Supplementary Information for the article:
Roughness of a transmembrane peptide reduces lipid membrane
dynamics
Marie Olšinová, Piotr Jurkiewicz, Jan Sýkora, Ján Sabó, Martin Hof, Lukasz Cwiklik and Marek Cebecauer*
J. Heyrovsky Institute of Physical Chemistry, Czech Academy of Sciences, Prague, Czech Republic
* Corresponding author: [email protected]
June 9th, 2017
Contents
Supplementary Discussion ................................................................................................................................... 2
Section 1. Model system - Fluid membranes with a simple α-helical transmembrane peptide ..................... 2
Section 2. Molar concentration of proteins in cell membranes – the estimation........................................... 2
Section 3. Transmembrane peptides influence the local mobility of phospholipids ...................................... 2
Section 4. Altered distribution of cholesterol in membranes containing LW21 peptide ................................ 3
Section 5: Rough surface of protein transmembrane domains ....................................................................... 4
Section 6: Possible consequences for cellular membranes ............................................................................. 4
Supplementary Figures ........................................................................................................................................ 5
Supplementary Tables ....................................................................................................................................... 21
Supplementary Movies ...................................................................................................................................... 23
References ......................................................................................................................................................... 24
1
Supplementary Discussion
Section 1. Model system - Fluid membranes with a simple α-helical transmembrane peptide
The transmembrane domains (TMDs) of integral membrane proteins with their rough surface are in close
contact with lipids and, therefore, could directly modulate lipid behavior in cellular membranes. To analyze a
direct impact of the rough peptide surface on membranes, we have selected the LW21 peptide
(GLLDSKKWWLLLLLLLLALLLLLLLLWWKKFSRS; 21 hydrophobic residues underlined) in DOPC (dioleoyl
phosphatidylcholine) bilayers based on a number of prerequisites: i) The peptide is monomeric in DOPC
membranes (Supplementary Fig. 4). The observation is in agreement with previously published results for
similar model systems with WALP family peptides1 and indicates that LW21 does not aggregate in the tested
membranes. ii) Previous work2 provides evidence that there is no hydrophobic mismatch between the length
of the hydrophobic part of the LW21 peptide and the thickness of DOPC membrane with or without
cholesterol. The peptide incorporates into membranes composed of DOPC with and without cholesterol with
comparable efficiency which confirms these results (Supplementary Fig. 12). iii) Our previous study
demonstrates that LW21 peptide (variant used in this work) adapts a transbilayer orientation in DOPC
membranes.3 Flanking lysines in our LW21 peptide were used previously to anchor transmembrane peptide
in lipid bilayers.3 We have added another 4-5 residues from CD247 molecule at both ends to further stabilize
the transbilayer orientation of the peptide. A stable transbilayer orientation of a peptide is important for our
study since partial or peripheral association of the peptide with membranes could affect the experiments
and reduce validity of the study focused on direct impact of TMDs on membranes. All atom MD simulations
suggest minimal impact of flanking residues on herein studied effect of the rough surface of LW21 on
membrane lipids (Supplementary Fig. 13) iv) DOPC with its low melting point of -18.3°C provided highly fluid
lipid environment in all tested vesicles in the absence and presence of 25% cholesterol.4 No formation of
lipid domains, including nanodomains, was detected in such membranes previously.5 All these properties
were important prerequisites to investigate a direct impact of peptide roughness on lipid membrane
dynamics (in the absence of other potential effects, e.g. obstacles or lipid domains).
In addition, we could support LW21 as a valid TMD model of plasma membrane integral proteins by efficient
surface expression of LW21-GFP fusion protein (Supplementary Fig. 14).
Section 2. Molar concentration of proteins in cell membranes – the estimation
Proteins were found to form approximately one half of total mass in cellular membranes.6 To our knowledge,
no molar concentrations were experimentally determined for lipids and proteins in cell membranes to date.
Here, we estimate the molar concentration of proteins in cell membranes by taking into account a small size
of lipids (~1kDa) and an average size of proteins (~40 kDa; ref. 7). These values indicate that cell membranes
contain approximately 2.5 mol% of proteins. Even though this is a very crude estimate, it validates our
studies, which were performed using peptide concentrations in the range of 0-3 mol%.
Section 3. Transmembrane peptides influence the local mobility of phospholipids
The TRES (time-resolved emission spectra) analysis of the membrane environment-sensitive probe Laurdan
provides two parameters: integrated relaxation time τr referring to local mobility of molecules and total
emission shift Δν reflecting hydration of tested membranes as described in the method section. Fig. 2b
shows increased local viscosity in membranes containing increasing amounts of the peptide, as indicated by
increasing relaxation time values. Interestingly, Δν values remained unchanged under all tested conditions
2
(Supplementary Table 2) indicating that the presence of the LW21 peptide does not disturb the structural
integrity of lipid bilayer. Similar trends were observed for Laurdan/TRES measurements of membranes with
25 mol% cholesterol. Laurdan probe indicates changes at the carbonyl level of phospholipids or, in other
words, at the interface of hydrophilic and hydrophobic parts of the membrane (Supplementary Fig. 5).
Another membrane environment-sensitive probe used in this work, DPH (diphenylhexatriene), is located
closer to the midplane of membranes (Supplementary Fig. 5). The results demonstrate similar effect of the
peptide in this part of tested membranes. Namely, DPH rotation in the bilayer interior was restricted, which
is manifested in the elevated order parameter in the presence of peptide. This effect was preserved in
cholesterol-containing membranes (Table 1).
Interestingly, analyses of membrane properties using the two environment sensitive probes provided slightly
different outputs. The impact of the peptide observed at the level of carbonyl groups using Laurdan probe
was stronger in the presence of cholesterol compared to its absence (Fig. 2b). The opposite effect was found
at the level of acyl chains (closer to the center of the bilayer) using DPH probe (Table 1). On its own,
cholesterol alters the interior of membranes and the parts closer to the water interface differently. It
stabilizes a lipid membrane by ordering its hydrophobic interior, increasing lipid packing and sealing it
against water-soluble molecules (Fig. 2b, Table 1 and Supplementary Table 2).8 These effects are primarily
executed by the stiff steroid moiety of cholesterol located in the lipid backbone region (Supplementary Fig.
10). Cholesterol impact on the lipid headgroup parts is weaker. The highly ordered interior of cholesterolcontaining membranes is thus less susceptible to further ordering but the presence of peptide still caused an
increase of order as measured by DPH. Importantly, this increase was much smaller than the one obtained in
pure phospholipid membranes (Table 1). Further, studies are required to understand distribution of impacts
of the peptide across the membrane in more detail.
Section 4. Altered distribution of cholesterol in membranes containing LW21 peptide
All atom MD simulations of the LW21 peptide in DOPC membranes containing cholesterol indicate
heterogeneous distribution of lipids. Cholesterol exhibits a tendency to avoid contact with the rough surface
of the peptide (Fig. 3c-e). As a consequence, this probably leads to its accumulation in parts of the
membrane with low presence of the peptide. The organization of phospholipids in the vicinity of the peptide
and the trapping of their acyl chain on the rough surface of the peptide remain unaffected in the absence
and presence of cholesterol. This observation is supported by detailed analysis of the acyl chain order
calculated from MD simulations where the lipids from the peptide annulus and those in the bulk (nonannular) can be analyzed separately (Supplementary Fig. 15). The data suggest that the overall mobilityreducing effect of peptides in the presence of cholesterol is probably caused by complex events: i) the rough
surface of the peptide causes lipid acyl chain trapping and locally reduced lateral mobility of phospholipids
(Figs. 3a, b and Supplementary Fig. 6); ii) cholesterol presence is strongly reduced in the peptide annulus
(Figs. 3c-d and Supplementary Fig. 9; see also refs. 9-11), which leads to iii) increased content of cholesterol in
the non-annular lipid membrane (Fig. 3c,d). The property under i) applies for both membranes, with and
without cholesterol. The data in the Supplementary Fig. 15 can be also interpreted that the peptide has
ordering effect on phospholipid acyl chains in the absence but local disordering effect in the presence of
cholesterol. As mentioned above, such disordering effect does not disturb the structural integrity of the lipid
bilayer and fluid lipids adapt to the presence of this ‘imperfect’ membrane component (Supplementary Table
2).10 Therefore, due to the segregation of cholesterol, MD simulations indicate a different impact of the
peptide on membranes with and without cholesterol. Further studies are required for more detailed
understanding of this phenomenon. It is also important to mention that the acyl chain order parameter
calculated from the angles between C-C bonds in the lipid acyl chain and the membrane normal (MD
simulations) reflects directly the geometry of the acyl chain rather than its freedom to move.
3
Section 5: Rough surface of protein transmembrane domains
Apart from glycine, side residues of amino acids form a rough surface in helical peptides (Supplementary Fig.
1a-c). Their combinations fold into various grooves of different shape but rarely cause smoothening of the
surface (Supplementary Fig. 1d-f). Indeed, transmembrane parts of a majority, if not all, of studied integral
membrane proteins of different origin exhibit severe surface roughness independent of whether these are
formed by one or more TMDs (for examples see refs. 12-17). It is, therefore, not an exclusive property of the
LW21 peptide.
Non-specific interactions of lipid acyl chains with the surface of a membrane protein were observed recently
in 2D crystals of aquaporin-0.15 Acyl chains transiently associate with the grooves formed by the TMDs and
laterally co-diffused with the protein.14,18 In other work, specific and tight interaction of sphingomyelin with
p24 protein of the COPI vesicular machinery was demonstrated to be a headgroup- and acyl chaindependent.19 Our work indirectly supports a universal property of phospholipids to transiently interact with a
rough surface of TMDs of integral membrane proteins. Whether there is any preference of particular lipids or
fatty acids (acyl chains) for TMDs remains to be evaluated.
Section 6: Possible consequences for cellular membranes
Cellular membranes are primarily composed of a variety of phospholipids and, in the case of the plasma
membrane and selected intracellular membrane compartments, sterols.20 The presence of large amounts of
integral membrane proteins6 with their transmembrane domains forms an environment reminiscent of the
most complex model studied in this work: phospholipid membrane containing 1-3 mol% of transmembrane
peptides and 25 mol% of cholesterol. Indeed, cellular membranes are highly heterogeneous21,22 and reduced
lateral diffusion was observed in the areas of plasma membrane with higher protein density.23 Therefore, the
impact of a simple α-helical transmembrane peptide on lipid membranes demonstrated in this work may
contribute to the understanding of such molecular processes in cell membranes.
Our data also support the findings that integral proteins and peptides prefer highly fluid membranes.24,25
According to our results (Fig.2a), an increased presence of integral proteins in more rigid membranes could
lead to a dramatic reduction of local diffusion and immobilization of molecules. Such a scenario is in conflict
with biological processes that are driven preferentially by highly mobile molecules.26,27 Immobilized
membrane molecules are rapidly eliminated from the system by endocytosis.28
In our study, we demonstrate the tendency of cholesterol to avoid contact with transmembrane peptides
(Fig. 3c-e). This, in principle, could lead to a segregation of the two molecules into different membrane
domains or areas. Indeed, reported electron microscopy images of plasma membrane sheets reproducibly
indicate the existence of protein-rich and protein-low areas. It remains to be determined whether
cholesterol distributes unequally between these two regions of the plasma membrane.
4
Supplementary Figures
5
Supplementary Figure 1. Cartoons of the rough surface of helical peptides composed of monogenic or
polygenic amino acid sequence. Poly-glycine, poly-alanine and poly-isoleucine artificial helical peptides
(upper panel) represent diverse sequences composed of single amino acid species. Except of poly-glycine, all
helices exhibit highly rough surface. Glycine alone does not support helical structure. Combinations of amino
acids AILS (upper panel, right) indicates that non-homogenous amino acid sequence drives even more
extensive roughness. Surface representations for LW21 peptide and 2 transmembrane domains (EpoR – PDB
ID: 2MV612 and nicastrin – PDB ID: 2N7R13) are shown for comparison (lower panel). The structure of helical
peptides was modeled using surface visualization in PyMOL software. The structures of artificial peptides do
not represent calculated surfaces and are shown solely to indicate roughness of the surface for any
combination of amino acids.
6
Supplementary Figure 2. The toy models of cylinder-like structures with annual lipids. The toy models M1M3 (yellow-orange; as in Fig. 1c) were generated using coarse grain force field and embedded to DOPC lipid
bilayer (see Online Methods for more details). The snapshot indicates lipids (grey) with at least one MARTINI
bead of lipid acyl chains in within 0.5 nm distance from the surface of the toy models.
7
Supplementary Figure 3. Model membrane system. Schematic illustration of the model membrane system
used in the experimental part of the work. Giant unilamellar vesicles (GUVs) containing DOPC (grey), lipid
tracer (DiD; yellow) and LW21 peptides (green, amino acid sequence:
GLLDSKKWWLLLLLLLLALLLLLLLLWWKKFSRS) were attached to the optical surface via surface streptavidin
binding to biotinylated lipids (blue) present in the membranes.
8
Supplementary Figure 4. LW21 peptide is monomeric and non-aggregated in tested membranes. a. Model
of LW21 peptide with labelled tryptophan residues, which were used to investigate the oligomerization state
of LW21 peptide in DOPC membranes (as in b). b. Time resolved fluorescence anisotropy decays of LW21, C2LW21 and melittin peptides in DOPC membranes demonstrate the monomeric state of LW21 peptide at both
concentrations, 1 and 5 mol%. Melittin represents a monomeric control, whereas C2-LW21 dimerizes due to
the presence of free cysteine in its N terminus (see Chemicals and peptides in the Online Materials and
Methods).
9
Supplementary Figure 5. Schematic illustration of the membrane with environment-sensitive fluorescent
probes Laurdan and DPH. Laurdan senses mobility and hydration at the level of phospholipid carbonyl group
(blue), DPH probe determines rotational freedom of acyl chains close to the center of lipid bilayer (green). In
the experiments, both leaflets were labeled symmetrically.
10
Supplementary Figure 6. Retardation of phospholipids in the vicinity of the LW21 peptide surface – maps
for all leaflets presented. a. Lateral diffusion maps of lipids as resolved by all-atom MD simulations in the
absence (upper row) and presence of cholesterol (bottom row) for the individual membrane leaflets
(left/right column). The maps are centered to an average position of the LW21 peptide. The plots show the
average distance travelled by a lipid starting at a particular lateral position in a bilayer over 100 ns time. b.
Lipid-lipid contact autocorrelation function calculated for DOPC and DOPC/cholesterol bilayers in the
absence and presence of LW21 peptide. Contacts between acyl chain carbons were taken into account with
the cut-off of 0.75 nm.
11
Supplementary Figure 7. Quantitative analysis of contacts of DOPC, cholesterol and water with individual
amino acids of the LW21 peptide. Average number of contacts between the peptide backbone and: sn-1 and
sn-2 acyl chains of DOPC, cholesterol, and water per individual residues calculated from the MD simulation
of LW21 in the DOPC membrane with 25 mol% of cholesterol. The cut-off value of 0.75 nm was employed for
the contact definition. Error bars represent standard deviation. No specific interaction of lipids with
individual amino acids is observed. The non-uniform contact distributions of both cholesterol (elevated close
to the membrane mid-plane) and water (exclusively at the membrane-water interfaces) resemble the
density profiles of these species in the membrane. Similarly, lower contact numbers between the peptide
and acyl chains close to the peptide termini are due to decreased presence of lipid acyl chains therein.
12
Supplementary Figure 8. Sustained interaction of phospholipid acyl chains with the peptide surface.
Autocorrelation functions of peptide backbone contacts with individual acyl chains, cholesterol and water in
the membranes without and with 25 mol% of cholesterol. The cut-off of 0.75 nm was employed for the
definition of contacts.
13
Supplementary Figure 9. Lateral distribution of DOPC and cholesterol with respect to the LW21 peptide.
Lateral distribution maps of cholesterol (red) and DOPC (green), and a merge map are presented in artificially
colored manner. The peptide density is shown in the merged map in blue. Each panel represents an average
view of the simulation box in the membrane plane and corresponds to the area with size of approximately
6.8 × 6.8 nm. Values in the maps were calculated for one membrane leaflet employing the trajectory
centered the position of the peptide and with all rotations removed. A tendency of DOPC to reside close to
the peptide and that of cholesterol to occupy non-annular regions is visible.
14
Supplementary Figure 10. Planar structure of cholesterol. Cholesterol structure (blue) forms a planar
interface which cannot fill the grooves at the surface of the LW21 peptide (white). Structures of both
molecules are shown as vdW (ball) representations using VMD software. A typical snapshot from the MD
trajectory is presented with one representative cholesterol molecule residing in the vicinity of the peptide.
Remaining cholesterol molecules, phospholipid (DOPC) molecules and water were removed for clarity.
15
Supplementary Figure 11. Cholesterol does not affect the effect of rough structures on lipid membrane
dynamics. The autocorrelation curves for the contacts between lipid tails and the surface of model
structures M1-M3 (see Fig. 1c) embedded in DOPC-cholesterol membrane with increasing roughness.
16
Supplementary Figure 12. The incorporation of the LW21 peptide into membranes composed of lipids with
different thickness – acyl chain length - in the absence (a) or presence (b) of 25 mol% cholesterol. The values
were calculated as the average fluorescence intensity measured for the fluorescent LW21 peptide (see
Online Materials and Methods for more detail).
17
Supplementary Figure 13. The LW21 flanking residues do not influence the contacts of DOPC, cholesterol
and water with the peptide. The graphs present the average number of contacts formed by phospholipid
acyl chains (sn-1 and sn-2), cholesterol and water with LW21 peptide backbone. Data for DOPC membranes
containing 25 mol% cholesterol (membranes are shown for LW21 either without (a) and with (b) flanking
residues. The results are virtually the same for both versions of the peptide
(GLLDSKKWWLLLLLLLLALLLLLLLLWWKKFSRS; short version underlined). The data show negligible impact of
flanking residues on lipid-peptide contacts. Note that the data in panel a correspond to those presented in
Fig. 3e. Error bars represent error of the mean calculated employing the block averaging method.
18
Supplementary Figure 14. The plasma membrane expression of LW21-GFP protein. Jurkat T cells were
transfected with expression plasmid encoding LW21-GFP protein and imaged using confocal microscopy of
GFP 18 hours later. Sorting of LW21-GFP to the plasma membrane is well visible in the GFP channel (lefthand panel). Right-hand panel presents the brightfield image of the cell. Scale bar represents 5 µm.
19
Supplementary Figure 15. Acyl chain ordering in membranes containing the LW21 peptide were calculated
from MD simulation data (see also Supplementary Table 4). The graph represents average deuterium order
parameter SCD of sn-2 acyl chains of DOPC in the absence (left) or presence (right) of 25 mol% cholesterol.
Peptide-free membranes are compared to annular and non-annular lipids of the LW21 peptide. Error bars
represent error of the mean calculated based on the block averaging method. The ordering of acyl chains
increases with SCD.
20
Supplementary Tables
Supplementary Table 1. Diffusion coefficients and standard deviations of fluorescently labelled peptides and
lipid tracers measured by z-scan FCS method on top of GUVs. Ratio of diffusion coefficients with and without
cholesterol for specific peptide content is also included.
Unlabelled
LW21
peptide
0%
1%
2%
3%
DAtto488LW21 [m2/s]
0%
25%
cholesterol cholesterol
10.5 ± 1.2
8.8 ± 1.1
8.3 ± 0.6
7.8 ± 1.2
7.5 ± 1.2
5.5 ± 1.4
6.6 ± 1.3
2.9 ± 0.8
D25/D0
0%
cholesterol
13.2 ± 0.5
11.3 ± 0.9
10.1 ± 1.1
8.8 ± 1.0
0.8 ± 0.2
0.9 ± 0.2
0.7 ± 0.3
0.4 ± 0.2
DDiD [m2/s]
25%
cholesterol
9.1 ± 1.3
8.6 ± 0.8
6.3 ± 1.8
3.7 ± 1.1
D25/D0
0.7 ± 0.1
0.8 ± 0.1
0.6 ± 0.3
0.4 ± 0.2
Supplementary Table 2. Total spectral shift of Laurdan embedded in LUVs composed of either pure DOPC
or DOPC:Chol (3:1, molar ratio) measured at 25°C. Total spectral shift is directly proportional to the polarity
of the dye environment. Error bars give intrinsic uncertainty of the method.
Content of unlabelled LW21
peptide (mol%)
0
1
2
3
DOPC (cm-1)
DOPC+cholesterol (cm-1)
4080 ± 50
4080 ± 50
4100 ± 50
4070 ± 50
4160 ± 50
4140 ± 50
4140 ± 50
4090 ± 50
Supplementary Table 3. Composition of the systems characterized using fully atomistic MD simulations.
Each bilayer was hydrated with ~6700 water molecules. Four Cl- counterions were added in the case of
LW21-containing bilayers in order to neutralize the system.
System
# lipids
# cholesterol
# LW21
DOPC
128
0
0
DOPC/CHOL
128
40
0
DOPC/LW21
128
0
1
DOPC/CHOL/LW21
128
40
1
DOPC/CHOL/LW21(long) 128
40
1
21
Supplementary Table 4. Acyl chain deuterium order parameter SCD (MD simulations).
System
<order parameter>
SCD
error
DOPC
0.113
0.004
DOPC/LW21 annular
0.127
0.011
DOPC/LW21 non-annular
0.119
0.004
DOPC/CHOL
0.161
0.004
DOPC/CHOL/LW21 annular
0.138
0.013
DOPC/CHOL/LW21 non-annular
0.164
0.004
22
Supplementary Movies
Supplementary Movie 1. The last 100 ns of the 500 ns-long MD simulation of the DOPC/LW21 system. Side
view (parallel to the bilayer midplane) of the simulation box is shown. The movie was created based on the
trajectory sampled each 100 ps with the smoothing employed for each three frames. The trajectory was
centered with respect to the peptide center of geometry. Rotations with respect to the helix were removed.
Phosphorous atoms of DOPC are shown as white balls and DOPC chains are presented as grey lines. The
peptide is depicted using NewCartoon representation with coloring based on the secondary structure (purple
– helical, white – non-helical). The remaining atoms and molecules, including water, are not shown for clarity.
The movie was prepared employing MovieMaker plugin of VMD. H264-MPEG-4 encoding was used.
Supplementary Movie 2. The last 100 ns of the 500 ns-long MD simulation of the DOPC/CHOL/LW21 system.
Side view (parallel to the bilayer midplane) of the simulation box is shown. The movie was created based on
the trajectory sampled each 100 ps with the smoothing employed for each three frames. The trajectory was
centered with respect to the peptide center of geometry. Rotations with respect to the helix were removed.
Phosphorous atoms of DOPC are shown as white balls and DOPC chains are presented as grey lines. Cholesterol
is shown in red employing the licorice representation. The peptide is depicted using NewCartoon
representation with coloring based on the secondary structure (purple – helical, white – non-helical). The
remaining atoms and molecules, including water, are not shown for clarity. The movie was prepared employing
MovieMaker plugin of VMD. H264-MPEG-4 encoding was used.
Supplementary Movie 3. The last 100 ns of the 500 ns-long MD simulation of the DOPC/CHOL/LW21 system
as in Movie S2 but highlighting the annular lipids. Only the acyl chains of DOPC molecules with at least one of
its atoms at the distance <0.21 nm from any peptide atom are depicted (using grey CPK representation).
Smoothing every three frames was not used for lipid and peptide CPK representation to better visualize the
dynamics of annular lipids and peptide side chains. Phosphorous atoms of DOPC are shown as white balls. The
peptide is depicted using both NewCartoon representation with coloring based on the secondary structure
(purple – helical, white – non-helical) and CPK representation (green). The remaining atoms and molecules,
including water and cholesterol, are not shown for clarity. The movie was prepared employing MovieMaker
plugin of VMD. H264-MPEG-4 encoding was used.
23
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