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doi:10.1016/j.jmb.2004.11.036
J. Mol. Biol. (2004) xx, 1–9
A Study of the Membrane–Water Interface Region of
Membrane Proteins
Erik Granseth, Gunnar von Heijne and Arne Elofsson*
Stockholm Bioinformatics
Center, AlbaNova, SE-106 91
Stockholm, Sweden
The most conspicuous structural characteristic of the a-helical membrane
proteins is their long transmembrane a-helices. However, other structural
elements, as yet largely ignored in statistical studies of membrane protein
structure, are found in those parts of the protein that are located in the
membrane–water interface region. Here, we show that this region is
enriched in irregular structure and in interfacial helices running roughly
parallel with the membrane surface, while b-strands are extremely rare.
The average amino acid composition is different between the interfacial
helices, the parts of the transmembrane helices located in the interface
region, and the irregular structures. In this region, hydrophobic and
aromatic residues tend to point toward the membrane and charged/polar
residues tend to point away from the membrane. The interface region thus
imposes different constraints on protein structure than do the central
hydrocarbon core of the membrane and the surrounding aqueous phase.
q 2004 Elsevier Ltd. All rights reserved.
*Corresponding author
Keywords: membrane protein; protein structure; bioinformatics; interface
helix
Introduction
a-Helical membrane proteins account for
approximately 20% of all proteins in a typical
genome.1 Over the past few years, several studies
have been performed on the structural characteristics of a-helical membrane proteins. These studies
have mainly focused on the transmembrane
a-helices, describing their amino acid composition
and packing interactions in considerable detail.2–6
In contrast, the parts of the proteins located in the
membrane–water interface region have received
little attention, and we are not aware of any detailed
studies of the structural constraints imposed on
proteins by this region of the lipid bilayer. Here, we
present a first analysis of this interface region in a
non-redundant set of a-helical membrane proteins
of known structure.
Our data set consists of 56 chains from 27 PDB
files, Table 1. No one chain is more than 30%
identical in sequence to any other chain in the data
set. In total, the data set contains 221 transmembrane helices and 78 interfacial helices (see
Materials and Methods for a precise definition of
Abbreviations used: TM; transmembrane; PDB; Protein
Data Bank.
E-mail address of the corresponding author:
[email protected]
the different types of secondary structure). For
comparison, we also include five peripheral
membrane proteins in the study.
The global view
In agreement with earlier findings,2 the secondary structure composition in the G10 Å central
region of the membrane is almost 100% helix,
Figure 1(a). The helix content steadily decreases
from the center of the membrane to a minimum at
around G35 Å. The small amount of irregular
structure in the central region originates from ion
channels and aquaporin-like proteins that contain
so-called re-entrant loops that extend part of the
way across the membrane. Outside the central
G10 Å region, the amount of irregular structure
increases to a peak around G25 Å, and then
decreases again. Appreciable amounts of b-strucstructure are found only G35 Å or more from the
center of the bilayer.
The distribution of different categories of amino
acid residues is shown in Figure 1(b). As expected,
the central region contains mainly aliphatic
residues, but also a significant proportion of polar
residues (30%). Polar residues, Gly and Pro in
particular, are most frequent in the G20–30 Å
region where most of the transmembrane helices
end. Another noticeable feature is a peak of Trp and
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Table 1. Description of proteins used in this study, chains in bold contain interface helices, chains in italic contain reentrant loops
TM helix protein
PDB code
Organism
Chains
Cytochrome c oxidase
Cytochrome bc1
Potassium channel
Bacteriorhodopsin
Halorhodopsin
ba3 cytochrome c oxidase
Calcium ATPase
Rhodopsin
Ubiquinol oxidase
Aquaporin
Glycerol-conducting channel
Sensory rhodopsin
Multidrug efflux transporter
Photosystem I
Formate dehydrogenase
Light-harvesting protein
Fumarate reductase
ABC transporter
Light-harvesting complex
Mechanosensitive channel
Mechanosensitive channel
Cytochrome c oxidase
1AR1
1BGY
1BL8
1C3W
1E12
1EHK
1EUL
1F88
1FFT
1FQY
1FX8
1H68
1IWG
1JB0
1KQF
1KZU
1L0V
1L7V
1LGH
1MSL
1MXM
1OCC
Paracoccus denitrificans
Bos taurus
Streptomyces lividans
Halobacterium salinarum
Halobacterium salinarum
Thermus thermophilus
Oryctolagus cuniculus
Bos taurus
Escherichia coli
Homo sapiens
Escherichia coli
Natronomas pharaonis
Escherichia coli
Synechoccus elongatus
Escherichia coli
Rhodopseudomonas acidophila
Escherichia coli
Escherichia coli
Rhodospirrilum molischianum
Mycobacterium tubercolosis
Escherichia coli
Bos taurus
Potassium channel
Photosyntetic reaction center
Lactose permease
G3P transporter
Fumarate reductase
Monotopic membrane protein
Monoamine oxidase B
Cyclooxygenase-2
Fatty acid amide hydrolase
Squalene-hopene cyclase
Coagulation factor V
1P7B
1PRC
1PV6
1PW4
1QLA
Burkholderia pseudomallei
Rhodopseudomonas viridis
Escherichia coli
Escherichia coli
Wolinella succinogenes
1GOS
1CX2
1MT5
3SQC
1CZS
Homo sapiens
Mus musculus
Rattus norvegicus
Alicyclobacillus acidocaldarius
Homo sapiens
B
C,D,E,G,J,K
A
A
A
A,B,C
A
A
A,B,C
A
A
A
A
B,F,I,J,L,M
B,C
A,B
C,D
A
A,B
A
A
A,B,C,D,G,I,
J,K,L,M
A
H,L,M
A
A
C
Number of
membrane
helices
2
13
2
7
7
15
10
7
14
6
6
7
12
18
5
2
6
10
2
2
3
23
2
11
12
12
5
Number of
interface
helices
8
1
3
2
2
2
4
21
4
2
2
2
1
3
1
12
2
2
4
A
A
A
A
A
The chain abbreviation is from the corresponding PDB structure.
Tyr2 residues at G15 Å. The “positive inside” rule7
is also apparent, in that there is a clear increase in
the frequency of Lys and Arg residues on the
cytoplasmic side of the membrane. Asp and Glu, in
contrast, seem to have no clear preference for any
particular side of the membrane.
The membrane–water interface region
As indicated above, the parts of the proteins
located in the membrane–water interface region
G15–25 Å from the center of the membrane have
distinct structural properties compared both to the
parts located in the central region and those totally
exposed to the aqueous phase. Lack of highresolution structural data has so far made it difficult
to study this region, but the database of known
structures is now big enough to make a first
characterization possible. As shown in Figure 2(a),
even when all transmembrane helices are removed
there are still many hundreds of residues in the
interface region. It is interesting to note that the total
number of residues in successive 1 Å thick slabs is
lower in the middle of the membrane than in the
interface region (Figure 2(a), thick lines), indicating
that helix bundle membrane proteins tend to have a
“waist” near the center of the membrane. This waist
is still seen even if cofactor-containing proteins are
removed (data not shown).
Figure 2(b) shows the distribution of secondary
structure when the transmembrane helices have
been removed. It can be seen that the interface
region consists mainly of irregular structure
(w70%) and interfacial a-helices (w30%). The
helical content drops from G15 Å to G25 Å, while
the fraction of irregular structure is maximal at
G25 Å. There is a conspicuous lack of b-strand
residues in the interface region, while further away
from the membrane the b-strand content is similar
to that of globular proteins.8 An example of a
protein that contains many interfacial helices is the
photosynthetic reaction center shown in Figure 3.
The average length of interfacial helices resembles
the average length of helices in globular proteins,9
while transmembrane helices are much longer,
Table 3.
The functional roles of interface helices are not
well understood, and probably differ from protein
to protein. The helices may be needed for structural
reasons, such as to help positioning the
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Figure 1. (a) Distribution of secondary structure in the K50 to 50 Å region. (b) Distribution of amino acid residues.
Hydrophobic residues are V, A, F, I, L, M, polar C, G, P, H, N, Q, S, T, aromatic W, Y, basic R, K and acidic D and E. The
positive Z-coordinate is towards the periplasm/intermembrane side.
Figure 2. (a) Amount of residues in 1 Å slabs before and after removal of transmembrane helices. (b) Distribution of
secondary structure with transmembrane helices removed. The G12 Å region is removed due to small amounts of
residues.
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Figure 3. The photosynthetic reaction center
(1PRC.pdb) chain M containing five TM helices (gray)
and five interface helices (dark gray). The top of the
picture is inside the cytoplasm and the C-terminal loop is
not shown. The picture was made using PyMOL.29
transmembrane helices. In photosystem I, interface
helices are thought to shield cofactors from the
aqueous phase.10 Another functional role can be
found in the MscS mechanosensitive channel,
where an interfacial helix is thought to be involved
in channel gating by transferring mechanical force
from a “sensor” domain to a transmembrane helix
in the ion channel.11 The KirBac 1.1 inwardly
rectifying potassium channel also has an interface
helix which is believed to slide parallel with the
membrane in order to regulate the gating of the
channel.12
Three main types of secondary structures
Table 2. Distribution of amino acid residues in the
G15–25 Å region
Amino acid
W
Y
C
G
P
H
N
Q
S
T
V
A
F
I
L
M
D
E
R
K
WCY
CCGCPCHCNCQCSCT
VCACFCICLCM
DCE
RCK
Irregular
structure
(%)
Interface
helix
(%)
TM
helix
(%)
1.9
3.1
0.7
13.5
8.1
3.1
6.0
3.0
7.3
6.2
3.9
6.9
5.8
4.0
6.7
2.3
5.6
3.4
4.5
3.9
5.0
47.9
29.6
9.0
8.4
7.3
6.2
1.1
3.9
2.7
1.6
3.6
3.9
5.2
5.0
7.3
8.3
6.0
6.0
9.3
3.1
3.7
5.0
5.5
5.0
13.5
27.0
40.0
8.7
10.5
2.9
4.7
0.2
4.5
4.9
3.8
2.9
3.5
5.1
5.4
6.6
9.6
5.7
6.0
12.1
2.6
3.5
4.7
6.5
4.8
7.6
30.3
42.6
8.2
11.3
characterize the interface region: protruding ends
of transmembrane helices, interface helices, and
irregular structure. Table 2 shows that the three
different types of interface structures differ in amino
acid composition. The interface and transmembrane helices consist mainly of hydrophobic
and polar aromatic (Trp, Tyr) residues; the latter are
particularly abundant in the interface helices. Gly
and Pro are enriched in the irregular structure,
together with other polar residues such as Asn and
Ser. These residues have been shown to be efficient
turn-promoters when placed in the middle of long
transmembrane segments,13 and also serve as helixbreaking or helix-capping residues in globular
proteins.14
It has been reported that polar residues near the
ends of transmembrane helices tend to point away
from the center of the membrane, while apolar
residues tend to point towards the center.15 We have
carried out a similar analysis for the interfacial
secondary structure elements by calculating the
angle f of the Ca–Cb bond relative to the membrane
normal for each residue in the interface region.
Residues were classified into three categories: those
pointing towards the membrane (fR1208), those
pointing away from the membrane (f!608), and
those parallel with the membrane (608%f!1208).
The results are shown in Figure 4. Hydrophobic
and aromatic residues tend to point towards the
membrane, while polar and charged residues tend
to point away from the membrane in all three
classes of interfacial secondary structure. This
tendency is strongest for the irregular structures,
where 46% of the hydrophobic and 41% of TrpCTyr
point towards the membrane. Two additional
features are worth noting. First, the fraction of
residues that are parallel with the membrane is
highest for the TM helices (59%). Intuitively, sidechains of transmembrane helices should tend to be
parallel with the membrane, since the helices are
approximately perpendicular to the membrane
plane. Second, the charged and polar amino acid
residues in the interface helices are more frequently
pointing away from the membrane (41–45%) than in
the other secondary structure types (25–36%).
The direction of the Ca–Cb bond is only a rough
guide to the direction of long Tyr and Trp sidechains relative to the membrane plane, therefore we
studied this in more detail using the angle between
the Ca atom and the last polar atom on the sidechain. The Tyr and Trp side-chains tend to be
directed towards the outside of the membrane
when they are located below the membrane–water
interface region around 15 Å; when located further
out, they more frequently have their side-chains
directed towards the membrane, see Figure 5(a).
Interestingly, for Trp, this effect comes mainly from
those residing in interface helices, 53% of the
tryptophan residues that have their polar atom
more than 1 Å closer to the lipid bilayer than their
Ca atom are from interface helices. The turning of
the polar group of Tyr in the 15–20 Å region, on the
other hand, does not come from tyrosine residues in
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Figure 4. Direction of Ca–Cb for residues in interface helices, transmembrane helices and in irregular structures in the
15–25 Å region.
interface helices, but from those in irregular
structures which makes up 44% of those that are
directed towards the middle of the membrane. Lys
and Arg have long aliphatic side-chains with a
positively charged amine or guanidinium group at
the end. The aliphatic hydrocarbon part prefers to
be localized in the hydrophobic region of the bilayer
while the positively charged group prefers the more
polar interface region, something described as
snorkeling.16,17 The snorkeling effect of Lys and
Arg can be observed, since the charged moiety of
the side-chain in the 10–15 Å region is significantly
higher than further away from the membrane
region, see Figure 5(b). This effect is most prominent
for Lys, with an average snorkeling of the polar
atom of 2.5 Å, compared with 1.6 Å for Arg. The
acidic Asp and Glu showed no statistically significant snorkeling, but were most often directed away
from the membrane.
Interface helices have a lot in common with
amphipathic peptides residing in the membrane–
water interface region. Snorkeling of Arg and Lys is
a means of allowing the peptide to penetrate deeper
inside the membrane and subsequently more
strongly bound to it.18 Trp is capable of forming
hydrogen bonds with its NH group, but it also has
the largest non-polar surface of all amino acid
residues. Tryptophan residues inside the membrane
prefer to direct the polar NH group towards the
aqueous region, while those in interface helices
prefer to direct the NH group towards the lipid
headgroup. This might lead to the possibility of
burying the more hydrophobic six-membered ring
inside the hydrophobic bilayer.
Recently, Chamberlain et al.15 made a similar
study of the snorkeling behavior of different
amino acid residues in transmembrane helices in
the 0–15 Å region. The above results are in agreement with their study, but also extend it to the 15–
25 Å region. The most striking result of the
extension of the studied region is the change of
direction of the polar atoms of Trp and Tyr when
they enter the membrane–water region, and that
the snorkeling of Lys and Arg occurs mainly in the
10–15 Å region.
Loops in the interface region are responsible for
connecting TM helices and interface helices. As seen
in Table 3, the average lengths of loops between
TM helices and interface helices are on average two
residues shorter than those connecting two TM
helices. It is rare (only seen 14 times, nine of them
occurring in 1JB0) for an interface helix to be
followed by another interface helix. Most interface
helices thus connect two TM-helices.
Longer loops connecting two transmembrane
helices often contain interface helices, see Figure 6.
The shortest loop containing an interface helix is
eight residues, allowing for one turn of the helix
and two short loops, but the majority are significantly longer, on average 27 residues, see Table 3.
The longest loop without any interface helix is 31
residues, but most loops connecting two TM helices
are short, on average nine residues and two-thirds
are shorter than ten residues. In contrast, an
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Figure 5. Average distance between Ca atom and polar atom for different regions for (a) Trp and Tyr and for (b) Arg
and Lys. A positive value means that the side-chain is directed away from ZZ0. The error bars are the standard error.
Table 3. Average lengths (residues) for secondary structures close to the interface helix
Secondary structure category
Loops between helices
TM helix–loop–interface helix
TM helix-loop–TM helix
Interface helix–loop–interface helix
Helices
Interface helix
TM helix
Length between TM helices
Loop
Interface helix
Average length
Min. length
Max. length
7.0
9.0
8.6
1
1
1
35
31
19
8.9
26.0
4
15
19
43
9.0
27.2
1
8
31
69
interface helix between two TM helices separates
the helices by more than 31 residues one-third of the
time.
As seen in Figure 6, long loops do not imply that
the helices are spatially distant. In fact, for loops
shorter than 20 residues, it is always loops lacking
interface helices that separate the TM helices the
most. For longer loops, the separation of TM helices
varies significantly. Thus, interface helices might
play a role as anchoring points for the positioning of
the transmembrane helices.
Monotopic membrane proteins
Monotopic membrane proteins are soluble proteins or protein domains that bind to the surface of
the membrane. As a comparison, we have analyzed
four proteins listed as monotopic in the Membrane
Protein Resource† plus the C2 domain of human
coagulation factor V.19 The latter has a b-sandwich
fold and contains b-strands and short connecting
loops near its membrane-binding surface. The other
monotopic proteins all have amphiphilic a-helices
residing in the proposed membrane-binding interface.20–22 Fatty acid amide hydrolase and monoamine oxidase B both contain one predicted
transmembrane helix but have their active sites
and most of their mass in a globular, membraneinteracting domain (see the Membrane Protein
Resource for further details).
An examination of the amino acid distribution
in the membrane-interacting regions (as defined
in the original publications) reveals that the
† http://blanco.biomol.uci.edu/MemPro_resources.
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Figure 6. Figure showing the distance (in Å) between the end/beginning of two TM helices against the number of
residues connecting them. Filled squares are loops containing interface helix and small filled circles are loops without
interface helices.
membrane-binding parts of monotopic proteins by
and large are similar to the interface structures
discussed above. Some details are different, however. Acidic amino acid residues are half as
common in the interface regions from the monotopic proteins (4% versus 9% in the transmembrane
proteins) and basic residues are 50% more common
compared to the interface structures in integral
membrane proteins (15% versus 10% in the transmembrane proteins), strongly, suggesting that
electrostatic interactions with negative charged
lipid headgroups help stabilize membrane association.23 The most common amino acid in the
interface regions from the monotopic proteins is
Leu, which alone makes up 21% of the residues.
The distribution of the Ca–Cb angles relative to
the membrane normal show that most of the amino
acid residues lie parallel with the membrane with
the exception of Trp and Tyr (data not shown).
In all, 64% of the Trp and Tyr residues are directed
towards the membrane, which is a considerably
higher fraction than for the interfacial structures in
helix-bundle proteins, see Figure 4. This further
emphasizes the importance of Leu, Trp and Tyr as
membrane anchors, since monotopic proteins lack
the rigid anchoring that transmembrane helices
provide.
interface region imposes clear structural constraints
on integral membrane proteins. Of the residues
in this region, w70% have irregular secondary
structure, and the remaining 30% form interfacial
a-helices oriented roughly parallel with the membrane plane. There is essentially no b-structure in
the interface region. Irrespective of the type of
secondary structure, aliphatic and aromatic residues tend to point towards the center of the
membrane while polar and charged residues tend
to point away from the center. The same general
features are seen in the membrane-binding parts of
monotopic membrane proteins, suggesting that
common evolutionary forces shape all different
kinds of protein surfaces exposed to the membrane–
water interface.
Materials and Methods
Data set and analysis of known 3D structures
PDB files for helix bundle membrane proteins of known
structure were downloaded from the Membrane Protein
Structure database†24 and directly from the Protein Data
Bank.25 No chain with more than 30% sequence identity
to another was included, resulting in a total of 56 nonhomologous chains from 27 PDB files, Table 1.
To orient all membrane proteins, the first three and last
Conclusions
In summary, we find that the membrane–water
† http://blanco.biomol.uci.edu/MemPro_resources.
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three Ca atoms from each TM helix in the biological unit
defined a vector. These vectors were then used to
calculate an average vector. The structure was finally
rotated so that the Z-axis corresponds to this average
vector and was therefore perpendicular to the plane of the
membrane. The positive direction of the Z-axis was
directed towards the periplasm/intermembrane side.
The GES hydrophobicity scale26 was used to generate a
hydrophobicity profile for each protein by first calculating
the mean hydrophobicity for all residues with their Ca
Z-coordinate located within the same 1 Å slice perpendicular to the membrane and then applying a 5 Å wide
hat-shaped smoothing window. Thus, for a slab at
Z-coordinate i, the smoothed average hydrophobicity
profile was calculated as:
hHðiÞi ¼
1
ð3hðiÞ þ 2ðhði K 1Þ þ hði þ 1ÞÞ
9
2.
3.
4.
5.
6.
þ hði K 2Þ þ hði þ 2ÞÞ
The ZZ0 plane was defined as the minimum on the
resulting hydrophobicity profile. The same kind of
averaging was used for making Figures 1 and 2.
Secondary structure classifications were made using
STRIDE.27 Helices were re-classified as irregular structure
if they were shorter than three residues and not flanking
any other helix. One-residue b-bridges were also reclassified as irregular structure.
V, A, F, I, L and M were classified as hydrophobic, C, G,
P, H, N, Q, S and T as polar, W and Y as polar aromatic, R
and K as basic, and D and E as acidic.
Residues were classified into three categories: those
pointing towards the membrane (fR1208), those pointing
away from the membrane (f!608), and those parallel
with the membrane (608%f!1208).
The polar atom used for snorkeling analysis was Nz for
Lys, N3 for Trp, Oh for Tyr and the mean coordinate of N3,
Nh1 and Nh2 for Arg.
The structures used for the analysis of the monotopic
membrane proteins are listed in Table 1. Each structure
was rotated using the Chimera visualization tool28 to
have its Z-axis oriented perpendicular to the suggested
membrane surface given in the corresponding articles.
A hydrophobicity profile was then calculated for each
protein in the same way as described above for the
integral membrane proteins, and the protein was translated along the Z-coordinate until its hydrophobicity
profile matched the averaged profile for the integral
proteins as well as possible. Only one subunit of each of
the structures was used for analysis and each chain had
less than 30% sequence identity to each other. Amino acid
distributions and side-chain directions were calculated as
described above.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
Acknowledgements
18.
This work was supported by grants from the
Swedish Natural Sciences Research Council and the
foundation for Strategic Research to A.E. and G.vH.
19.
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Edited by J. Thornton
(Received 22 September 2004; received in revised form 16 November 2004; accepted 16 November 2004)