angles, helix hydrophobic-to-hydrophilic vector projections, and the average number of interhelix hydrophilic (serine–serine) contacts lining the pore of the transmembrane channel. To analyze dynamical properties, we calculated the rotational autocorrelation function of each helix and the cross-correlation of the rotational velocity between adjacent helices. The observed structural and dynamical characteristics show that higher order bundles containing four to six peptides are composed of multiple lower order bundles of one to three peptides. For example, the LS2 channel was found to be stable in a tetrameric bundle composed of a “dimer of dimers.” In addition, we observed that there is a minimum of two strong hydrophilic contacts between a pair of adjacent helices in the dimer to tetramer systems and only one strong hydrophilic interhelix contact in helix pairs of the pentamer and hexamer systems. We believe these results are general and can be applied to more complex ion channels, providing insight into ion channel stability and assembly.     

Activation and Desensitization of TRPV1 Channels under the Influence of Capsaicin

Neurophysiology - The work was carried out on primary cultures of neurons (1- to 2-day-long cultivation) isolated from DRGs of 8- to 12-day-old rats; fluorescent microscopy was used for evaluation...     

Molecular Dynamics Simulations of a DMPC Bilayer Using Nonadditive Interaction Models

We present a polarizable force field based on the charge-equilibration formalism for molecular dynamics simulations of phospholipid bilayers. We discuss refinement of headgroup dihedral potential parameters to reproduce ab initio conformational energies of dimethylphosphate calculated at the MP2/cc-pVTZ level of theory. We also address the refinement of electrostatic and Lennard-Jones (van der Waals) parameters to reproduce ab initio polarizabilities and water interaction energies of dimethylphosphate and tetramethylammonium. We present results of molecular dynamics simulations of a solvated dimyristoylphosphatidylcholine bilayer using this polarizable force field as well as the nonpolarizable, fixed-charge CHARMM27 and CHARMM27r force fields for comparison. Calculated atomic and electron-density profiles, deuterium order parameters, and headgroup orientations are found to be consistent with previous simulations and with experiment. Polarizable interaction models for solvent and lipid exhibit greater water penetration into the lipid interior; this is due to the variation of water molecular dipole moment from a bulk value of 2.6 Debye to a value of 1.9 Debye in the membrane interior. The reduction in the electrostatic component of the desolvation free-energy penalty allows for greater water density. The surface dipole potential predicted by the polarizable model is 0.95 V compared to the value of 0.8 V based on nonpolarizable force-field calculations. Effects of inclusion of explicit polarization are discussed in relation to water dipole moment and varying charge distributions. Dielectric permittivity profiles for polarizable and nonpolarizable interactions exhibit subtle differences arising from the nature of the individual component parameterizations; for the polarizable force field, we obtain a bulk dielectric permittivity of 79, whereas the nonpolarizable force field plateaus at 97 (the value for pure TIP3P water). In the membrane interior, both models predict unit permittivities, with the polarizable models contributing from one to two more units due to the optical dielectric (high-frequency dipole fluctuations). This contribution is a step toward the continuing development of a CHARMM (Chemistry at Harvard Molecular Mechanics) polarizable force field for simulations of biomacromolecular systems.     

Inhibitory Effects of Capsaicin on Voltage-Gated Potassium Channels by TRPV1-Independent Pathway

Previously we observed that capsaicin, a transient receptor potential vanilloid 1 (TRPV1) receptor activator, inhibited transient potassium current (I_A) in capsaicin-sensitive and capsaicin-insensitive trigeminal ganglion (TG) neurons from rats. It suggested that the inhibitory effects of capsaicin on I_A have two different mechanisms: TRPV1-dependent and TRPV1-independent pathways. The main purpose of this study is to further investigate the TRPV1-independent effects of capsaicin on voltage-gated potassium channels (VGPCs). Whole cell patch-clamp technique was used to record I_A and sustained potassium current (I_K) in cultured TG neurons from trpv1 knockout (TRPV1^?/?) mice. We found that capsaicin reversibly inhibited I_A and I_K in a dose-dependent manner. Capsaicin (30 μM) did not alter the activation curve of I_A and I_K but shifted the inactivation–voltage curve to hyperpolarizing direction, thereby increasing the number of inactivated VGPCs at the resting potential. Administrations of high concentrations capsaicin, no use-dependent block, and delay of recovery time course were found on I_K and I_A. Moreover, forskolin, an adenylate cyclase agonist, selectively decreased the inhibitory effects of I_K by capsaicin, whereas none influenced the inhibitions of I_A. These results suggest that capsaicin inhibits the VGPCs through TRPV1-independent and PKA-dependent mechanisms, which may contribute to the capsaicin-induced nociception.     

Fusion Peptide Interaction with Lipid Bilayer: Modeling with Monte Carlo Simulation and Continuum Electrostatics Calculation

Abstract

Conformation of 20-residue peptide E5, an analog of the fusion peptide of influenza virus hemagglutinin, was explored by Monte-Carlo technique starting with the fully buried in the membrane ideal α-helix. The lipid bilayer (of 30 Å width) together with surrounding water were modeled by the atomic solvation parameters. During the simulation, residues 2–18 of the peptide retained α-helical conformation, and the peptide was found to be partially immersed into the bilayer. In the resulting low-energy conformers, the N-terminus was buried inside the membrane, its position with respect to the bilayer surface (Z_NT) being varied from 2.5 to 7.5 Å, and the orientation of the helical axis relative to the membrane plane (Θ) – from 10 to 35°. The low-energy conformers (below -200kcal/mol) were clustered in the space (Z_NT, Θ) into 4 groups. To select low-energy states of the peptide compatible with NMR data, we calculated pKa values of E5 ionizable groups and compared them with the experimental values. It was shown that the best correlation coefficient (0.87) and rmsd (0.68 in pH units) were obtained for the group of states which is characterized by Θ = 15–19° and Z_NT = 3.5–4.5Å.     

Decrypting the Heat Activation Mechanism of TRPV1 Channel by Molecular Dynamics Simulation

As a prototype cellular sensor, the TRPV1 cation channel undergoes a closed-to-open gating transition in response to various physical and chemical stimuli including noxious heat. Despite recent progress, the molecular mechanism of heat activation of TRPV1 gating remains enigmatic. Toward decrypting the structural basis of TRPV1 heat activation, we performed extensive molecular dynamics simulations (with cumulative simulation time of ～11 μs) for the wild-type channel and a constitutively active double mutant at different temperatures (30, 60, and 72°C), starting from a high-resolution closed-channel structure of TRPV1 solved by cryo-electron microscopy. In the wild-type simulations, we observed heat-activated conformational changes (e.g., expansion or contraction) in various key domains of TRPV1 (e.g., the S2-S3 and S4-S5 linkers) to prime the channel for gating. These conformational changes involve a number of dynamic hydrogen-bond interactions that were validated with previous mutational studies. Next, our mutant simulations observed channel opening after a series of conformational changes that propagate from the channel periphery to the channel pore via key intermediate domains (including the S2-S3 and S4-S5 linkers). The gating transition is accompanied by a large increase in the protein-water electrostatic interaction energy, which supports the contribution of desolvation of polar/charged residues to the temperature-sensitive TRPV1 gating. Taken together, our molecular dynamics simulations and analyses offered, to our knowledge, new structural, dynamic, and energetic information to guide future mutagenesis and functional studies of the TRPV1 channels and development of TRPV1-targeting drugs.     

Peptide Aggregation and Pore Formation in a Lipid Bilayer: A Combined Coarse-Grained and All Atom Molecular Dynamics Study

We present a simulation study where different resolutions, namely coarse-grained (CG) and all-atom (AA) molecular dynamics simulations, are used sequentially to combine the long timescale reachable by CG simulations with the high resolution of AA simulations, to describe the complete processes of peptide aggregation and pore formation by alamethicin peptides in a hydrated lipid bilayer. In the 1-μs CG simulations the peptides spontaneously aggregate in the lipid bilayer and exhibit occasional transitions between the membrane-spanning and the surface-bound configurations. One of the CG systems at t = 1 μs is reverted to an AA representation and subjected to AA simulation for 50 ns, during which water molecules penetrate the lipid bilayer through interactions with the peptide aggregates, and the membrane starts leaking water. During the AA simulation significant deviations from the α-helical structure of the peptides are observed, however, the size and arrangement of the clusters are not affected within the studied time frame. Solid-state NMR experiments designed to match closely the setup used in the molecular dynamics simulations provide strong support for our finding that alamethicin peptides adopt a diverse set of configurations in a lipid bilayer, which is in sharp contrast to the prevailing view of alamethicin oligomers formed by perfectly aligned helical alamethicin peptides in a lipid bilayer.     

Molecular Dynamics Simulation Study of the Interaction of Piscidin 1 with DPPC Bilayers: Structure-Activity Relationship

Abstract

To study structure-activity relationship of antimicrobial peptides and to design novel antimicrobial peptides with selectivity for bacterial cells, we have performed molecular dynamics simulations of the interaction of Piscidin (Pis1) and its two analogues (Pis1-AA and Pis1-PG) with dipalmitoylphosphatidylcholine (DPPC) bilayer through 45 ns. Our results inform us of the detailed location and orientation of the peptide with respect to the bilayer as well as provide about hydrogen-bond-formation patterns and electrostatics interactions. Simulations show that Pis1 and Pis-AA form the most hydrogen bonds and Pis-PG forms the fewest hydrogen bonds with lipid. Thus, Pis1 and Pis-AA should have stronger interactions with the lipid head group when compared to Pis-PG. Experimental studies have shown that Pis1 and Pis1-AA have a high antimicrobial and hemolytic activities, and Pis1-PG has low hemolytic activity while keeps potent antimicrobial activity. Our results complement the previous experimental studies. According to our MD results and previous experimental studies, Pis1 and Pis1-AA are more effective at the zwitterionic bilayer comparing Pis1-PG. These properties of Pis1-PG could be accordance with its low hemolytic activities.     

A Molecular Dynamics Investigation of Lipid Bilayer Perturbation by PIP2

Phosphoinositides like phosphatidylinositol 4,5-bisphosphate (PIP₂) are negatively charged lipids that play a pivotal role in membrane trafficking, signal transduction, and protein anchoring. We have designed a force field for the PIP₂ headgroup using quantum mechanical methods and characterized its properties inside a lipid bilayer using molecular dynamics simulations. Macroscopic properties such as area/headgroup, density profiles, and lipid order parameters calculated from these simulations agree well with the experimental values. However, microscopically, the PIP₂ introduces a local perturbation of the lipid bilayer. The average PIP₂ headgroup orientation of 45° relative to the bilayer normal induces a unique, distance-dependent organization of the lipids that surround PIP₂. The headgroups of these lipids preferentially orient closer to the bilayer normal. This perturbation creates a PIP₂ lipid microdomain with the neighboring lipids. We propose that the PIP₂ lipid microdomain enables the PIP₂ to function as a membrane-bound anchoring molecule.     

Molecular Dynamics Simulation of Model Lipid Membranes: Structural Effects of Impurities

Abstract

The gel to fluid phase transition or ordered to disordered phase transition observed in biological membranes are simulated by using constant energy Molecular Dynamics. The surface part of the membrane is modelled as a two-dimensional matrix formed by the head groups of the phospholipid molecules. Head molecules which are modelled as three spheres fused with three force centers, interact with each other via van der Waals and Coulomb type interactions. The -so called- impurity or foreign molecule embedded in the surface represents the protein type molecule which is present in biological membranes and control its activity. It is modelled as a pentagon having one force centers in each corner. It also interacts with the surface molecules again via van der Waals and Coulomb type interactions. The surface density is kept constant in the simulations of the systems with or without impurity. Structural and orientational changes due to impurity were observed and proved by monitoring two-dimensional order parameter. It has been shown that melting of the surface or breakage of the ordering of the surface molecules becomes easier and ordered to disordered phase transition temperature was lowered by 100 K if the impurity is present.     

On the Electroporation Thresholds of Lipid Bilayers: Molecular Dynamics Simulation Investigations

Electroporation relates to the cascade of events that follows the application of high electric fields and that leads to cell membrane permeabilization. Despite a wide range of applications, little is known about the electroporation threshold, which varies with membrane lipid composition. Here, using molecular dynamics simulations, we studied the response of dipalmitoyl-phosphatidylcholine, diphytanoyl-phosphocholine-ester and diphytanoyl-phosphocholine-ether lipid bilayers to an applied electric field. Comparing between lipids with acyl chains and methyl branched chains and between lipids with ether and ester linkages, which change drastically the membrane dipole potential, we found that in both cases the electroporation threshold differed substantially. We show, for the first time, that the electroporation threshold of a lipid bilayer depends not only on the “electrical” properties of the membrane, i.e., its dipole potential, but also on the properties of its component hydrophobic tails.     

Kinetics of the Opening and Closing of Individual Excitability-Inducing Material Channels in a Lipid Bilayer

The kinetics of the opening and closing of individual ion-conducting channels in lipid bilayers doped with small amounts of excitability-inducing material (EIM) are determined from discrete fluctuations in ionic current. The kinetics for the approach to steady-state conductance during voltage clamp are determined for lipid bilayers containing many EIM channels. The two sets of measurements are found to be consistent, verifying that the voltage-dependent conductance of the many-channel EIM system arises from the opening and closing of individual EIM channels. The opening and closing of the channels are Poisson processes. Transition rates for these processes vary exponentially with applied potential, implying that the energy difference between the open and closed states of an EIM channel is linearly proportional to the transmembrane electric field. A model incorporating the above properties of the EIM channels predicts the observed voltage dependence of ionic conductance and conductance relaxation time, which are also characteristic of natural electrically excitable membranes.     

Partitioning of Nonsteroidal Antiinflammatory Drugs in Lipid Membranes: A Molecular Dynamics Simulation Study

Using the potential of mean constrained force method, molecular dynamics simulations with atomistic details were performed to examine the partitioning and nature of interactions of two nonsteroidal antiinflammatory drugs, namely aspirin and ibuprofen, in bilayers of dipalmitoylphosphatidylcholine. Two charge states (neutral and anionic) of the drugs were simulated to understand the effect of protonation or pH on drug partitioning. Both drugs, irrespective of their charge state, were found to have high partition coefficients in the lipid bilayer from water. However, the values and trends of the free energy change and the location of the minima in the bilayer are seen to be influenced by the drug structure and charge state. In the context of the transport of the drugs through the bilayer, the charged forms were found to permeate fully hydrated in contrast to the neutral forms that permeate unhydrated.     

Comparative Molecular Dynamics Simulation Studies of Protegrin-1 Monomer and Dimer in Two Different Lipid Bilayers

Antimicrobial peptides interact specifically with the membrane of a pathogen and kill the pathogen by releasing its cellular contents. Protegrin-1 (PG-1), a β-hairpin antimicrobial peptide, is known to exist as a transmembrane monomer in a 1,2-dilauroylphosphatidylcholine (DLPC) bilayer and shows concentration-dependent oligomerization in a 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) bilayer. To examine its structure, dynamics, orientation, and interaction in membranes, we performed comparative molecular dynamics simulations of PG-1 monomer and dimer in DLPC and POPC bilayers for a total of 840 ns. The PG-1 monomer exhibits larger tilting in DLPC than in POPC due to a hydrophobic mismatch. PG-1 tilting is dependent on its rotation angle. The specific orientation of PG-1 in membranes is governed by the interactions of its aromatic residues with lipid headgroups. The calculated ¹⁵N and ¹³CO chemical shifts of Val¹⁶ in DLPC reveal that there are different sets of tilt and rotation angles that satisfy the experimental values reasonably, suggesting that more experiments are needed to determine its orientation. The dimer simulations show that the dimer interface is better preserved in POPC than in DLPC because POPC's greater hydrophobic thickness causes reduced flexibility of the C-terminal strands. Both monomer and dimer simulations show membrane thinning around PG-1, largely due to arginine-lipid interactions.     

Ion Transport Through Excitability-Inducing Material (EIM) Channels in Lipid Bilayer Membranes

Two different methods were used to determine the relative permeability and the voltage-dependent conductance of several different cations in excitability-inducing material (EIM)-doped lipid bilayers. In one method, the conductances of individual channels were measured for Li, Na, K, Cs, NH₄, and Ca, and in the other method biionic potentials of a membrane with many channels were measured for Li, Na, K, Cs, and Rb. The experimental results for the two methods are in agreement. The relative permeabilities are proportional to the ionic mobilities in free aqueous solution. The voltage dependence of the conductance is the same for all cations measured.