Studies on viral fusion peptides: the distribution of lipophilic and electrostatic potential over the peptide determines the angle of insertion into a membrane.

The oblique insertion of type 1 viral fusion peptides into the cell membrane of the host cell has been shown previously to be an essential element of viral fusion. The actual physical explanation of the cause of the oblique insertion has been the subject of speculation. In this study the physical properties of the fusion peptide surface have been determined computationally and compared to the tilt angles determined both experimentally and by the use of molecular dynamics. It has been shown that the relationship between the distribution of lipophilic potential over the peptide surface and the peptide geometry control the tilt angle of the peptide in a biomimetic DMPC bilayer whereas the depth of penetration into the bilayer appears to be determined by the electrostatic potential and hydrogen bonding at the C-terminus.

Chemical reactions in 2D: self-assembly and self-esterification of 9(10),16-dihydroxypalmitic acid on mica surface.

9(10),16-Dihydroxypalmitic acid (diHPA) is a particularly interesting polyhydroxylated fatty acid (1) because it is the main monomer of cutin, the most abundant biopolyester in nature, and (2) because the presence of a terminal and a secondary hydroxyl group in midchain positions provides an excellent model to study their intermolecular interactions in a confined phase such as self-assembled layers. In this study we have combined atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), attenuated total reflection Fourier transform infrared (ATR-FT-IR) spectroscopy, as well as molecular dynamics (MD) simulations to conclude that the self-assembling of diHPA molecules on mica is a layer by layer process following a Brunauer-Emmett-Teller (BET) type isotherm and with the first layer growing much faster than the rest. Interactions between secondary hydroxyls reinforce the cohesive energy of the monolayer, while the presence of the terminal hydroxyl group is necessary to trigger the multilayered growth. Besides, XPS and ATR-FT-IR spectroscopies clearly indicate that spontaneous self-esterification occurs upon self-assembling. The esterification reaction is a prerequisite to propose a self-assembly route for the biosynthesis of cutin in nature. Molecular dynamics simulations have shown that internal molecular reorganization within the self-assembled layers provides the appropriate intermolecular orientation to facilitate the nucleophilic attack and the release of a water molecule required by the esterification reaction.

On a novel rate theory for transport in narrow ion channels and its application to the study of flux optimization via geometric effects.

We present a novel rate theory based on the notions of splitting probability and mean first passage time to describe single-ion conduction in narrow, effectively one-dimensional membrane channels. In contrast to traditional approaches such as transition state theory or Kramers theory, transitions between different conduction states in our model are governed by rates which depend on the full geometry of the potential of mean force (PMF) resulting from the superposition of an equilibrium free energy profile and a transmembrane potential induced by a nonequilibrium constraint. If a detailed theoretical PMF is available (e.g., from atomistic molecular dynamics simulations), it can be used to compute characteristic conductance curves in the framework of our model, thereby bridging the gap between the atomistic and the mesoscopic level of description. Explicit analytic solutions for the rates, the ion flux, and the associated electric current can be obtained by approximating the actual PMF by a piecewise linear potential. As illustrative examples, we consider both a theoretical and an experimental application of the model. The theoretical example is based on a hypothetical channel with a fully symmetric sawtooth equilibrium PMF. For this system, we explore how changes in the spatial extent of the binding sites affect the rate of transport when a linear voltage ramp is applied. Already for the case of a single binding site, we find that there is an optimum size of the site which maximizes the current through the channel provided that the applied voltage exceeds a threshold value given by the binding energy of the site. The above optimization effect is shown to arise from the complex interplay between the channel structure and the applied electric field, expressed by a nonlinear dependence of the rates with respect to the linear size of the binding site. In studying the properties of current-voltage curves, we find a double crossover between sublinear and superlinear behaviors as the size of the binding site is varied. The ratio of unidirectional fluxes clearly deviates from the Ussing limit and can be characterized by a flux ratio exponent which decreases below unity as the binding site becomes wider. We also explore effects arising from changes in the ion bulk concentration under symmetric ionic conditions and the presence of additional binding sites in the hypothetical channel. As for the experimental application, we show that our rate theory is able to provide good fits to conductance data for sodium permeation through the gramicidin A channel. Possible extensions of the theory to treat the case of an asymmetric equilibrium PMF, fluctuations in the mean number of translocating ions, the case of fluctuating energy barriers, and multi-ion conductance are briefly discussed.

Simulation studies of the interactions between membrane proteins and detergents.

Interactions between membrane proteins and detergents are important in biophysical and structural studies and are also biologically relevant in the context of folding and transport. Despite a paucity of high-resolution data on protein-detergent interactions, novel methods and increased computational power enable simulations to provide a means of understanding such interactions in detail. Simulations have been used to compare the effect of lipid or detergent on the structure and dynamics of membrane proteins. Moreover, some of the longest and most complex simulations to date have been used to observe the spontaneous formation of membrane protein-detergent micelles. Common mechanistic steps in the micelle self-assembly process were identified for both alpha-helical and beta-barrel membrane proteins, and a simple kinetic mechanism was proposed. Recently, simplified (i.e. coarse-grained) models have been utilized to follow long timescale transitions in membrane protein-detergent assemblies.

Molecular simulations and lipid-protein interactions: potassium channels and other membrane proteins.

Molecular dynamics simulations may be used to probe the interactions of membrane proteins with lipids and with detergents at atomic resolution. Examples of such simulations for ion channels and for bacterial outer membrane proteins are described. Comparison of simulations of KcsA (an alpha-helical bundle) and OmpA (a beta-barrel) reveals the importance of two classes of side chains in stabilizing interactions with the head groups of lipid molecules: (i) tryptophan and tyrosine; and (ii) arginine and lysine. Arginine residues interacting with lipid phosphate groups play an important role in stabilizing the voltage-sensor domain of the KvAP channel within a bilayer. Simulations of the bacterial potassium channel KcsA reveal specific interactions of phosphatidylglycerol with an acidic lipid-binding site at the interface between adjacent protein monomers. A combination of molecular modelling and simulation reveals a potential phosphatidylinositol 4,5-bisphosphate-binding site on the surface of Kir6.2.

Kv channel S6 helix as a molecular switch: simulation studies.

Ion channels form pores of nanoscopic dimensions in biological membranes and play a key role in the physiology of cells. The majority of ion channels are gated, i.e. they contain a molecular switch that allows a transition between a closed (functionally 'off') and open (functionally 'on') state. Comparison of crystal structures of potassium channels suggest that the gating mechanism of voltage-gated potassium (Kv) channels involves a key role for the pore-lining S6 helix. There is a conserved PVP sequence motif in the S6 helix. Molecular dynamics simulations are used here to explore the conformational dynamics of the S6 helix hinge in models of fragments of a Kv channel, namely an S5-P-S6 monomer and an (S5-P-S6)4 tetramer. The latter is a model of the complete pore-forming domain of a Kv channel. All models were simulated embedded in an octane slab (a simple membrane mimetic). The results of these simulations indicate that the PVP motif may form a molecular hinge, even when the S6 helix forms part of a more complex model. The conformational dynamics of S6 are modulated by the remainder of protein, but it remains flexible. These simulation results are compatible with a channel gating model in which S6 bends in the vicinity of the PVP motif in addition to the region around the conserved glycine (G466) that is N-terminal to the PVP motif. This model is supported by comparison of the Kv S6 models with the S6 helix of the bacterial KvAP channel crystal structure. Thus, K channel gating may depend on a complex nanoswitch with three rigid helical sections linked by two molecular hinges.

Pores formed by the nicotinic receptor m2delta Peptide: a molecular dynamics simulation study.

The M2delta peptide self-assembles to form a pentameric bundle of transmembrane alpha-helices that is a model of the pore-lining region of the nicotinic acetylcholine receptor. Long (>15 ns) molecular dynamics simulations of a model of the M2delta(5) bundle in a POPC bilayer have been used to explore the conformational dynamics of the channel assembly. On the timescale of the simulation, the bundle remains relatively stable, with the polar pore-lining side chains remaining exposed to the lumen of the channel. Fluctuations at the helix termini, and in the helix curvature, result in closing/opening transitions at both mouths of the channel, on a timescale of approximately 10 ns. On average, water within the pore lumen diffuses approximately 4x more slowly than water outside the channel. Examination of pore water trajectories reveals both single-file and path-crossing regimes to occur at different times within the simulation.

Bundles consisting of extended transmembrane segments of Vpu from HIV-1: computer simulations and conductance measurements.

Part of the genome of the human immunodeficiency virus type 1 (HIV-1) encodes for a short membrane protein Vpu, which has a length of 81 amino acids. It has two functional roles: (i) to downregulate CD4 and (ii) to support particle release. These roles are attributed to two distinct domains of the peptide, the cytoplasmic and transmembrane (TM) domains, respectively. It has been suggested that the enhanced particle release function is linked to the ion channel activity of Vpu, with a slight preference for cations over anions. To allow ion flux across the membrane Vpu would be required to assemble in homooligomers to form functional water-filled pores. In this study molecular dynamics simulations are used to address the role of particular amino acids in 4, 5, and 6 TM helix bundle structures. The helices (Vpu(6-33)) are extended to include hydrophilic residues such as Glu, Tyr, and Arg (EYR motif). Our simulations indicate that this motif destabilizes the bundles at their C-terminal ends. The arginines point into the pore to form a positive charged ring that could act as a putative selectivity filter. The helices of the bundles adopt slightly higher average tilt angles with decreasing number of helices. We also suggest that the helices are kinked. Conductance measurements on a peptide (Vpu(1-32)) reconstituted into lipid membranes show that the peptide forms ion channels with several conductance levels.

Amantadine blocks channel activity of the transmembrane segment of the NB protein from influenza B.

NB is short auxiliary protein with ca. 100 amino acids, encoded in the viral genome of influenza B. It is believed to be similar to M2 from influenza A and Vpu from HIV-1 in that it demonstrates ion channel activity. Channels formed by the protein can be blocked by amantadine. We have synthesized the putative transmembrane segment of NB (IRG S20 IIITICVSL I30 VILIVFGCI A40 KIFI (NB, Lee)). Reconstituted in a lipid bilayer, the peptide shows channel activity. The addition of amantadine leads to dose-dependent loss of channel activity. Channel blocking is reversible. Channel behaviour of the peptide in the presence of amantadine is in accordance with findings for the intact channel. Thus, the synthetic transmembrane peptide captures the ion channel activity of the intact NB protein.

Electrostatics studies and molecular dynamics simulations of a homology model of the Shaker K+ channel pore.

A homology model of the pore domain of the Shaker K+ channel has been constructed using a bacterial K+ channel, KcsA, as a template structure. The model is in agreement with mutagenesis and sequence variability data. A number of structural features are conserved between the two channels, including a ring of tryptophan sidechains on the outer surface of the pore domain at the extracellular end of the helix bundle, and rings of acidic sidechains close to the extracellular mouth of the channel. One of these rings, that formed by four Asp447 sidechains at the mouth of the Shaker pore, is shown by pK(A) calculations to be incompletely ionized at neutral pH. The potential energy profile for a K+ ion moved along the central axis of the Shaker pore domain model selectivity filter reveals a shallow well, the depth of which is modulated by the ionization state of the Asp447 ring. This is more consistent with the high cation flux exhibited by the channel in its conductance value of 19 pS.

Proline-induced hinges in transmembrane helices: possible roles in ion channel gating.

A number of ion channels contain transmembrane (TM) alpha-helices that contain proline-induced molecular hinges. These TM helices include the channel-forming peptide alamethicin (Alm), the S6 helix from voltage-gated potassium (Kv) channels, and the D5 helix from voltage-gated chloride (CLC) channels. For both Alm and KvS6, experimental data implicate hinge-bending motions of the helix in an aspect of channel gating. We have compared the hinge-bending motions of these TM helices in bilayer-like environments by multi-nanosecond MD simulations in an attempt to describe motions of these helices that may underlie possible modes of channel gating. Alm is an alpha-helical channel-forming peptide, which contains a central kink associated with a Gly-x-x-Pro motif in its sequence. Simulations of Alm in a TM orientation for 10 ns in an octane slab indicate that the Gly-x-x-Pro motif acts as a molecular hinge. The S6 helix from Shaker Kv channels contains a Pro-Val-Pro motif. Modeling studies and recent experimental data suggest that the KvS6 helix may be kinked in the vicinity of this motif. Simulations (10 ns) of an isolated KvS6 helix in an octane slab and in a POPC bilayer reveal hinge-bending motions. A pattern-matching approach was used to search for possible hinge-bending motifs in the TM helices of other ion channel proteins. This uncovered a conserved Gly-x-Pro motif in TM helix D5 of CLC channels. MD simulations of a model of hCLC1-D5 spanning an octane slab suggest that this channel also contains a TM helix that undergoes hinge-bending motion. In conclusion, our simulations suggest a model in which hinge-bending motions of TM helices may play a functional role in the gating mechanisms of several different families of ion channels.

The structure of the HIV-1 Vpu ion channel: modelling and simulation studies.

Vpu is an 81 amino acid auxiliary protein in HIV-1 which exhibits channel activity. We used two homo-pentameric bundles with the helical transmembrane segments derived from FTIR spectroscopy in combination with a global molecular dynamics search protocol: (i) tryptophans (W) pointing into the pore, and (ii) W facing the lipids. Two equivalent bundles have been generated using a simulated annealing via a restrained molecular dynamics simulations (SA/MD) protocol. A fifth model was generated via SA/MD with all serines facing the pore. The latter model adopts a very stable structure during the 2 ns of simulation. The stability of the models with W facing the pore depends on the starting structure. A possible gating mechanism is outlined.

Potassium and sodium ions in a potassium channel studied by molecular dynamics simulations.

We have performed simulations of both a single potassium ion and a single sodium ion within the pore of the bacterial potassium channel KcsA. For both ions there is a dehydration energy barrier at the cytoplasmic mouth suggesting that the crystal structure is a closed conformation of the channel. There is a potential energy barrier for a sodium ion in the selectivity filter that is not seen for potassium. Radial distribution functions for both ions with the carbonyl oxygens of the selectivity filter indicate that sodium may interact more tightly with the filter than does potassium. This suggests that the key to the ion selectivity of KcsA is the greater dehydration energy of Na(+) ions, and helps to explain the block of KcsA by internal Na(+) ions.

Amino acid distributions in integral membrane protein structures.

Advances in structure determination of membrane proteins enable analysis of the propensities of amino acids in extramembrane versus transmembrane locations to be performed on the basis of structure rather than of sequence and predicted topology. Using 29 available structures of integral membrane proteins with resolutions better than 4 A the distributions of amino acids in the transmembrane domains were calculated. The results were compared to analysis based on just the sequences of the same transmembrane alpha-helices and significant differences were found. The distribution of residues between transmembrane alpha-helices and beta-strands was also compared. Large hydrophobic (Phe, Leu, Ile, Val) residues showed a clear preference for the protein surfaces facing the lipids for beta-barrels, but in alpha-helical proteins no such preference was seen, with these residues equally distributed between the interior and the surface of the protein. A notable exception to this was alanine, which showed a slight preference for the interior of alpha-helical membrane proteins. Aromatic residues were found to follow saddle-like distributions preferring to be located in the lipid/water interfaces. The resultant 'aromatic belts' were spaced more closely for beta-barrel than for alpha-helical membrane proteins. Charged residues could be shown to generally avoid surfaces facing the bilayer although they were found to occur frequently in the transmembrane region of beta-barrels. Indeed detailed comparison between alpha-helical and beta-barrel proteins showed many qualitative differences in residue distributions. This suggests that there may be subtle differences in the factors stabilising beta-barrels in bacterial outer membranes and alpha-helix bundles in all other membranes.

Channel gating: twist to open.

Recent studies of the bacterial mechanosensitive channel MscL have combined a number of different approaches to come up with a model for the channel gating mechanism.

Membrane proteins: Aquaporins--channels without ions.

Recently determined structures have shed new light on the way that aquaporins act as passive, but selective, pores for the transport of small molecules--such as water or glycerol--across membranes.

Side-chain ionization states in a potassium channel.

KcsA is a bacterial K+ channel that is gated by pH. Continuum dielectric calculations on the crystal structure of the channel protein embedded in a low dielectric slab suggest that side chains E71 and D80 of each subunit, which lie adjacent to the selectivity filter region of the channel, form a proton-sharing pair in which E71 is neutral (protonated) and D80 is negatively charged at pH 7. When K+ ions are introduced into the system at their crystallographic positions the pattern of proton sharing is altered. The largest perturbation is for a K+ ion at site S3, i.e., interacting with the carbonyls of T75 and V76. The presence of multiple K+ ions in the filter increases the probability of E71 being ionized and of D80 remaining neutral (i.e., protonated). The ionization states of the protein side chains influence the potential energy profile experienced by a K+ ion as it is translated along the pore axis. In particular, the ionization state of the E71-D80 proton-sharing pair modulates the shape of the potential profile in the vicinity of the selectivity filter. Such reciprocal effects of ion occupancy on side-chain ionization states, and of side-chain ionization states on ion potential energy profiles will complicate molecular dynamics simulations and related studies designed to calculate ion permeation energetics.

Voltage-dependent insertion of alamethicin at phospholipid/water and octane/water interfaces.

Understanding the binding and insertion of peptides in lipid bilayers is a prerequisite for understanding phenomena such as antimicrobial activity and membrane-protein folding. We describe molecular dynamics simulations of the antimicrobial peptide alamethicin in lipid/water and octane/water environments, taking into account an external electric field to mimic the membrane potential. At cis-positive potentials, alamethicin does not insert into a phospholipid bilayer in 10 ns of simulation, due to the slow dynamics of the peptide and lipids. However, in octane N-terminal insertion occurs at field strengths from 0.33 V/nm and higher, in simulations of up to 100 ns duration. Insertion of alamethicin occurs in two steps, corresponding to desolvation of the Gln7 side chain, and the backbone of Aib10 and Gly11. The proline induced helix kink angle does not change significantly during insertion. Polyalanine and alamethicin form stable helices both when inserted in octane and at the water/octane interface, where they partition in the same location. In water, both polyalanine and alamethicin partially unfold in multiple simulations. We present a detailed analysis of the insertion of alamethicin into the octane slab and the influence of the external field on the peptide structure. Our findings give new insight into the mechanism of channel formation by alamethicin and the structure and dynamics of membrane-associated helices.