Computational and Theoretical Polymer Science 10 (2000) 97–102
Molecular dynamics simulations of hydrophobic and amphiphatic proteins
interacting with a lipid bilayer membrane
J.-H. Lin, A. Baumgaertner*
Forum Modellierung, Forschungszentrum, 52425 Jülich, Germany
Received 19 April 1999; received in revised form 21 June 1999; accepted 27 June 1999
Abstract
Molecular dynamics simulations of polypeptides at high dilution near a fully hydrated bilayer membrane have been performed. In contrast
to previous theoretical predictions, Monte Carlo simulations and conclusions from experiments a spontaneous insertion of amphiphatic or
hydrophobic proteins into a membrane is not observed. Rather it is found that an amphiphatic chain has the tendency to remain in proximity
to the membrane surface, whereas the location of a hydrophobic chain is more unbound. This is shown using two proteins, melittin and
polyleucine. The conformation of the proteins and their orientation with respect to the membrane surface are discussed. q 2000 Elsevier
Science Ltd. All rights reserved.
Keywords: Molecular dynamics simulation; Hydrophobicity; Membrane; Proteins
1. Introduction
The adhesion at and partitioning of proteins into
membranes and their subsequent folding are fundamental
processes in biological cells. It has been known for a long
time that short proteins, in many cases toxins, adsorb and
translocate spontaneously into membranes [1,2]. Despite
many experimental facts indicating spontaneous adsorption
and insertion under certain experimental conditions, our
theoretical understanding of this translocation process is
still poor [3–11]. In the present work we report on some
results from molecular dynamics simulations of two
proteins, melittin and polyleucine, interacting at high dilution with a fully hydrated lipid bilayer.
Melittin is 26 amino acids long including the C terminus
and the N terminus. It has the sequence (H2N-Gly-Ile-Gly pAla-Val-Leu-Lys p -Val-Leu-Thr-Thr-Gly-Leu-Pro-AlaLeu-Ile-Ser-Trp-Ile-Lys p -Arg p -Lys p -Arg p -Gln-GlnCONH2) where charged amino acids are indicated by an
asterisk. Melittin is the major protein component of the
venom of the European honey bee Apis mellifera. It has a
hemolytic activity. The N terminus part (Ile-Gly-Ala-ValLeu) is more hydrophobic, whereas the anchor sequence
* Corresponding author. Tel.: 1 49-2461-61-4074; fax: 1 49-2461-612983.
E-mail address: [email protected] (A. Baumgaertner).
(Lys p-Arg p-Lys p-Arg p-Gln-Gln) is positively charged and
hence strongly hydrophilic.
Polyleucine (Leu25) consists of 25 identical side chains of
the amino acid leucine. The C terminus and the N terminus
are acetyl group and amine group, respectively, and therefore this peptide is completely uncharged. Leucine is one of
the most hydrophobic amino acids. According to the
Roseman scale [12] or the Eisenberg scale [13] the average
hydrophobicity of Leu25 is 275 kcal/mol and that of melittin
is about 2 13 kcal/mol. This energy scale is the relative free
energy change of transport from an aqueous to a nonpolar
environment.
Since Leu25 and melittin are very hydrophobic, one would
expect that these proteins would spontaneously insert into
the membrane. This view has been supported by Monte
Carlo simulations using a simplified membrane model
[4,5,9]. However, as it will be shown by the present work,
more detailed atomistic models do not support the evidence
of a spontaneous insertion. The main reason of previous
shortcomings is probably the inadequate modeling of the
water–membrane interface. This interface essentially
prevents the proteins to come into close contact with the
nonpolar core of the bilayer membrane.
It is important to note that our findings are in agreement
with observations and experiments on analogs of melittin,
like alamethicin [6] and others [14], at very high dilution. At
higher concentrations, however, experiments indicate insertion of melittin in a probably aggregated form [15].
1089-3156/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S1089-315 6(99)00062-8
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Fig. 1. The distance z(t) of the center-of-mass of melittin and polyleucine
with respect to the midplane between two adjacent membrane surfaces as a
function of time t.
2. Model and simulation techniques
The membrane consists of a bilayer of 2 × 100 lipids of
the type POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine). Each lipid consists of 134 atoms. Carbon
atoms with hydrogen atoms are modeled as united-atoms,
which totally reduces the number of atoms of one lipid
molecule from 134 for an all-atom model to 52 for the
united atom model. The partial charges of the head group
are adapted from previous studies [16,17]. The initial
configuration of the lipids in their liquid-crystalline phase
was equilibrated [17] together with 10 951 TIP3P water
molecules [18]. The Particle–Mesh–Ewald method was
used for the Coulomb interaction calculation. The total
system consists of 40 467 atoms for the melittin case and
40 509 atoms for the case of polyleucine. More details on
the preparation of the membrane–water system and the
conformational properties of the lipids can be found elsewhere [17].
We used periodic boundary conditions in all three directions. Therefore, it should be noted that in the z-direction the
periodic boundary condition leads to a periodic stack of
bilayer membranes. At equilibrium the distance between
the membranes corresponds to the width of the water
layer which is about 46.7 Å.
The initial structure of melittin was taken from Protein
Data Bank (PDB code: 2mlt). This peptide together with six
Cl 2 counter ions was solvated with 6509 water molecules.
After 500 ps MD simulation at 300 K and 1 bar, the melittin
lost part of its a-helicity and became more disordered.
Melittin, along with Cl 2 ions and the water molecules
within the box defined by the outermost positions of Cl 2
ions, were then transplanted into the water layer of the
POPC membrane system by removing almost the same
size of box of water molecules. The concentration of melittin corresponds approximately to 10 mM.
The initial conformation of Leu25 was taken to be similar
to that of melittin, where the backbone structure of Leu25
was taken from that of melittin, and the side chains of
melittin were replaced by that of leucine. Since the new
polypeptide was totally uncharged at all, the six Cl 2 ions
were removed from the model system.
Molecular dynamic simulations were performed by the
SANDER program in amber [19]. The simulations were
performed at T ˆ 300 K in the NPT ensemble. The lateral
pressure and the perpendicular pressure were P ˆ 1 bar.
Since explicit water molecules were included in the simulation as solvent, no distance-dependent dielectric constant
was used. The atomic coordinates were saved every 1 ps
and the atomic velocities were saved every 10 ps to reduce
the cost for the need to rerun some part of the simulation. It
takes about 0.1 h for 1 ps run on the CRAY T3E using 32
PEs.
Since polyleucine is totally uncharged, the partial charges
and other force field parameter modifications were expected
to be unnecessary. Similar treatment was reported from a
translocation study of a shorter polyleucine peptide across
the hexane–water interface [20,21]. On the other hand, the
positive charged residues of melittin on the N-terminal end,
GLY and LYS, would be likely to be deprotonated upon
entering the oily phase. But since melittin was still in the
aqueous phase during the whole simulation, the change of
the protonation state was not expected.
3. Results and discussions
3.1. Adhesion versus insertion
Fig. 2. Density profiles of melittin, water, and lipid heads.
The locations z(t) of the two proteins with respect to the
membrane surface as a function of time are presented in Fig.
1. Since the protein is trapped between two adjacent
surfaces of a stack of membranes, we have located our
fixed coordinate system at the midplane between these
two surfaces. The locations of the upper and the lower
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99
Fig. 3. Snapshot of melittin. The picture was prepared by using MolScript v2.1 [30].
membrane surfaces are characterized by the average z-coordinate of the phosphate groups, PO4, of the lipid heads of the
POPC lipids. According to Fig. 1 the average distance
between the surfaces is < 47 Å. This distance is sufficiently
large to accommodate the proteins which may adopt a maximum length in the helical state of about 32 Å. The locations
of the two proteins are characterized by the z-components of
their centers of mass.
From our results of z(t) obtained during MD simulations
up to 1 ns, we found no indications towards the onset of
insertion into the membrane. Even strong perturbations of
the membrane surface were not observed. Rather, the
center-of-mass positions of the proteins fluctuate around
the midplane of the water region. Direct contact of the
molecules with the membrane is very weak, which can be
seen from the average density profile of melittin as shown in
Fig. 2 where the overlap between the profiles of melittin and
the PO4 groups of the lipid heads is very small. Although
melittin is located in proximity to only one membrane
surface rather than fluctuating between both sides, it is not
conclusive whether this indicates an affinity or even adhesion to one of the membrane surfaces. It is equally conceivable that this is an effect from the limited simulation time;
for much longer time one could expect a shift or broadening
of the density profile. In summary, it should be noted that
the melittin is not buried in the interface as expected from
experiments [22,23]. There it was estimated that about 40%
of the melittin surface is embedded in the hydrophobic part
of planar POPC bilayers.
In addition, our findings are in contrast to previous Monte
Carlo studies on the insertion process of amphiphatic
proteins [4,5,9]. These studies have indicated that melittin
at very high dilution must be expected to insert into the
hydrophobic core of the membrane, basically driven by
the hydrophobic effect, as predicted by the two-state
model [1,2]. The main reason for the discrepancy is basically due to the concept of a two-state phenomena which
neglects the kinetics of the translocation process. The
kinetics, however, is determined by the subtleties of the
interfacial barrier near the lipid heads which separate the
hydrophobic core of the bilayer from the aqueous medium.
It is noted that our result shows different behavior of
polyleucine at such a membrane–water interface from that
of a previous study for polyleucine at the hexane–water
Fig. 4. The helicity h(t) of melittin and polyleucine as a function of time t.
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Fig. 5. The bending V (t) of melittin and polyleucine as a function of time t.
interface [20,21]. It was reported that from the beginning
polyleucine moved rapidly towards the hexane–water interface at a speed of 11 Å/ns. It is very clear from our simulation that the head groups of phospholipids form a very
different interface from the hexane–water interface. During
the course of our simulation, the hydrophobic peptide has
not yet recognized the preferred hydrophobic phase in the
acyl chain region.
3.2. Conformational properties
Melittin and Leu25 are comparably short proteins which
would serve perfectly as single transmembrane-spanning ahelices. In fact, melittin is expected to have a predominant
helical structure with a kink approximately at the position
14 caused by proline which do not form hydrogen bonds
[24]. In the aqueous medium this structure is not changed
significantly. Since melittin is fairly short, it cannot fold
properly in order to exhibit a compact tertiary structure
which could shield the hydrophobic side chains from
contact with water. A totally coiled structure in water is
not expected.
The conformation of melittin is remarkably stable over
the whole time scale of 1 ns. One snapshot is presented in
Fig. 3. The snapshots indicate that melittin has a triangular
or open hairpin-like conformation where the hairpin is
formed by two a-helices separated by a kink induced by
Pro14.
The opening angle V < 45 between the two helical axes
of the hairpin is remarkably stable over the whole period of
simulation and is shown in Fig. 5.
The a-helicity of the peptide, h(t), is determined by the
O(i) to H–N…i 1 4† distances. According to the amber ‘91
force-field hydrogen bond parameters for these two atom
types, we found it is quite adequate to define hydrogen
bond length as 2.5 Å. Thus a hydrogen bond between residues i and i 1 4 is formed when the O(i) to H–N…i 1 4†
distance is less than or equal to 2.5 Å. Since there are 26
residues in melittin, a perfect a-helix of melittin could form
22 hydrogen bonds, therefore we can define the total ahelicity h of melittin as the number of hydrogen bonds in
this peptide divided by 22. The helicity of melittin as a
function of time, h(t), is presented in Fig. 4. It is found
that the average helicity of melittin is about khl < 0:75
and does not change significantly as a function of time.
Similar observations as for melittin are found for Leu25.
Starting with a disordered conformation, this polypeptide
assumes after 400 ps an a-helix with a high degree of
order. The helicity as function of time is shown in Fig. 4.
The bending angles of melittin and polyleucine as a function
of time are shown in Fig. 5 and the snapshot after 700 ps is
depicted in Fig. 6. The conformational transition from a
disordered to a perfect helical structure can also be inferred
from the opening angle V (Fig. 5) which is defined by the
angle between the two helical axes pointing from the middle
of the chain to both ends.
After 500 ps the molecule is stretched exhibiting an opening angle close to 1808. The interesting observation is that
Fig. 6. Snapshot of Leu25.
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101
A similar procedure can be applied to Leu25. But in this
case u is defined by the angle between membrane surface
normal and the helical axis. From our data the average angle
is < 908 which indicates a parallel orientation to the
membrane surface.
4. Remarks and conclusions
Fig. 7. The orientation u (t) of melittin and polyleucine with respect to the
membrane normal as a function of time t.
although Leu25 has only hydrophobic side chains, its helical
structure is not very much perturbed. This perturbation
could be induced by the competition of forming hydrogen
bonds either internally or with the surrounding water molecules which could lead to a less rigid helical structure and
eventually lead for a much longer helix to a coiled structure.
3.3. Orientation
The orientation of melittin at lipid bilayers is controversial. From IR spectroscopy and CD studies [25,26] it was
concluded that in dry or partially hydrated (97% relative
humidity) bilayers the a-helical portion of melittin is preferentially oriented parallel to the acyl chains. Similarly from
polarized ATR-IR spectroscopy of dry phospholipid multibilayers [27]. Magnetic resonance experiments [28,29] indicate a location of melittin on the membrane surface with
only the hydrophobic residues buries in the lipid bilayer. It
is unclear whether the discrepancy of the different reports on
the orientation of melittin interacting with membranes originates from the techniques or from the different type of
model membrane preparation which was used for its determination.
The orientation of the melittin’s hairpin and the orientation of the helical axis of Leu25 with respect to the surface
can be estimated from our MD results.
In the case of melittin we define two vectors, aN and aC,
for the two branches of the hairpin pointing from residue
Pro14 to the N terminus and the C terminus, respectively.
The orientational vector is defined as
d ˆ …aN 1 aC †=uaN 1 aC u:
…1†
The orientaional angle u between d and the surface normal
is shown in Fig. 7 as a function of time.
From the data shown in Fig. 7 it can be concluded that the
hairpin has a stable orientation with respect to the
membrane surface. The orientation fluctuates around
kul < 40 ^ 108.
It should be noted that the validity of our present findings
relies heavily, amongst other reasons, on the adequacies of
our applied force fields. But this is the current situation and
we are looking forward to seeing whether the present force
fields have to be modified in the future. Of course, in this
situation it would be a great achievement if the reported
simulations could be extended in a more systematic way
by varying some parameters of the force fields or some
external conditions, such as type of lipid head, applied pressure, temperature, among others, in order to elucidate the
conditions under which protein insertion takes place. This,
however, is a long-term research program because several
10 4 atoms with long range interactions needs a considerable
amount of computer time, even on a CRAY T3E. But it is
very likely that the future will see such a kind of systematic
approach to the lipid–protein interaction using molecular
dynamics simulations.
Acknowledgements
It is a pleasure for us to dedicate the present contribution
to Ueli Suter, who has made so many important contributions to polymer science, in particular, in the area of simulations of macromolecules. J.L. is supported by the doctoral
fellowship program of the Forschungszentrum Jülich
(www.fz-juelich.de/mod/biophys).
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