5 An Alamethicin Channel in a Lipid Bilayer

D. P. Tieleman, H. J. C. Berendsen and M. S. P. Sansom. An alamethicin channel in a bilayer:

molecular dynamics simulations. Biophysical Journal, in press.

D. P. Tieleman, J. Breed, H. J. C. Berendsen and M. S. P. Sansom. 1998. Alamethicin channels in

a membrane: molecular dynamics simulations. Faraday Discussions 111, in press.

5 An alamethicin channel

SummaryWe present the results of 2 ns molecular dynamics simulations of ahexameric bundle of Alm helices in a POPC bilayer. These simulationsexplore the dynamic properties of a model of a helix bundle channel in acomplete phospholipid bilayer in an aqueous environment. We study thestability and conformational dynamics of the bundle in a phospholipidbilayer. We also investigate the effect of the ionisation state of the ringof Glu18 sidechains on bundle stability. If all of the Glu18 sidechains areionised, the bundle is unstable; if none of the Glu18 sidechains is ionisedthe bundle is stable. pKa calculations suggest that either zero or one ion-ised Glu18 is present at neutral pH, correlating with the stable form ofthe helix bundle. The structural and dynamic properties of water in thismodel channel were examined. As in earlier in vacuo simulations [122]the dipole moments of water molecules within the pore were alignedantiparallel to the helix dipoles. This contributes to the stability of thehelix bundle.

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5.1 Introduction

5.1 Introduction

Channels formed by integral membrane proteins enable ions to move pass-ively across lipid bilayers. They are important in numerous cellular processes,ranging from electrical signalling [133] to facilitating the uncoating of viralgenomes [193]. They form transbilayer pores through which selected ionsmay move at high rates (∼107 ions s−1 channel−1). In order to understandthe physical basis of their functional properties we need to characterise theirstructural and dynamic properties. However, this is far from easy. As ionchannels are membrane proteins, relatively little is known about their threedimensional structures. Indeed, although membrane proteins make up ∼20to 30% of most genomes [153,154], we know the high resolution structure ofonly a handful of such proteins, including a bacterial K+ channel [194].

Given such relative ignorance, the study of simple model membraneproteins can provide valuable information on membrane protein structureand dynamics. This has proved to be the case for ion channels, wherestudies of a simple channel forming peptide, gramicidin A, have providedunique insights into the structural basis of channel function. However,the structural peculiarities of gramicidin are such that it is useful to alsoexamine other peptide models that more closely mimic ion channel proteins.Many ion channels contain a central pore lined by a bundle of approximatelyparallel α-helices. Such channels range in complexity from the M2 proteinof influenza A (∼100 amino acids per subunit) to the nicotinic acetylcholinereceptor (∼500 amino acids per subunit). Given the importance of thisstructural motif in channel proteins, it is important to have a simple yetdetailed model system for channels formed by α-helices.

Alamethicin (Alm) is a 20 residue peptide which forms ion channels inlipid bilayers. The properties of the channels and the structural and spectro-scopic properties of Alm have been studied in considerable detail [161–163].Alm forms multi-conductance channels in a voltage-dependent manner.The alamethicin molecule adopts a largely α-helical conformation in thecrystal, in non-aqueous solvents, and in the presence of lipid bilayers. Thisconformation is stabilised by the presence of a large number of Aib residuesin the sequence of Alm:

Ac-Aib-Pro-Aib-Ala-Aib-Ala-Gln7-Aib-Val-Aib-Gly-Leu-Aib-Pro14-Val-Aib-Aib-Glu18-Gln-Phol

The multi-conductance behaviour of Alm channels is generally explainedin terms of the “barrel stave” model [164, 195–197]. In this model, multiple

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5 An alamethicin channel

Alm molecules form a helix bundle surrounding a central pore. Different con-ductance levels correspond to different numbers of helices in the bundle. Thismodel is in accord with a large body of experimental data [162], includingneutron scattering studies from Alm in lipid bilayers [198]. A key feature ofthe model is that the helices are oriented parallel (rather than antiparallel)to one another. The mutual repulsion of the parallel helix dipoles is com-pensated for by their favourable interaction with the electrostatic field acrossthe bilayer and with the water inside the transbilayer pore [122]. This pictureis supported by the asymmetry of voltage-activation of Alm channels: whenAlm is added to one face (the cis face) of a bilayer, only cis-positive voltageswill result in channel formation. This supports a structure that is asymmetricrelative to the bilayer plane, i.e. a parallel helix bundle. A parallel helix bundleis also supported by the work of Woolley and his colleagues [199,200], whohave shown that channels formed by covalently coupled pairs of Alm helices(which are physically constrained to be parallel to one another) strongly re-semble channels formed by unmodified Alm.

Molecular models of channels formed by Alm helix bundles have been gen-erated by restrained molecular dynamics simulations in vacuo. These modelshave been refined by short MD simulations with water molecules inside thepore, using restraints on the helix bundle to maintain bundle integrity [148].The models can explain the change in stability of Alm channels when the Gln7sidechain is replaced by a smaller polar residue [201,202]. The pore dimen-sions of such models with different numbers of helices/bundle correlate withthe experimentally observed multiple conductances of Alm [147, 157, 162].Similar models of channels formed by Alm analogues have been used as thebasis of simple electrostatics calculations which predict the ionic-strength de-pendent non-linear current-voltage curves observed for such channels withreasonable accuracy [199]. Models of Alm helix bundles have been used inMD simulations to demonstrate that water within channels formed by paral-lel helix bundles is ordered and shows reduced translational and rotationalmobility relative to bulk water [122]. Finally, models of Alm channels havebeen used to explore changes in translational mobility of Na+ ions withinnarrow pores [215].

Recently, Zhong et al. have described simulations of two synthetic ionchannels in an octane slab between two water layers [208,209]. In this paperwe extend this approach and present the results of 2 ns MD simulations of ahexameric bundle of Alm helices in a POPC bilayer. We study the stability andconformational dynamics of the bundle in a phospholipid bilayer, the effectof the ionisation state of the ring of Glu18 sidechains on bundle stability, andthe structural and dynamic properties of water in this model channel.

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5.2 Methods

5.2 Methods

5.2.1 Starting model of the hexameric Alm bundle

An initial model of a hexameric Alm helix bundle was generated using re-strained MD in vacuo as described in refs. [148, 203]. During the final stageof the simulated annealing protocol used to build the model the restraintsapplied were: (i) intra-helix restraints, to maintain H-bonding of the backboneof each Alm monomer; and (ii) inter-helix restraints, between the N-terminalsegments of adjacent monomers of the bundle, in order to maintain the in-tegrity of the bundle. The model was solvated within and at either mouth ofthe pore and simulated for 300 ps [122], retaining the intra- and inter-helixrestraints. The Cα RMSD from the initial in vacuo model to the model at theend of 300 ps in water was ∼0.2 nm. The resultant model was desolvated andused as the starting model for the bilayer simulations.

5.2.2 Two simulation systems

Two simulations of a hexameric Alm helix bundle were run, differing in theionisation state of the Glu18 residues. Electrostatics calculations on rings ofglutamate residues at either mouth of a model of the pore domain of thenicotinic acetylcholine receptor [204] suggest that about 50% of the glutam-ates in the C-terminal ring are protonated at neutral pH. Calculations of thepKa of the glutamate sidechains in the hexameric Alm helix bundle showthat at pH 7, either none or just one of the six glutamate sidechains will beionised [211]. To investigate the effect of ionisation of glutamate sidechainson the dynamics and stability of the Alm helix bundle, we have chosen tosimulate two limiting states of the system. In one (N6) all six glutamates areionised, in the other (N6H) all six glutamates are protonated. The true stateof the system is probably closer to N6H than to N6.

The setup of the system will be described first for N6. An equilibratedPOPC bilayer with 128 lipid molecules was used. A cylindrical hole was madein the centre of the bilayer by removing lipids whose P atoms fell within1.55 nm of the central axis of the cylinder. A short MD simulation with aradially acting repulsive force was used to drive any remaining atoms out ofthe cylinder into the bilayer. The Alm N6 model was inserted into the cavitythus created. This system was solvated with SPC waters. Six Na+ ions replacedwater molecules at the positions of lowest Coulomb potential. The resultantsystem (6 Alm helices, 3528 waters, 6 Na+ ions, giving a total of 17000 atomsin a box of dimensions 6.1 × 6.1 × 7.0 nm) was simulated for 10 ps with no

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5 An alamethicin channel

(a)

(b)

Figure 5.1. A Starting configuration of the simulation system for an Alm hexamerichelix bundle inserted in a POPC bilayer. Water is not shown. The lipid headgroupatoms and the Alm molecules are shown in space-filling format. B Snapshot of theAlm N6H simulation showing the helix bundle and the rings of Gln7, Glu18 and Gln19sidechains, and water molecules within and just outside the pore.

restraints, with constant surface area, and with a constant pressure of 1 barin the z direction, followed by 5 ps with positional restraints on the peptideatoms relative to the starting N6 structure. The N6H system was created fromthe N6 system by protonating the six Glu18 residues, removing the Na+ ionsand energy minimizing. A snapshot of the N6H system is given in figure 5.1.

5.2.3 Simulation details

Molecular dynamics simulations were run using GROmacs [93, 145]. A twinrange cutoff was used: 1.0 nm for van der Waals interactions, 1.8 nm forelectrostatic interactions. The timestep was 2 fs, using LINCS to constrainbond lengths [260]. We used a constant pressure of 1 bar in all three directions(τp = 1.0 ps) and constant temperature of 300 K, coupling separately to water,lipid and protein (τT = 0.1 ps) [35]. Lipid parameters were as in previous MD

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5.3 Results

0 250 500 750 1000 1250 1500 1750 2000time (ps)

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

RM

SD

(nm

)

Alm 6Alm 6H

Figure 5.2. RMSDs vs. time for the Cα atoms for Alm N6 and Alm N6H.

studies of lipid bilayers [25,178]. The water model used was SPC [86], whichhas been shown to behave well in lipid bilayer/water simulations [32]. Proteinparameters were taken from GROMOS [12]. Simulations were run on an SGIOrigin 2000 195 MHz R10k processor and took ∼8 days per ns on a singleprocessor.

5.3 Results

5.3.1 Progress of the simulations

In figure 5.2 the CαRMSDs vs. time of the N6 and N6H simulations are plotted.The RMSD of N6 increases steadily up to ∼0.35 nm over the first 1200 ps andthen plateaus at this value. This suggests that substantial changes in thestructure of the N6 helix bundle occur over the course of the 2 ns simulation.In contrast the Cα RMSD for the N6H model reaches a peak of ∼0.25 nm after400 ps, and then settles at∼0.23 nm for the remainder of the simulation. Thissuggests that N6H shows substantially smaller structural drift than does N6,and thus that the former protonated state of the hexameric Alm helix bundleis a more stable structure in this MD simulation.

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5 An alamethicin channel

0

0.1

0.2

0.3

0.4

0 21 42 63 84 105

Cα

RM

SF

(nm

)

residue

H1 H2 H3 H4 H6H5

Figure 5.3. Residue-by-residue Cα RMS fluctuations about their average coordinates.Alm N6 (black line) and Alm N6H (grey line). The vertical broken lines delineate theextents of helices H1 (residues 0 to 20) to H6 (residues 105 to 125). (Note that inour residue numbering scheme, residues 0, 21, 42, 64, 84 and 105 correspond to theN-terminal acetyl groups of the Alm molecules).

5.3.2 Helix fluctuations and secondary structure

In figure 5.3 the CαRMS fluctuations (from the mean structure over the courseof the simulation) are shown as a function of residue number. The most strik-ing general trend is that the RMS fluctuations are greater for the C-terminalsegments of the helices than for the N-terminal segments. The magnitude ofthe fluctuations is greater for N6(H) than for a single helix either inserted in aPOPC bilayer or in solution in MeOH, but less than the fluctuations of a singleAlm helix in water. For a majority of the helices (i.e. H1, H2, H3, H4 and H5)the RMS fluctuations of the C-terminal segment are significantly greater forN6 than for N6H.

A similar pattern emerges from analysis of the secondary structures of theconstituent helices of the two bundles as functions of time (figure 5.4). Over-all, each peptide molecule retains a largelyα-helical conformation throughoutthe 2 ns. In all cases, there is greater deviation from α-helicity, and from afixed secondary structure, in the C-terminal segments than in the N-terminal

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5.3 Results

0 250 500 750 1000 1250 1500 1750 2000

H1

H2

H3

H4

H5

H6A

helix

# (

N6)

time (ps)

0 250 500 750 1000 1250 1500 1750 2000

H1

H2

H3

H4

H5

H6B

helix

# (

N6H

)

time (ps)

Figure 5.4. Secondary structure, as defined by DSSP (Kabsch and Sander, 1983), as afunction of time for: A Alm N6; and B Alm N6H. The greyscale is: white is coil, lightgrey turn, dark grey 3-10 helix and black α-helix. H1 to H6 denote the constituenthelices of the bundles.

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5 An alamethicin channel

0 2 4 6 8 10 12 14 16 18residue

−120

−90

−60

−30

0

30

Φ o

r Ψ

(de

gree

s)

N6 ΦN6 Ψ

0 2 4 6 8 10 12 14 16 18 20residue

N6H ΦN6H Ψ

A B

Figure 5.5. Time averaged values of backbone Φ and Ψ angles vs. residue number,for all of the helices of A Alm N6 and B Alm N6H.

segment of the helices. The overall level of fluctuation in secondary structureis possibly a little lower for N6 than for N6H, but the difference is not striking.Comparison with fluctuations in the secondary structure of an isolated Almhelix reveals that in the hexameric bundles, the fluctuations in the secondarystructure of the C-terminal segment are intermediate in magnitude betweenthose of the isolated helix in an apolar environment, and in an aqueous envir-onment. This mirrors the conclusion reached on the basis of the Cα RMSFs(see above). Looking in more detail at the secondary structure, there is someevidence for formation of 3-10 helix in the C-terminal half of the peptide mo-lecule (e.g. N6, helix H1, t ∼1000 ps and N6H, helix H5, t = 1200 to 2000 ps).There is obvious variation from helix to helix within a bundle (e.g. helix H4vs. H5 in N6; helix H3 vs. H4 in N6H), implying that the helix bundle does notexhibit exact six-fold symmetry. Again, comparison of N6 and N6H suggestsa somewhat greater degree of structural fluctuation in the former.

The time-averaged backbone dihedral angles (also averaged over the sixhelices of each bundle) for the two simulations are shown in figure 5.5. Inter-estingly, these show almost exactly the same pattern as for MD simulationsof an isolated Alm helix in MeOH or inserted in a POPC bilayer [78]. Althoughthe fluctuations of the C-termini of the helices are greater in the pore thanin isolated Alm in an apolar environment, the average conformation is thesame. This differs from isolated Alm in water where different values of themean dihedrals are seen.

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5.3 Results

Possible hinge-bending motions of Alm molecules within the hexamericbundles were explored in detail by combining essential dynamics [186] withthe DynDom analysis of Hayward and Berendsen [187] to reveal the majorcomponents of intramolecular motions. However, this analysis was somewhatinconclusive. Although it showed greater intramolecular mobility for the N6simulation than for the N6H, the analysis did not reveal the hinge-bendingtype motion that we have previously encountered within isolated Alm helices,both in MeOH and when inserted (not in bundles) in a POPC bilayer.

5.3.3 Bundle fluctuations

It seems that the C-terminal segments of the component helices show greaterintramolecular fluctuations for N6 than for N6H. Together with the differenceseen in the Cα RMSDs, this suggests that the stability of the bundle might alsodiffer between N6 and N6H. This is supported by visual examination of Cαtrace snapshots of the N6 and N6H bundles taken at 500 ps intervals alongeach trajectory (figure 5.6A-D). The N6 bundle expands somewhat as time pro-gresses. This is confirmed by comparison of the radius of gyration as functionof time for the two systems (figure 5.7). Changes are particularly evident forthe C-terminal segments of the helices, the packing of which is considerablydisrupted (relative to the starting model) in order to allow the charged Glu18sidechains to move away from one another. In particular, as a consequenceof such changes in packing, one helix (helix H4) no longer seems to have itsN-terminal segment packed with those of its neighbours. In contrast, in sim-ulation N6H, the N-terminal segments of the helices remain packed with oneanother much as in the initial model. The differences in the interactions canalso been seen in the orientations of the Glu18 sidechains (figure 5.6EF). InN6H these continue to point towards the lumen of the channel, whereas inN6 a number of the (charged) Glu18 sidechains are pointing away from thechannel lumen at the end of the simulation.

Overall, the N6H helix bundle is more stable than the N6 bundle over theduration of the simulation. We will now examine the nature of the H-bondinginteractions of the pore with the bilayer and with water, and the behaviourof water within the pore in more detail. We will focus on N6H, although somecomparisons will be made with N6 in order to assess the robustness of ourconclusions.

5.3.4 Bundle interactions

The amphipathic Alm helices in the bundle are in an anisotropic environ-ment, with their apolar sidechains directed towards the surrounding lipids

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5 An alamethicin channel

A B

C D

FE

Figure 5.6. A - D Cα traces of snapshots (at 0, 500, 1000, 1500 and 2000 ps) from theA,C Alm N6 and B,D Alm N6H simulations. In A,C the helix bundles are viewed downa perpendicular to the bilayer normal, with the C-termini of the helices uppermost.In B,D the view is from the C-terminal mouth of the pore, down the bilayer normal.E,F Structures of the E Alm N6 and F Alm N6H simulations at t = 2000 ps. Figuresdrawn using Molscript [79].

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5.3 Results

0 250 500 750 1000 1250 1500 1750 2000time (ps)

1.35

1.37

1.39

1.41

1.43

1.45

1.47

1.49

radi

us o

f gyr

atio

n (n

m)

N6N6H

Figure 5.7. Radius of gyration of the Alm bundle as a function of time for the AlmN6 and Alm N6H simulations.

and their polar sidechains towards the water within the pore. Furthermore,the polar residues at the C-termini of the helices (i.e. Glu(H)18, Gln19 andthe terminal hydroxyl of Phl20) are close to the lipid headgroups. Compar-ison of the numbers of H-bonds formed by the Alm helix bundle (simulationN6H) with water molecules (figure 5.8A) and with lipid molecules (figure 5.8B)show that bundle/water H-bonds predominate. However, a number of pep-tide/lipid H-bonds do occur, from the sidechains of GluH18 and Gln19, andthe terminal hydroxyl of Phl20 to the phosphate, glyceryl and acyl oxygensof the lipid. The number of these H-bonds rises during the first ∼800 ps ofthe simulation (figure 5.8D) until there is about one H-bond to lipid for eachAlm helix (a similar pattern is seen for the N6 simulation - data not shown).This is expected to contribute to the stability of the helix bundle, by helpingto “anchor” it into the lipid bilayer.

Given the preponderance of peptide/water H-bonds, and the crucial rolethese must play in stabilising a pore (as opposed to a “closed” bundle ofhelices), it is important to examine these in more detail to identify the re-lative contributions of the polar residues of the Alm helix to the lining ofthe pore. First we look at the H-bonds to water molecules of the Gln7 side-chains (figure 5.8C). Gln7 was first suggested to play a role in stabilising the

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5 An alamethicin channel

0.0

60.0

120.0

180.0

num

ber

peptide/water

Gln7/water

Gly11/water

0.0 500.0 1000.0 1500.0 2000.0time (ps)

Gln19/water

10.0

20.0

30.0

num

ber

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10.0

20.0

30.0

num

ber

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num

ber

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ber

peptide/lipid

Aib10/water

GluH18/water

Phl20/water

0.0 500.0 1000.0 1500.0 2000.0time (ps)

10.0

20.0

30.0

num

ber

0.0

10.0

20.0

30.0

num

ber

0.0

10.0

20.0

30.0

num

ber

0.0

A

C

E

G

B

D

F

H

Figure 5.8. Analysis of H-bonding in the Alm N6H simulation. Number of H-bondsas a function of time are shown for: A peptide to water; B peptide to lipid; C Gln7sidechains to water; D Aib10 carbonyl oxygen to water; E Gly11 carbonyl oxygen towater; F GluH18 sidechains to water; G Gln19 sidechains to water; and H the terminalhydroxyl of Phl20 to water.

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5.3 Results

Alm helix bundle when the X-ray structure of Alm was determined [164,197].More recently, combined experimental and computational studies have sup-ported a role for this sidechain in pore stabilisation [201,202]. Counting thenumber of H-bonds to water made by this sidechain reveals that between 20and 25 H-bonds per hexameric bundle (i.e. ∼4 H-bonds per sidechain) aremaintained throughout the N6H simulation. Similar behaviour is seen for N6(not shown). Moving along the Alm helix, it has been suggested [182] that theproline-induced helix kink exposes the backbone carbonyl oxygen atoms ofAib10 and Gly11, thus enabling these backbone groups to contribute to thepolar lining of the pore. Enumeration of H-bonds reveals that this is indeedthe case, although it seems that the Aib10 carbonyl (figure 5.8D) is rathermore solvent exposed than is that of Gly11 (figure 5.8E).

The (protonated) Glu18 sidechains maximise their H-bonding to pore wa-ter (figure 5.8F). The sidechains of Gln19 (figure 5.8G) also form a significantnumber of H-bonds to water. Taken together, these two rings of sidechainscontribute ∼30 H-bonds to water throughout the simulation. Finally, the C-terminal hydroxyl group of Phl20 (figure 5.8H) forms ∼two H-bonds to waterper Alm monomer. The numbers of sidechain/water H-bonds for the N6Hbundle do not vary greatly with respect to time, whereas the correspondingnumbers for the less stable N6 bundle (data not shown) show significant driftas the helices move apart.

5.3.5 The pore and its water

As seen in previous simulation studies (in the absence of a bilayer [122]) thereis a well defined column of water within the lumen of the Alm helix bundle. Atits narrowest region, in the vicinity of the Gln7 ring, this accommodates only4 or 5 water molecules. As the water within a pore is crucial to the permeationof ions through that pore, we have examined the nature of the pore and itswater in some detail. The pore radius profile has been determined every 50ps. The resultant time-averaged radius profiles for N6 and N6H are comparedin figure 5.9. Despite the lesser stability of the N6 bundle, the profiles arerelatively similar. That of N6H has a smaller minimum radius, as might beexpected given the expansion of the N6 bundle noted above. There are twoconstrictions, the narrower one being in the vicinity of the Gln7 ring and theslightly wider one in the vicinity of the Glu(H)18 ring. At its narrowest pointthe pore has a radius of ∼0.25 nm, and so partial dehydration of e.g. a K+ ion(ionic radius 0.13 nm) may occur in this region during permeation, althoughit should be remembered that the Gln7 sidechains may exhibit flexibility. Onthe basis of this pore radius one may obtain an approximate prediction ofthe pore conductance using the methods of Smart et al. [147]. For 1M KCl

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5 An alamethicin channel

0.0 1.0 2.0 3.0 4.0 5.0 6.0z (nm)

0.00

0.25

0.50

0.75

1.00

1.25

1.50

radi

us (

nm)

N6N6H

Figure 5.9. Pore radius profiles, evaluated using HOLE [205], for the Alm N6 and AlmN6H simulations. The profiles were averaged from structures saved every 50 ps. Errorbars represent standard errors.

this yields a predicted single channel conductance of 260 pS, which is inagreement with the corresponding experimental value of 280 pS [147,206].

The water diffusion coefficient along the pore axis was calculated fromtheir mean square displacement over a period of 5 ps (figure 5.10). A diffusioncoefficient was assigned to the local region on the pore axis correspondingto the position of the water molecule at the start of the 5 ps period. Notethat those waters within the pore are located between z = 1.5 and z = 4.5nm. The diffusion coefficients of waters within the pore are considerably re-duced relative to bulk water. In the narrowest region of the pore (z ∼2.8 nm,corresponding to the Gln7 ring) the water diffusion coefficient falls to ∼0.4× 10−9 m2 s−1, about one twelfth of its bulk value. A similar reduction inthe translational motion of water within narrow pores was found in simplersimulations [122]. The motion of water is also reduced close to the surfaceof the bilayer, i.e. at z ∼1.0 nm and z ∼5.0 nm [20,32].

The dipoles of the water molecules within the pore are oriented by thehelix dipoles (figure 5.11). Thus, within the pore the mean z component ofthe water dipoles is nearly 2.0 Debye, which should be compared with a dipolemoment of 2.3 Debye for a single SPC water. Much the same orientation of

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5.3 Results

0.0 1.0 2.0 3.0 4.0 5.0 6.0z (nm)

0.0

1.0

2.0

3.0

4.0

5.0

6.0

D (

10−

9 m2 s−

1 )

N6N6H

N C

Figure 5.10. Water diffusion coefficients (Dz) as a function of position along the pore(z) axis for the Alm N6 and Alm N6H simulations.

water dipoles within the pore is seen whether one analyses N6H (no ionisedsidechains) or N6 (six ionised sidechains). However, in the latter case thereis some local orientational polarization of the water dipoles in the vicinity ofthe Glu18 ring superimposed upon the overall orientational polarization dueto the helix dipoles. Given the differences in helix packing (less ordered) inthe N6 simulation, this suggests that this effect of the helix dipoles on thewater is robust to the details of the model, and so might be expected to be ageneral feature of those ion channels whose pores are formed by bundles ofapproximately parallel α-helices.

The observed degree of orientation of the water dipoles may be employedto calculate the local field experienced by water molecules within the pore.Thus, the maximum value of µz = 1.9 Debye in the middle of the pore (fig-ure 5.11) should be compared with µ0 = 2.3 Debye for SPC water (where µ0 isthe dipole moment of water and µz is its projection along the z (pore) axis).Using the Langevin equation,

µz = µ0

[coth

(µ0EzkBT

)−(kBTµ0Ez

)](5.1)

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5 An alamethicin channel

0.0 1.0 2.0 3.0 4.0 5.0 6.0z (nm)

−1.0

−0.5

0.0

0.5

1.0

1.5

2.0

µ z (D

)

N6N6H

N CE18

Figure 5.11. Projection of water dipole moments onto the pore axis for the Alm N6and Alm N6H simulations

where Ez is the z component of the electrostatic field due to the helix dipoles,and kB and T are the Boltzmann constant and temperature respectively, thisyields a field Ez = 3.4 109 Vm−1. (See [207] for a more detailed description ofthe theory of interaction ofα-helix bundle dipoles with pore water molecules).This “observed” field can be compared with an approximate prediction of theelectrostatic field generated by the bundle of aligned α-helix dipoles. An es-timate of the latter field was obtained by numerical differentiation of the elec-trostatic potential energy along the pore axis of an Alm helix bundle in vacuo.To obtain the electrostatic potential along the pore axis, just the backboneatoms (with Charmm partial atomic charges) were used, although inclusionof sidechain atoms did not make a significant difference as long as the negat-ive charges on the carboxylates of the glutamate residues were omitted. Notethat this approximate prediction is a simple Coulombic field, and does nottake into account the environment surrounding the helix bundle. The fieldestimated in this fashion had a maximum value in the centre of the pore of∼1.5 109 Vm−1. This is in good agreement with the ’observed’ field calculatedfrom the Langevin equation (given the approximations involved). This agree-ment confirms that there is indeed a strong interaction between water dipolesand aligned helix dipoles, which will contribute to the stability of the pore.

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5.4 Discussion

The importance of this interaction can be evaluated by comparing the time-averaged helix-helix electrostatic interaction energy within the N6H bundle(∼+140 kJ/mol) with the corresponding bundle-water electrostatic interactionenergy (∼-2950 kJ/mol). It is clear the water-bundle interactions more thancompensate for the unfavourable helix-helix interactions, as was suggestedby previous simulations [122].

5.4 Discussion

5.4.1 Scope of the simulations

The experimental properties of Alm channels are well known [162] and canbe compared to the results from the simulations. For example, the singlechannel conductance predicted on the basis of the simulated Alm N6H struc-ture is in good agreement with that determined experimentally. Furthermore,continuum electrostatics calculations have allowed us to pay attention to thelikely ionisation states of the Glu19 sidechains. In particular, the stability ofthe Alm N6H bundle during the simulations provides further evidence thatthis model may be a reasonable approximation to the true structure of (thisconductance level of) the alamethicin channel.

However, these simulations remain an approximation to the true prop-erties of the system. The conductance calculations are mainly based on thechannel geometry, which is a severe simplification. A more rigorous studyof the conductance of the Alm channels requires potential of mean forcefield calculations for ions passing through the channel. The current simula-tions span 2 ns, which is still a relatively short period of time, both in thecontext of lipid motions [19] and of the mean time it takes an ion to movethrough a channel (∼10 to 100 ns). Furthermore, the simulations have beenconducted in the absence of a transbilayer voltage difference. In this studythis is not a serious problem, as the work of e.g. He et al. [198] suggeststhat Alm helix bundles form in the absence of such a voltage difference, eventhough Alm pore formation is voltage-dependent at the low peptide:lipid ra-tios employed in electrophysiological studies. However, a number of recentsimulations [5,209,210] and theoretical [192] studies have explored the mod-elling of a transbilayer voltage difference, and it will be important to conductsimulations similar to those described in this paper in the presence of suchan external electrostatic field. Another limitation is that only a hexamericbundle of Alm molecules has been considered. Models of smaller (N = 4 and5) and larger (N = 7 and 8) Alm helix bundles have been generated in earlierstudies [148] and will form the basis of future MD simulations in an explicitbilayer plus water environment [211]. There are further limitations to the sim-

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ulations, such as the absence of electrolyte and the treatment of electrostaticinteractions. Typically, experimental studies of Alm channels are conductedusing at least 0.5 M electrolyte, corresponding to ∼50 ions in the system usedin this simulation. This is an area which future studies will have to address.The simulation protocol we have used has been shown to give reasonableagreement with experimental results for pure bilayer simulations [19] and toyield stable simulations for the porin OmpF in a POPE bilayer [78]. However,a number of studies have been concerned with the effects of different treat-ments of long-range electrostatic interactions [19,40]. It is likely that in thepresence of explicit charges long-range interactions should be treated moreaccurately.

5.4.2 Biological significance

Despite the limitations of the simulations, the properties of the Alm N6Hbundle and of its interactions with water and lipids are of some significance.In particular, the Alm helix bundle may be thought of as a paradigm of otherion channels formed by helix bundles, including the pore domains of the in-fluenza M2 protein proton pore [123,212] and of the nicotinic acetylcholinereceptor [189, 190, 213]. Furthermore, the recently determined X-ray struc-ture of a bacterial K+ channel [194] reveals a pore based upon an α-helixbundle motif, into which the pore-lining P-domain is inserted in order to con-fer greater ion selectivity. As it seems likely that this basic architecture willbe found in voltage-gated K+, Na+ and Ca2+ channels [213], the parallel helixbundle motif may prove to be widespread among a wide range of ion chan-nels. Simulations can be a useful tool to study such bundles [208,209].

One property which clearly emerges from the current study is the altereddynamics of water molecules within the pore. This has also been seen in com-parable simulations of the bacterial porin OmpF [78]. Significantly, an almostidentical effect on dynamics of water with Alm and other model pores was ob-served in previous simulations of a pore plus water system in the absence ofa bilayer model [122]. This is important in that it suggests such simpler simu-lations may capture the essence of the dynamics of water (and, by extension,of ions [214,215] in models of transbilayer pores. The other unusual propertyof water within Alm channels is the alignment of the water dipoles along thepore axis. This was also observed in the earlier simulations [122, 216], andcurrent calculations demonstrate rather conclusively that the water dipolesare aligned by the electrostatic field created by the parallel dipoles of theconstituent α-helices of the bundle. Such alignment of intra-pore water di-poles has important consequences for more mesoscopic treatments of chan-nel properties. As discussed by Sansom et al., alignment of the water dipoles

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by the helix dipoles means that if the water within the pore is modelled asa continuum a somewhat lower dielectric than that of bulk water should beused [207]. However, there are other, more fundamental problems in treatingnearly dielectrically saturated water as a simple dielectric continuum. Fur-thermore, as discussed above, helix/water dipolar interactions contribute tothe stabilisation of a parallel α-helix bundle.

A further way in which the Alm helix bundle may be of relevance toother ion channels is in the flexibility conferred on the helices by the pro-line residues at position 14. From the DSSP analysis, and from visualisationof superimposed structures, it is evident that even in the Alm N6H simula-tion the C-terminal segments of the Alm molecules undergo dynamic confor-mation changes. Comparison with simulations of an isolated Alm moleculein water, in MeOH and spanning a POPC bilayer [217] suggest that in N6Hthe degree of conformation flexibility is greater than that of the isolated Almmolecule in a trans-bilayer (POPC) or bilayer-mimetic (MeOH) environment,but is less than that of Alm in water. This is presumably because in N6Hthe Alm helices are exposed to water only on the pore-lining surface. Thus,the Alm channel is a dynamic assembly, with rapid structural fluctuationsaround its C-terminal mouth. This is also of interest in the context of K+

channels. The recent bacterial KcsA channel structure has a bundle of fourinner helices which appear to restrict the size of the intracellular channelmouth. The equivalent S6 helices of voltage gated K+ channels (e.g. Shaker)contain a Pro-Val-Pro motif, which in vacuo MD simulations suggested mightact as a hinge-bending motif. Recent MD simulations of isolated S6 helices ina POPC bilayer (Shrivastava, Sansom and Forrest - unpublished results) con-firm that hinge-bending motion occurs within a bilayer environment. Thus, K+

channels may exhibit fluctuations in their pore dimensions similar to thoseseen in Alm channels.

5.4.3 Future directions

This work has demonstrated that MD simulations of Alm including a lipidbilayer plus water enable unrestrained simulations of a channel forming helixbundle to be carried out in a realistic model of its natural environment. In thecontext of the large body of experimental data concerning Alm channels [162],there are a number of ways in which this work may be extended. Firstly,in order to understand better the energetics of pore/ion interactions, freeenergy profiles [68, 218–220] for the ion as it moves along the pore shouldbe calculated. In this context, the result in the paper suggesting that waterdynamics and orientation in the full bilayer simulation are very similar tothose in earlier, restrained and in vacuo simulations may be helpful. Secondly,

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simulations should be extended to models of other values of N helices/bundlein order to see whether the agreement between predicted and experimentalpore conductance found in the current study is maintained. Finally, the effectsof varying the helix bundle environment, by including a transbilayer voltageterm [5,209] and by using different phospholipids, should be explored.

Ultimately, it should be possible to link microscopic models such as thosein this paper to experimentally measured current-voltage curves and build acomplete picture of the dynamics and energetics of an ion channel, albeit asimple one.

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