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Biophys J, May 1999, p. 2460-2471, Vol. 76, No. 5
-Helices
*M. M. Shemyakin and Yu. A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Ul. Miklukho-Maklaya, 16/10, Moscow V-437, 117871 GSP, Russia; and #Université des Sciences et Technologies de Lille, Centre de Recherches et d'Etudes en Simulations et Modélisation Moléculaires, Bâtiment C8, 59655 Villeneuve d'Ascq Cedex, France
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHOD OF CALCULATION
RESULTS AND DISCUSSION
CONCLUSIONS
REFERENCES
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We describe application of the implicit solvation model
(see the first paper of this series), to Monte Carlo simulations of several peptides in bilayer- and water-mimetic environments, and in
vacuum. The membrane-bound peptides chosen were transmembrane segments
A and B of bacteriorhodopsin, the hydrophobic segment of surfactant
lipoprotein, and magainin2. Their conformations in membrane-like media
are known from the experiments. Also, molecular dynamics study of
surfactant lipoprotein with different explicit solvents has been
reported (Kovacs, H., A. E. Mark, J. Johansson, and W. F. van
Gunsteren. 1995. J. Mol. Biol. 247:808-822). The principal
goal of this work is to compare the results obtained in the framework
of our solvation model with available experimental and computational
data. The findings could be summarized as follows: 1) structural and
energetic properties of studied molecules strongly depend on the
solvent; membrane-mimetic media significantly promote formation of
-helices capable of traversing the bilayer, whereas a polar
environment destabilizes -helical conformation via reduction of
solvent-exposed surface area and packing; 2) the structures calculated
in a membrane-like environment agree with the experimental ones; 3)
noticeable differences in conformation of surfactant lipoprotein
assessed via Monte Carlo simulation with implicit solvent (this work)
and molecular dynamics in explicit solvent were observed; 4) in vacuo
simulations do not correctly reproduce protein-membrane interactions,
and hence should be avoided in modeling membrane proteins.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHOD OF CALCULATION
RESULTS AND DISCUSSION
CONCLUSIONS
REFERENCES
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Understanding the structure-function relationship
for transbilayer peptides constituting either membrane-bound domains in proteins or functioning autonomously represents an intriguing challenge
in the field of structural biology. It is now established that peptide
conformation is greatly influenced by the environment (e.g., Kelly,
1998
), and a number of chameleon-like molecules have been discovered
which, being placed in different surroundings, are able to change their
secondary and/or tertiary structure (Mihara and Takahashi, 1997).
Indeed, preferences of amino acid residues to form or destabilize
different types of secondary structure, which were characterized by
many researchers in globular proteins (e.g., Fasman, 1989), may differ
from those in the membrane-bound state (Deber and Goto, 1996). Unlike
water-soluble proteins, structural properties of membrane-embedded
systems are studied less because of limitations of current experimental
methods in working with such complex systems. In this situation,
important insights into the problem of peptide adsorption on the
bilayer, membrane insertion, and stability in the membrane-bound state
could be achieved through computer simulations. At the same time,
successful application of these techniques requires correct treatment
of solvent effects.
Various solvation models used in Monte Carlo (MC) and molecular
dynamics (MD) studies of membrane peptides and proteins, along with
their advantages and shortcomings, were discussed in the accompanying
article. Among them, the models with implicit consideration of membrane
effects are of special interest because of their computational efficiency and ability to account for principal trends in protein-lipid interactions. In this approximation the bilayer is usually treated as a
continuous medium whose properties vary along the membrane thickness,
and membrane insertion is simulated using either MC or MD methods.
Off-lattice (Milik and Skolnick, 1993) or full-atom (Ducarme et al.,
1998) representations of the peptide molecules are used. Comparison of
the results obtained in the framework of such models with experimental
data shows that the calculations give good predictions both for the
association state and peptide's orientation relative to the membrane
surface. Moreover, implicit solvent models provide a number of insights
into the mechanism of the peptides' insertion into membranes. At the
same time, during the simulations the peptide's conformation was often
fixed to the -helix, and no changes of the structure were allowed.
Hence, the problems related to conformational rearrangements, like
formation or destabilization of the secondary structure induced by
the environment, could not be addressed by these techniques.
Based upon formalism of atomic solvation parameters (ASP), in the first
article of this series we proposed an implicit solvation model that
mimics effects of membrane environment in simulations of peptides. In
this model, an additional (solvation) term was incorporated into the
ECEPP/2 potential and the resulting force field was tested in MC
simulations of a number of small peptides and 20-residue
homopolypeptides made of Leu, Val, Ile, and Gly. The main objective of
this study is to inspect the solvation model, along with the method of
exploring conformational space, by applying them to MC simulations of
rather complex systems: membrane-bound peptides. Apart from validation
of the approach, such computer experiments are able to provide new
interesting structural and functional information about the peptides in
a particular environment, which is difficult to access with the
experimental techniques. An important feature of the model is total
conformational variability of the molecules under study, which permits
investigation of solvent effects on conformational properties. The
first part of the paper deals with modeling of environmental effects
for transmembrane (TM) -helices A and B of bacteriorhodopsin (BRh),
whose structure in membrane-mimetic media is known from the experiment.
Then we describe conformational behavior of magainin2 and the
hydrophobic segment of surfactant lipoprotein (SP-C), which also were
extensively studied in the experiments, although the atomic-scale
structures are not yet available. The calculated properties are
compared with the experimental data and the results of MD simulations
in explicit solvents reported by Kovacs et al. (1995).
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METHOD OF CALCULATION |
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TOP
ABSTRACT
INTRODUCTION
METHOD OF CALCULATION
RESULTS AND DISCUSSION
CONCLUSIONS
REFERENCES
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The coordinates of TM segments A (BRh-A, residues 10-29) and B
(BRh-B, 34-65) of BRh were taken in the Brookhaven Protein Data Bank,
PDB (Bernstein et al., 1977), entry 1BRD. In the initial structures,
the following residues were in -helical conformation: 11-28 in
BRh-A and 34-65 in BRh-B. Before the calculations, hydrogen atoms were
added to the PDB models. TM segments of human SP-C (residues 6-35) and
magainin2 from Xenopus laevis (residues 83-105) were built
as -helices. This was done using the FANTOM program (von Freyberg
and Braun, 1991). All the peptides were taken with neutral N- and
C-termini.
The conformational space of peptides was explored in nonrestrained,
variable-temperature MC simulations in torsion angle space in vacuo and
with ASPs imitating water and hydrophobic core of a membrane, as
described in the first article of this series. Details of MC
simulations and analysis of the results can be also found there. The
simulation length was 2000 MC cycles. After each MC iteration, the
structures were subjected to conjugate gradient energy minimization.
SP-C was also simulated by the MC method during 14,000 steps with
ASPgc (gas-cyclohexane) and ASPgw (gas-water) at constant temperature (T = 300 K) and without energy
minimization. In this last case the starting conformation was that
found in the result of variable-temperature MC simulation in
cyclohexane. Identical starting conformations were used in simulations
of the same peptide in different environments. Accessible surface area and secondary structure were assessed using the DSSP program (Kabsch and Sander, 1983). Ribbon drawings of molecules were produced with the
MOLMOL program (Koradi et al., 1996).
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RESULTS AND DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHOD OF CALCULATION
RESULTS AND DISCUSSION
CONCLUSIONS
REFERENCES
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To date, spatial structures of a number of peptides in membrane-mimetic environments (micelles, mixtures of organic solvents) have been established in the experiment. This provides a good basis for testing and refinement of theoretical solvation models suitable for simulations of effects of the bilayer on structural properties of peptides and proteins. To check ASPs, we chose the following objects: TM helices BRh-A and BRh-B, SP-C, and magainin2.
Transmembrane segment A of bacteriorhodopsin
BRh is an integral membrane protein that pumps protons across the
cell membrane in response to light absorption. Electron cryomicroscopic
(Grigorieff et al., 1996) data obtained for the whole protein as well
as NMR studies of its TM components (e.g., Pervushin and Arseniev,
1992) reveal -helical conformation of the membrane moiety. Moreover,
some individual membrane-spanning peptides (e.g., BRh-A, BRh-B) retain
their helicity well being in the monomeric state in micelles or in
chloroform/methanol solvent (Pervushin and Arseniev, 1992; Lomize et
al., 1992). Here we explore conformational space of -helices BRh-A
and BRh-B in different environments through variable-temperature MC
conformational search.
Resulting distribution of the backbone (
, 
) and side chain
(
1) torsion angles in BRh-A conformations in
cyclohexane, water, and vacuum, are shown in Fig.
1. Only the low energy conformers (50%
of all accepted structures) were taken for the analysis. Ribbon
representation of the lowest-energy conformers is shown in Fig.
2. As seen in Fig. 1 A, in
simulations with ASPs imitating nonpolar environment (set
ASPgc), the initial -helical geometry is stable during
conformational search: the angles and retain the values
characteristic for -helix. None of the accepted conformers contains
residues in nonhelical conformation. The lowest-energy structure
reveals two side chain/backbone hydrogen bonds (H-bonds): Leu-13:O···H
1:Thr-17;
Met-20:O···H1:Thr-24. It is important to note that
these H-bonds are observed experimentally for BRh-A incorporated into
micelles (Pervushin and Arseniev, 1992).
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As seen in Fig. 1 B, simulations with ASPs imitating polar
solvent (ASPgw) reveal distortion of the -helical
conformation for residues Gly-16 and Leu-22: they are assigned to
H-bonded turn ("T") and five-residue bend ("S") types of
secondary structure. [Here after, identifiers of the secondary
structure are given in the notation of the DSSP algorithm (Kabsch and
Sander, 1983).] Interestingly, the helix unfolding was always observed
near the middle of the peptide, whereas the termini were relatively
stable. Simulation in vacuum (Fig. 1 C) reveals that only
residues Ala-14-Met-20, Thr-24, Tyr-26, and Phe-27 still retain
-helical conformation, whereas the others can adopt the different
ones (in the lowest-energy structure Ile-11, Trp-12, Leu-13, Leu-19,
and Met-20 are assigned to "T" conformation, while Leu-22 is
assigned "S"). In vacuum the helix is significantly distorted at
the ends and in the middle, and two helical fragments (Leu-15-Met-20
and Gly-23-Leu-28) are tightly packed (Fig. 2 C) resulting
in considerable decreasing of ASA. Thus, values of total ASA for the
lowest-energy conformers in cyclohexane, water, and vacuum are 2250, 2050, and 1770 Å2, respectively. The structure in vacuum
has two side chain/backbone (Leu-13:O···H1:Thr-17;
Trp-12:O···H1:Thr-24) and one side chain/side chain
(Thr-17:H1···O1:Thr-24) H-bonds.
Inspection of energies of the conformers accepted during the search in
different environments (Table 1) shows
that the all-helical structure in nonpolar solvent is present within a
wide range of energies: 
E = 28.9 kcal/mol, where
E is the difference between maximal and minimal energies
in the set of conformers, although the energy distribution is centered
near the lowest-energy value (
196.8 kcal/mol): the mean and standard
deviation are 193.4 and 5.8 kcal/mol, respectively. In polar media,
the all-helical state is observed only within a narrow interval of
energies (E = 5.4 kcal/mol). Other states with one
and two residues in nonhelical conformation have values of
E equal to 4.0 and 12.8 kcal/mol, respectively, and the
last one corresponds to the lowest energy minimum. The -helix is
rather less favorable in vacuum: a broad spectrum of conformers with a
helical content from 18 to 4 residues was obtained. Although the
lowest-energy state (E = 156.9 kcal/mol) reveals 11 residues in -helix (Fig. 2 C), several close states with
minimal energies 156.4, 156.3, and 155.2 kcal/mol have 5, 10, and
12 residues in -helix, respectively. Moreover, energy intervals of
these states overlap between each other, and therefore the peptide
could adopt multiple alternative conformations separated by low energy
barriers.
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As seen in Table 1, in all the cases the van der Waals term dominates
the others, whereas relative values of different energy contributions
depend on the solvent used. The ratio between van der Waals, solvation,
and H-bonding terms in cyclohexane is similar for all the conformers.
On the contrary, in water and vacuum the structure is determined by a
balance of the terms, thus leading to multiple states with close total
energies. For example, in water distortion of an initial all-helical
structure (with N
= 18, where
N is a number of residues in -helix) is
governed by decreasing EVDW and
Esolv, which is energetically more favorable than the accompanying breaking of C==O...H---N H-bonds and
increasing EH-bond. The resulting lowest-energy
structure reveals optimal packing (minimal EVDW)
and solvent exposure (minimal Esolv); nonpolar side chains are less accessible to polar solvent as compared with the
initial -helical structure. In vacuum, stable conformations are
tightly packed and demonstrate low values of
EVDW. A significant contribution of
EH-bond is caused by the formation of side
chain/side chain and side chain/backbone H-bonds.
Analysis of the distribution of side chain dihedral angles
1 leads to the following conclusions. 1) A total number
of rotamers for side chains of BRh-A is maximal in vacuum, where
several rotameric states were observed for residues Trp-10, 12, Ile-11,
Leu-15, 19, 22, 28, Thr-24, Tyr-26, Phe-27, Val-29. 2) In cyclohexane and water all residues (except Leu-22 in cyclohexane) have only one
1-rotamer. We should note, however, that analysis of all
accepted conformers shows a somewhat larger number of stable rotamers
in cyclohexane than in water (data not shown). 3) Rotameric states for
Trp-12 are different in polar and nonpolar solvents. To check how
correct the rotameric states extracted from MC simulations in nonpolar
environments are, we compared them with the rotamers obtained from NMR
data in organic solvent and in micelles (Pervushin and Arseniev, 1992;
Table 2). It is seen that the
calculated rotamers agree fairly well with those observed by NMR. Both
experimental and calculated structures are in -helical conformation,
and this is important for comparison of the rotamers because
conformation of the side chain depends on that of the backbone.
Interestingly, the starting structure of BRh-A used in the simulations
was derived from electron cryomicroscopic data, and the initial side
chain conformations deviated greatly from those found by NMR. But in the result, the only difference between the side chain conformations obtained with our solvation model and by NMR is a
1-rotamer of Trp-12. The calculated rotamer
(g
) rarely occurs in the middle of -helices. Most
likely, such a disagreement can be explained by proximity of this
residue to the N-terminus of BRh-A used in modeling, whereas the
experimental data were obtained for fragment 1-36 of BRh, where Trp-12
was displaced far away from the terminus.
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To check sensitivity of the results to the number of ASP types
(M, see details in accompanying paper), we have applied the same protocol to simulate BRh-A with only five instead of eight ASP
types derived from gas-cyclohexane energies of transfer. Analysis of
the low-energy conformers demonstrated that in general the results
resemble those obtained with M = 8, although the
convergence was slower. Thus, 1-rotamers of Leu-22 and
Val-29 were found corresponding to the electron microscopic, but not
NMR, structure. This confirms our choice of the optimal number of ASP
types M = 8 in the solvation model.
Transmembrane segment B of bacteriorhodopsin
The backbone and side chain dihedral angles of fragment
Lys-40-Gly-65 of BRh obtained in the MC conformational search are presented in Fig. 3. It is seen that, as
in the case of BRh-A, the -helix is well-retained in nonpolar
solvent: only Val-49, which is followed by Pro, is not in the helical
but in bend ("S") conformation. This causes a kink of the helix
with the angle ~32° (27° in the NMR-derived models). Calculated
structure in water is quite similar: the helix does not unfold during
long-term MC run, except Val-49 (identifier "S" for the secondary
structure). At the same time, fluctuations of the backbone dihedrals
are somewhat higher in water in comparison with cyclohexane. In both
solvents the lowest-energy conformers reveal approximately the same
values of ASA: 3036 Å2 in cyclohexane and 2997 Å2 in water. On the contrary, the lowest-energy structure
in vacuum is different: the helix is broken on residues Ile-45-Val-49
(secondary structure: "TTSS-"), and the two helical fragments
Ala-39-Ala-44 and Pro-50-Leu-62 are tightly packed, resulting in
compact structure with considerably lower accessible surface (2525 Å2). The peptide's termini are also partially unfolded.
Comparison of 1-rotamers of BRh-B in cyclohexane with
those observed by NMR spectroscopy in micelles (Table
3) demonstrates fairly good agreement
between the calculated and experimental structures for most of the
residues, except Met-60 and Tyr-64. The main difference is a larger
flexibility of the experimental structures, where multiple rotameric
states were found for a number of residues.
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As in the case of BRh-A, the lowest-energy conformers of BRh-B reveal
several side chain/backbone H-bonds that involve H and
O atoms of Ser-35, Ser-59, Thr-47, and Thr-55.
Interestingly, polar side chains of such residues as Ser and Thr are
often found on lipid-exposed faces of TM segments in integral membrane
proteins (e.g., Samatey et al., 1995), where they are stabilized by
forming H-bonds with the helix backbone. As follows from NMR data
(e.g., Pervushin and Arseniev, 1992; Lomize et al., 1992), similar
effects are also observed for monomeric peptides in micelles. The
results obtained with our solvation model also reproduce this
phenomenon: the most energetically favorable conformers of BRh-A and
BRh-B in a membrane-like environment demonstrate stabilization of OH groups of Ser and Thr via H-bonding to the backbone.
To summarize, the structures of BRh-A and BRh-B calculated using
parameter set ASPgc reveal reasonable overall agreement
with the conformations obtained in micelles and
CH3OH/CHCl3 solvent by NMR spectroscopy. The
implicit solvation model with membrane-mimetic parameters reproduces a
strong helix-promoting effect induced by an apolar environment known
from the experiment (e.g., Deber and Goto, 1996). In addition, unlike
other simulations of peptides in bilayers (e.g., Milik and Skolnick,
1993; Ducarme et al., 1998), in our model the molecules were not
restrained to any predefined conformation (e.g., -helix), and the
effect of solvent on intramolecular energy terms was taken into
account. The approach could be efficiently employed for peptides that
traverse a bilayer and stay immersed in the nonpolar core of the
membrane: their conformational, H-bonding, etc. properties (some
deviations from the experiment might occur on the termini exposed to
polar environment) are well described by the ASPgc set.
Surfactant lipoprotein
SP-C is a 35-residue polypeptide essential for the function of
surfactants used for therapy of infant respiratory distress (Johansson
et al., 1995). It has been shown that SP-C could insert into lipid
bilayers in TM orientation (Johansson et al., 1994, 1995 and references
therein). As follows from NMR data (Johansson et al., 1995), in
micelles or mixed organic solvent it forms a highly regular -helix.
Because SP-C contains stretches of seven and four consecutive valine
residues that have prominent 
-sheet forming propensities in water,
it is interesting to assess its conformation in environments of
different polarity. These questions were recently addressed in the work
of Kovacs et al. (1995) who explored behavior of the -helical SP-C
via MD simulations in explicit solvent environments of chloroform,
methanol, and water. It was found that the -helix is stable in water
and methanol, while some destabilization appears in chloroform. Also,
the polyvalyl part (residues 15-21) remains intact even at elevated
temperature, and the valines do not disrupt the -helical
conformation. Therefore, availability of experimental indications on
the secondary structure in micelles and results of explicit
solvent simulations make SP-C a convenient model to check our method
and investigate helix-forming propensities in different environments in
detail. To accomplish this, two series of simulations were performed.
We explored the peptide's conformational space in the vicinity of the
-helical conformation using either variable-temperature MC search
with minimization of the conformers or constant-temperature MC runs without minimization.
Variable-temperature MC conformational search
The results of the variable-temperature MC run with ASPs for nonpolar and polar solvents, as well as in vacuo, are shown in Figs. 2, D-F, 4, and 5. As seen in Fig. 2, the lowest-energy conformers found with ASPgc and ASPgw sets and in vacuo are remarkably different. The initial-helical conformation
is stable in hydrophobic media (Figs. 2 D, 4 A), while in
water and vacuum it is distorted in the central part. In the
lowest-energy structures found in water, nonhelical conformation was
attributed to residues Leu-14-Val-24. Interestingly, unfolding of
initial all-helical structure in water occurs already at the stage of
minimization before executing MC protocol and then it extends, leading
to large conformational changes of SP-C. As was reasonable to expect
based on low helix-forming propensities of valine in aqueous solution,
in water the helix is broken in the region containing seven consecutive
valine residues, and the remaining two -helical segments are packed
together, leading to significantly decreasing total ASA. In the
lowest-energy conformers the values of total ASA in cyclohexane, water,
and vacuum are 3086, 2645, and 2338 Å2, respectively. In
vacuum the helix is not completely destroyed between valines 15-21: it
is partially transformed into 310-helix and reveal a break
on residues Val-15 and Val-16. In this case, loss of the helical
structure is also observed for 4-residue polyvalyl segment 24-26.
These results are different from MD simulations in explicit solvents
(Kovacs et al., 1995), where initial -helical conformation was found
to be well-retained in water and, to a lesser degree, in chloroform.
Unfortunately, no experimental structural data on SP-C in water are
available, and thus it is difficult to judge which model better
reproduces the peptide's behavior in aqueous solution. However,
numerous experimental (e.g., Lyu et al., 1991) and computational (e.g.,
Tobias and Brooks, 1991) data (including those obtained with explicit
and implicit solvents) point to destabilization of the -helix by
valine residues, and therefore agree with our results.
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, , and 1 of the
conformers accumulated during the simulation. As seen from the analysis of and angles, their deviations from standard values for
-helix increase in the order cyclohexane 
water vacuum. In
the nonpolar environment the deviations of angle were observed only
for Leu-14 (N-terminal Cys-6 was not counted) although corresponding
conformers were not the most energetically favorable. In water, large
deviations of and were obtained for residues 14-24, whereas
-helical conformation was retained for residues 7-15 and 25-34. In
vacuum, stable -helix was detected for residues 7-14, 20-21, and
27-33, and helix 310 was found for residues 17-19.
Interestingly, helix distortion in water and vacuum was observed for
stretches of valines in the central part of SP-C, although in nonpolar
media this part was observed only in -helical conformation.
Additional insight into structural properties of SP-C in
membrane-mimetic media is provided by analysis of distributions of resulting conformers over their energies (E). Fig. 5 shows
total energies of conformers combined into several groups according to
their helicity (N) and structural similarity
(in terms of root-mean-square deviation (r.m.s.d.) values). It is seen
that the rod-like helical structures able to traverse the bilayer and containing either 28 (groups 1 and 2) or 27 (group 3) residues in the
-helix demonstrate the lowest energies on a wide interval (~12
kcal/mol). The neighboring group of conformers (group 4, N = 27) is separated by the energy gap of
~3.5 kcal/mol and reveal a pronounced kink on residue Leu-9. This
structure is realized only within small intervals of E (E 
1.7 kcal/mol). Other structures with N = 26, 25, 24, etc. have energies ~5 kcal/mol higher than the
maximal-energy conformers from groups 1-3.
In general, the numbers of 1-rotamers of SP-C in
nonpolar and polar media are close to each other, and in vacuum it is
higher (Fig. 4). At the same time, the rotameric states of valine
residues are different in cyclohexane and water. Thus, in cyclohexane
the 180° rotamer is noticeably populated (except Val-17). This agrees well with the experimental data on SP-C in apolar organic solvent (Johansson et al., 1994). Small fractions of +70° and 60°
rotamers were also found for Val-15, Val-20, and Val-23. In water,
1 angles of valines 15-21 fall close to 60°,
although Val-15 and -19 also have +60° rotamers. On the contrary, the
second stretch of valines (23-26), Val-8, and Val-28 have dominant
rotamers with 1 angles near 180°. In vacuum, most
valines demonstrate two rotamers. Some differences in rotameric states
in nonpolar and polar solvents were observed for leucines 14, 22, and
32: in cyclohexane, 1 angles for all of them are 180°,
while in water they are 60°, 60°, and 180°/60°.
Interestingly, rotamer distributions on the N-terminus (residues 6-13)
are almost similar in both environments.
The side chain rotameric states of SP-C reported above are different
from those obtained in the result of MD simulations with explicit
solvents (Kovacs et al., 1995). Thus, for the 1-ns simulations, 1 angles for valines 15-21 were similar in nonpolar
(chloroform) and polar (methanol) solvents, revealing only slight
dominance of the 65 ± 5° rotamer, and the 180° rotamer was
populated only for Val-8 and Val-15 in the chloroform simulation. For
these two valines we found the same rotamers in cyclohexane, although
the MD data for other valines agree with our results obtained in water, but not in cyclohexane. In general, the results of MD simulations with
explicit solvent and MC conformational search with ASPs are close to
each other, except the valines. Thus, in the case of MD, conformational
freedom of the valine side chains is larger in nonpolar than in polar
solvent, whereas our MC results show that the corresponding numbers of
rotamers are quite similar. It should be noted, however, that the
rotamers found in this work were subjected to energy minimization,
while those reported in the MD study were not.
Constant-temperature MC simulations
Exhaustive conformational search in water shows that the initial-helical structure does not represent the deepest minimum on the
potential energy hyper-surface. Raising temperature combined with
energy minimization permits the system to migrate into the lower energy
subspace, which does not correspond to all-helical structure. It is
interesting to note that these regions of conformational space were not
explored in MD simulations with explicit solvent where the initial
-helical structure was found to be stable. We suppose this is due to
limited length of MD trajectory (1 ns) used by Kovacs et al. (1995),
which did not allow the system to pass the energy barriers separating
all-helical states from the others. To check behavior of the -helix
of SP-C in the vicinity of all-helical conformation and to compare
these data with those obtained using explicit solvent models in more
detail, we performed MC simulation in cyclohexane and water under
"mild" conditions (at constant temperature and without energy
minimization) resembling those used in explicit solvent MD.
Fig. 6 shows distribution of backbone and
side chain dihedral angles of the conformers obtained in MC simulations
with ASPs imitating cyclohexane (A) and water
(B). In both solvents the initial -helix was stable, but
fluctuations of and angles are somewhat larger in water than in
cyclohexane. Conformational mobility of side chains is quite similar in
both cases, although the total number of different rotameric states is
a bit higher in water (Fig. 7). Thus,
maximal r.m.s.d. values between coordinates of backbone atoms of the
starting and successive conformers obtained in the MC runs after 100 steps were 0.65 and 0.99 in cyclohexane and water, respectively.
Although the helix was also stable in 1 ns MD, somewhat larger r.m.s.d.
values were obtained in chloroform than in water. The largest
fluctuations of the backbone and side chain dihedrals were always
observed in the C-terminal part. In general, results of MC simulation
of SP-C agree better with MD data than the results of the more
exhaustive variable-temperature MC conformational search presented in
the previous section. It seems that neither 14,000 MC steps nor 1 ns MD
trajectories were unable to trap the low-energy regions of the
conformational space that are accessible to SP-C in water and which
correspond to distorted -helical structure.
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-helical
conformation of valine-containing peptides in nonpolar media as compared to simulations with explicit solvent. (We should remind that
MD data were collected in chloroform, while in our model we employed
ASPs for cyclohexane which is more hydrophobic.) However, in water our
model demonstrates considerably lower helix-forming propensities for
Val residues than those obtained with explicit solvent. These
inferences are in good accord with the experimental data on
membrane-promoting -helix stability (Deber and Goto, 1996) and
helix-distortion in aqueous solutions (e.g., Lyu et al., 1991; Tobias
and Brooks, 1991).
Magainin2
Magainin2 is a 23-residue antimicrobial peptide from
Xenopus skin that binds to the cell membrane (Zasloff,
1987). The peptide contains many charged and polar residues that are
rarely found in hydrophobic membrane environment. NMR studies have
shown that magainins reveal high helical content in detergent micelles
and are unfolded in aqueous solution (Bechinger et al., 1991;
Bechinger, 1997). Recent experimental studies (see Shai, 1995 for a
review) suggest that peptides of this class bind parallel to the
membrane surface and do not penetrate deeply into the hydrophobic core ("carpet-like" model). The limited degree of self-association might
also occur, leading to formation of transbilayer pores
("barrel-stave" model). Although the mode of action of magainins is
not yet well understood, it strongly depends on the peptide-membrane
interactions (Shai, 1995), and therefore studies of solvent effects on
structure and behavior of magainin2 are of particular importance.
MC simulations of magainin2 in different solvents and in vacuo show
similar tendencies to the peptides considered above. Thus, the
lowest-energy conformers in nonpolar media are totally -helical (Fig. 2 G), while those in water (N = 17) and in vacuum (N = 16) are unordered
in the middle part and near the termini (Fig. 2, H and
I). -Helix in cyclohexane is retained within a wide interval of energies (E 22 kcal/mol). In water,
conformers with N = 21, 20, and 17 were
observed. Corresponding values of E are 7.1, 8.5, and
10.4 kcal/mol, respectively. As for other peptides described above,
multiple conformational states with close energies but different
secondary structure (16 
N 21) were
obtained in vacuum. Finally, very similar tendencies in behavior of the
backbone and side chain dihedrals were observed for magainin2 (data not
shown) as compared with BRh-A, BRh-B, and SP-C. The principal result
for magainin2 is in qualitative agreement with the experimental data,
i.e., in the nonpolar environment the amphiphilic peptide containing
many charged groups retains -helicity and does not fold into compact structure.
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CONCLUSIONS |
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TOP
ABSTRACT
INTRODUCTION
METHOD OF CALCULATION
RESULTS AND DISCUSSION
CONCLUSIONS
REFERENCES
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MC simulations were employed to explore conformational space of
several membrane-binding peptides in environments of different polarity
and in vacuum. The solvent effects were treated using an ASP-based
implicit solvation model. Nonpolar solvent imitating membrane
environment and aqueous solution were represented by ASPs for
gas-cyclohexane and gas-water transfer, respectively. It is important
that unlike many previous studies, the simulations were done for
all-atom models of peptides that were not restrained to any
predetermined conformation. The results emphasize that the -helical
conformation is promoted by nonpolar solvent and exists in a wide
energy range. The lowest-energy structures found for BRh-A and BRh-B
agree fairly well with those derived in NMR experiments. Conformational
properties of SP-C and magainin2 in the membrane-like environment were
also found to be in accord with available experimental data.
On the contrary, simulations in water reveal helix distortion for the
peptides under study. Our results for SP-C do not confirm preference of
all-helical structure for SP-C in water, which was reported in an MD
study with explicit solvent (Kovacs et al., 1995); although MC
simulation at constant temperature shows retention of the helical
conformation, exhaustive MC search with variable temperature reveals
that the helix might be unfolded in the middle part (corresponding to
polyvalyl segment Val-15-Val-21), and the structure is tightly packed,
thus reducing its exposure to polar solvent. This observation is
consistent with known helix-destabilizing properties of valines in
aqueous solution. Unlike explicit solvent calculations, no significant
restriction of 1-rotamer sampling was obtained in a
nonpolar solvent compared to a polar one. The solvation model proposed
here gives more realistic results than simulations in vacuum, where the
helical structure undergoes rapid distortion and demonstrates large
conformational freedom for the side chains. In addition, multiple
conformational states (local minima) separated by low-energy barriers
and having different secondary structure and packing were found in
vacuum. A conclusion was made that simulations in vacuo could not be
used to model membrane-bound peptides.
We should outline that the restricted MC search presented here permits exploring only the potential energy hyper-surface in the vicinity of starting experimentally observed structure. Recently, we have performed detailed exploration of the conformational space of BRh-A in nonpolar solvent, starting from random coil conformation (Efremov et al., in preparation). During the simulation, numerous local minima corresponding to unordered structures and conformations containing helical segments of different length were trapped. In the result, the lowest-energy conformers were found to be all-helical and correspond well to the structure in micelles obtained by NMR. This implies that the ASP approach provides a realistic description of environmental effects induced by a membrane and permits selection of the native state, among many others.
Our current work is pursued to study helix-forming properties of other peptides in membrane-like surroundings through detailed exploration of their conformational space in MC simulations. Hopefully, this will provide important insight into the structural behavior of peptides in lipid bilayer and will permit development of algorithms designed to structure prediction of membrane proteins based on their sequences. Simulations in bulk solvent do not address questions concerning adsorption and/or insertion of peptides, water/bilayer partitioning, and peptides' orientation with respect to the bilayer. For this to be done the heterogeneous nature of membranes, namely polar layers separated by a hydrophobic core, should be taken into account. The work combining ASPs for cyclohexane and water in the framework of such a three-phase solvation model is in progress now.
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ACKNOWLEDGMENTS |
|---|
We thank Dr. W. Braun for providing us with the FANTOM program.
This work was supported in part by NATO Linkage Grant HTECH.LG.951401 and Russian Foundation for Basic Research (RFBR) Grants 98-04-48823 and 96-04-49788. We are grateful to anonymous referees for their criticism and useful comments.
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FOOTNOTES |
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Received for publication 15 July 1998 and in final form 13 February 1999.
Address reprint requests to Dr. Roman G. Efremov, M. M. Shemyakin and Yu. A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Ul. Miklukho-Maklaya, 16/10, Moscow V-437, 117871 GSP, Russia. Tel.: 7-095 335 51 55; Fax: 7-095 335 50 33; E-mail: efremov{at}nmr.ru.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHOD OF CALCULATION
RESULTS AND DISCUSSION
CONCLUSIONS
REFERENCES
|
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-helix.
Biochemistry.
33:6015-6023[Medline].-Helix stabilization by natural and unnatural amino acids with alkyl side chains.
Proc. Natl. Acad. Sci. USA.
88:5317-5320[Abstract].-to- transitions.
Curr. Opin. Struct. Biol.
7:501-508[Medline].-helices.
Proc. Natl. Acad. Sci. U.S.A.
92:4577-4581[Abstract].-helix initiation in alanine and valine peptides.
Biochemistry.
30:6059-6070[Medline].
Biophys J, May 1999, p. 2460-2471, Vol. 76, No. 5
© 1999 by the Biophysical Society 0006-3495/99/05/2460/12 $2.00
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