Structure and topology of the transmembrane domain 4 of the
divalent metal transporter in membrane-mimetic environments
Hongyan Li
1,2
, Fei Li
1
, Zhong Ming Qian
2
and Hongzhe Sun
1
1
Department of Chemistry and Open Laboratory of Chemical Biology, The University of Hong Kong, China;
2
Department of
Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hong Kong, China
The divalent metal transporter (DMT1) is a 12-transmem-
brane domain protein responsible for dietary iron uptake in
the duodenum and iron acquisition from transferrin in
peripheral tissues. The transmembrane domain 4 (TM4)
of DMT1 has been shown to be crucial for its biological
function. Here we report the 3D structure and topology of
the DMT1-TM4 peptide by NMR spectroscopy with
simulated annealing calculations in membrane-mimetic
environments, e.g. 2,2,2-trifluoroethanol and SDS micelles.
The 3D structures of the peptide are similar in both envi-
ronments, with nonordered and flexible N- and C-termini
flanking an ordered helical region. The final set of the 16
lowest energy structures is particularly well defined in the
region of residues Leu9–Phe20 in 2,2,2-trifluoroethanol,
with a mean pairwise root mean square deviation of
0.23 ± 0.10 A

cytes and also for transporting iron across the endosomal
membrane in the transferrin cycle [7–9]. The DMT1 consists
of 561 amino acids with 12 putative transmembrane
domains [1]. The DMT1 gene encodes two messenger
RNAs produced by alternative splicing of two 3¢ exons that
show different 3¢ untranslated regions containing an iron
response element (isoform I) and no iron response element
(isoform II), as well as distinct C-terminal protein sequences
[7–10]. Recently, DMT1 mRNA expression has also been
detected in the kidney [11].
Direct metal transport studies in Xenopus laevis oocytes
have demonstrated that DMT1 (isoform I) is a pH-
dependent divalent metal transporter with broad substrate
specificity including Fe
2+
,Mn
2+
,Co
2+
,Ni
2+
,Cu
2+
,Zn
2+
,
and toxic metals Cd
2+
and Pb
2+

bank ( />(Received 29 January 2004, revised 16 March 2004,
accepted 23 March 2004)
Eur. J. Biochem. 271, 1938–1951 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04104.x
Although numerous studies have been carried out to
explore the molecular biology aspects of DMT1 since the
discovery of this gene, there has so far been no structural
characterization of either this integral protein or a segment
of it. Analysis of the structure of membrane proteins either
by NMR spectroscopy or crystallography has proven
difficult, because the native structures of these integral
proteins are largely dependent on the associated membrane.
Recently model peptides, which mimic the sequence of a
segment or a subunit of membrane proteins, have been
widely used to investigate structure and function in several
integral membrane proteins [15–20]. This approach has
proved to be very successful in providing qualitative
structural information and in guiding complete structure
determination [21,22]. For example, it has enabled 3D
structural models of lactose permease, a 12-transmembrane
helix bundle that transduces free energy, to be derived
recently, based on its transmembrane topology, secondary
structure, and numerous interhelical contacts without using
crystals [23].
We have previously investigated the secondary structure
of the TM4 of DMT1 in various membrane-mimetic
environments, such as 2,2,2-trifluoroethanol (TFE), deter-
gent micelles and phosphate lipids [24]. We showed that the
DMT1-TM4 peptide assumed predominately an a-helical
conformation in these environments. In the present study,
we have used NMR spectroscopy and a molecular dynamic

and
2,2,2,-trifluoroethanol-d
3
99.94% (TFE), methanol-d
4
99.6%, deuterium oxide 99.96%, and sodium dodecyl-d
25
sulfate were purchased from Cambridge Isotope Laborat-
ories (Cambridge, MA, USA). Palmitol(doxyl)-stearoyl-
phosphatidylcholine (doxylPtdCho) lipids containing the
nitroxide spin label on C12 were purchased from Avanti
Polar Lipids (Alabaster, AL, USA).
Circular dichroism spectroscopy
CD experiments were performed on a Jasco J-720 spectro-
polarimeter at ambient temperature. Cells with path lengths
of 0.1 and 1.0 mm were employed for sample solutions
containing final peptide concentrations of 6, 12, 23, 47, 94,
188, 375 and 750 l
M
in TFE. Spectra were recorded from
190 to 260 nm at a scan rate of 50 nmÆmin
)1
with a respond
time of 0.25 s, step resolution of 0.1 nm and band width of
1 nm. Each spectrum was obtained from the average of four
scans. Prior to calculation of final ellipiticity, all spectra were
corrected by subtraction of background and were smoothed
using a fast Fourier transform filter.
NMR spectroscopy
The samples used for NMR studies were prepared as

1D experiments were acquired using 32 768 data points and
processed with 0.3 Hz line broadening. The NOESY [27,28]
experiments were recorded at mixing times of 50, 150, 200
and 250 ms, and the TOCSY spectra employed the MLEV-
17 pulse sequence [29] with mixing periods of 50–100 ms.
The relaxation delay was 1.8 s in the TOCSY experiments
and 2.0 s in the NOESY experiments. Typically, 40–80
transients were collected for each increment of F1 in the
NOESY experiments, and 80–120 in the TOCSY experi-
ments. All 2D experiments were collected using 2048 data
points in F2, 256–512 increments in F1. All 2D Spectra were
acquired in the phase sensitive mode using States-time-
proportional phase incrementation in F1 dimension.
Spectral data were processed on a computer using
standard Bruker software (
XWINNMR
Version 3.1). Data
were zero-filled to 2048 points in F1 dimension and then
transformed with a shifted sine-bell squared window
function in both dimensions. Base line correction was also
carried out.
Structure calculations
Distance constraints were obtained from NOESY spectra
recorded with a mixing time of 200 ms in SDS micelles and
150 ms in TFE at 298 and 305 K, respectively. In the case
of severe spectral overlap, the corresponding NOEs were
excluded from the set used for the structure calculations.
Both NOE intensities and chemical shifts were extracted
using the
SPARKY

water shell of 8 A
˚
in
AMBER
7 [33,34].
From these calculated structures, 16 conformers with the
lowest energy were selected to represent the NMR struc-
tures. The quality of the final structures was accessed using
the program of
PROCHECK
-
NMR
[35]. Further analysis and
visualization of the conformers including calculation of root
mean square deviations (rmsds) and identification of
H-bonds was performed using the molecular graphics
program
MOLMOL
[36].
Paramagnetic broadening experiments
Samples containing 2 m
M
DMT1-TM4 and 300 m
M
SDS-
d
25
in 0.6 mL 90% H
2
O/10% D

O
before being added to the sample. The experiments were
performed with concentrations of paramagnetic metal ions
of 0.1, 0.2, 0.4 and 1.0 m
M
. The TOCSY spectra were
again recorded in the presence of different amounts of
paramagnetic metal ions at different pH values (e.g. 7.4, 5.5
and 4.0).
Hydrogen exchange experiments
In the TFE system, 3 mg of DMT1-TM4 was directly
dissolved in 0.6 mL TFE-d
3
. Fast exchange amide protons
were monitored by subsequently recording a series of one-
dimensional
1
H-NMR spectra at 10, 30, 60, 90, 120 and
360 min until no further changes were observed in the
spectra. The TOCSY spectrum (mixing time 50 ms) was
then acquired in a total time of 19 h, and those protons
which showed cross-peaks in the H
a
–H
N
region of TOCSY
spectrum were regarded as slowly exchanging amide
protons.
In SDS micelles, 0.6 mL D
2

pH 6.0 were chosen for sequential assignments and structural
calculations, as it is close to the biological function pH ( 5.5)
of its integral protein. Moreover, the spectra at this pH were
relatively well resolved compared with those at other pH
values. Figure 1 shows the fingerprint region of the 600 MHz
NOESY spectra of DMT1-TM4 in 300 m
M
SDS-d
25
at
pH 6.0 (298 K) and in TFE-d
2
(305 K). It can be seen that the
peptide exhibited sufficient chemical shift dispersions in both
environments, allowing unambiguous assignments of most
proton frequencies. The
1
H resonance assignment was
straightforward, based on a standard procedure [38]. The
complete spin systems of the individual amino acid residues
were identified using the TOCSY spectra with mixing times of
50 and 100 ms. The backbone sequential connectivities were
established by following the H
a
and H
N
cross-peaks of
adjacent amino acids in the fingerprint and the H
N
–H

and Val17, which are overlapped together in TFE (Fig. 2),
indicative of a helical conformation in this region. This is
in agreement with our previous CD studies, which demon-
strated high helical contents in the DMT1-TM4 peptide
[24]. Furthermore, we also observed that the chemical shift
for threonine H
b
is greater than that of H
a
for both Thr11
and Thr15, indicating that both threonines are situated in
the helical region. Evaluation of the secondary structure
from backbone coupling constants was hampered due to
extensive line broadening both in the TFE and SDS micelle
environments, which retards determination of these coup-
ling constants.
Structure calculations and description
Distance constraints were obtained from NOESY spectra
recorded with a mixing time of 200 ms measured in 90%
H
2
O/10% D
2
O (v/v) containing 2 m
M
peptide and 300 m
M
SDS-d
25
at pH 6.0, and 150 ms in TFE-d

=H
N
iþ4
was also observed
for Val8–Phe20 in SDS and Val8–Lys23 in TFE, indicative
of a well-structured peptide in helical conformation over
each span [41,42]. The absence of medium-range NOEs
at the N-terminus suggested no defined structure in this
segment. However, in the C-terminal segment, NOEs
between H
a
i
=H
N
iþ2
and H
N
i
=H
N
iþ2
, which are characteristic
of 3
10
-helix [38], were also detected in TFE. No long-range
NOEs were observed over the full peptide, indicating that
the peptide does not form tertiary folds.
Fig. 1. Fingerprint region of the 600 MHz
NOESY spectra of DMT1-TM4. (A) 200 ms
NOE spectrum of 2 m

CYANA
[31]. Under this protocol, 200 randomized starting struc-
tures were energy minimized under the NMR constraints
and the 30 structures with no violations > 0.2 A
˚
for the
distance constraint and > 5° for the angle constraint, as well
as with the lowest target function were selected in either
SDS or TFE for further energy minimization. The structural
statistics showed that the structures of DMT1-TM4 in both
membrane-mimetic environments were well defined by
NMR data, as indicated by the low values of the target
function (Table 1). The backbone / and w dihedral angles
were also uniformly well-defined, as judged from an angular
order parameter of  1.0 in the span of Leu9–Phe20 [43].
These structures were subjected to an energy minimization
using the program
AMBER
7 [33,34] in the
AMBER
force field
[32]. The final 16 lowest energy structures of DMT1-TM4 in
both SDS (pH 6.0) and TFE were chosen to represent the
solution structures of the peptide, as shown in Fig. 3.
The quality of the final structures was assessed using the
program
PROCHECK
-
NMR
[35]. In the range of well-defined

pattern of sequential and medium-range NOEs and the
Fig. 2. Summary of NMR spectroscopy data for secondary structure prediction for DMT1-TM4 peptide. (A) In SDS micelles at pH 6.0, 298 K and
(B) in TFE at 305 K. The NOE connectivities, amide proton exchange rates, chemical shift index values as well as numbers of NOE constraints per
residue for DMT1-TM4 are shown. Slowly and rapidly exchanging amide protons are represented as filled and open circles, respectively. The NOEs
of intra, sequential and medium range are indicated as white, light gray and dark gray bars, respectively.
1942 H. Li et al.(Eur. J. Biochem. 271) Ó FEBS 2004
prediction based on the chemical shift index. However, the
pairwise rmsds between these structures and mean struc-
tures in the range Arg1–Tyr24 were significantly increased
for the backbone and all heavy atoms in both SDS and TFE
(Table 1), which suggested that the N-terminus was poorly
defined compared with the C-terminus in both SDS and
TFE, consistent with the fewer medium range NOEs
observed in this region. This is probably due to some
flexibility in this region. Although the C-terminal region
(Leu21–Tyr24) does not fold into a typical helical structure, it
is relatively ordered compared with the N-terminal region. In
particular, it is extremely close to a-helical folding in TFE,
judging from both angular order parameters ( 1.0) for
backbone / and w dihedral angles and Ramachandran space
analysis from
PROCHECK
-
NMR
. When the structures of
DMT1-TM4 in both SDS and TFE were superimposed
over the backbone atoms of Leu9–Phe18 for a best fit, we
noticed that the lower part of the helices and the C-terminus
were differently oriented in SDS compared with that in TFE.
The C-terminus was bent towards the helical core in SDS

the peptide becomes less structured as pH values increase.
However, residue Leu19 gave rise to a different pattern.
Figure 4 shows a summary of the intra- and inter-residual
NOE connectivities for the peptide in SDS micelles at
pH 4.0 and 7.5. From the pattern of NOEs, a well-defined
a-helical region comprised of residues Val8–Lys23, and
Gly7–Phe18 at pH 4.0 and 7.5, respectively, was proposed,
while the N-terminus was probably in an extended confor-
mation at both pH values. The structures of DMT1-TM4 at
both pH 4.0 and 7.5 were calculated subsequently using
molecular dynamics in torsion angle space, using a simu-
lated annealing protocol as described above.
From a total of 328 (pH 4.0) and 340 (pH 7.5) NOE
assignments, 222 (31 medium range, 103 intraresidue and 88
sequential NOEs, pH 4.0) and 232 (39 medium range, 103
intraresidue and 90 sequential NOEs, pH 7.5) nonredun-
dant upper-limit constraints were obtained for the structural
calculations. Sixteen structures with lowest target functions
were selected for each pH value, and were superimposed
over the backbone atoms of Ile10–Val17 (Fig. 5). The
structures of DMT1-TM4 at both pH 4.0 and 7.5 were
well characterized by NMR data with no distance violations
larger than 0.2 A
˚
. At pH 4.0, the pairwise backbone
rmsds over residues Leu9–Lys23 were 0.67 ± 0.20 and
1.52 ± 0.30 A
˚
for backbone atoms and all heavy atoms,
respectively; while at pH 7.5, the pairwise backbone

However, a longer helix was formed at lower pH, e.g. Leu9–
Lys23 at pH 4.0 vs. Leu9–Val17 at pH 7.5, indicating that
the C-terminal end is more susceptible to unfolding as the
pH value increases. At pH 4.0, in the segment of Leu9–
Lys23, H
N
iþ4
! CO
i
hydrogen bonds indicative of a-helices
were observed for the majority of structures out of the 16
conformers. However, H-bonds between Val17 and Leu21
were not detected in any of the 16 conformers, while the
H-bonds between Asp14 and Phe18 were missing in the
majority of the 16 conformers, indicating that the structures
may be distorted in this region. Similarly, H-bonds charac-
teristic of a-helices were also found in most of the structures
of the 16 conformers at pH 7.5 in the segment of Leu9–
Val17, except Ile12 and Phe16 in some of the conformers.
Table 1. Structural statistics for DMT1-TM4 in the presence of SDS at
pH 6.0 and in TFE.
Parameter
SDS
(pH 6.0, 298 K)
TFE
(305 K)
Target function (A
˚
2
) 0.03 ± 0.01 0.12 ± 0.02

Additional allowed regions 8.9 8.3
Generously allowed regions 0 0
Disallowed regions 0 0
a
Analyzed using
PROCHECK
-
NMR
.
Ó FEBS 2004 Structure and topology of TM4 of DMT1 (Eur. J. Biochem. 271) 1943
Moreover, some structures displayed H
N
iþ3
! CO
i
hydrogen
bonds indicative of 3
10
helices between Ala13 and Phe16,
and also between Ile12 and Thr15.
The structures of DMT1-TM4 in TFE were super-
imposed with those in SDS micelles at pH 4.0 over the
backbone atoms of Leu9–Phe20 for comparison (Fig. 5B).
We noticed no significant difference between the structures
in these environments, and even the orientations of the side
chains were remarkably similar.
Amide proton exchange
Backbone proton-deuterium exchange experiments have
long been used to verify whether amide protons are involved
in hydrogen bonds or are largely shielded from solvent

(data not shown), which is consistent with the formation of
H-bonds. Similarly, both the N- and C-terminal residues
lost their intensities within 30 min after addition of D
2
Otoa
lyophilized sample containing SDS-d
25
at pD 5.5. Residues
involved in the formation of the helix mostly retained their
intensities in the first 2 h, except Thr11 and Asp14. A
NOESY spectrum recorded after 8 h showed that residues
Ile10, Ile12, Asp13, Phe16, Val17 and Leu19–Leu21 were
observable in this period of time (H. Li, F. Li, Z. M. Qian &
H. Sun, unpublished observation), indicative of slowly
exchanging protons (Fig. 2). Nearly all cross-peaks in the
NOESY spectrum vanished after 12 h and no amide
protons were observable in 1D
1
H spectrum after 20 h
(data not shown). The amide proton exchange experiments
presented here suggest that hydrogen bonds play an
important role in the stabilization of DMT1-TM4 confor-
mations both in TFE and SDS micelles. The faster exchange
of amide protons of Thr11 and Asp14 in SDS micelles
indicates that there is probably some solvent accessibility in
the peptide between the micellar and aqueous environments.
Paramagnetic broadening studies
12-DoxylPtdCho. Information from
1
H line-broadening

(red) over the backbone atoms of Leu9–
Phe20.
Ó FEBS 2004 Structure and topology of TM4 of DMT1 (Eur. J. Biochem. 271) 1945
peaks of Ile10, Ile12 and Val17. This suggested that the
peptide was inserted in the interior of micelles. However,
little effect was observed for Asp22, Lys23 and Tyr24,
indicating that the C-terminus is probably exposed outside
the micelles. The N-terminus residues (Val2, Leu4 and Tyr5)
surprisingly decreased their intensities significantly in the
presence of 12-doxylPtdCho, implying that the N-terminus
may also be located inside the micelles.
Mn
2+
and Gd
3+
broadening. It has been shown previ-
ously that paramagnetic metal ions particularly affect
resonances of water and the surface of SDS micelles [49].
The paramagnetic broadening effects of Mn
2+
and Gd
3+
on the peptide resonances were studied by comparing 1D
1
H and 2D TOCSY or NOESY spectra in the presence and
absence of the paramagnetic metal ions. The amplitudes of
the spectra in the presence of the paramagnetic metal ions
were normalized to the least affected cross-peaks. The
paramagnetic metal ions were titrated into the samples
containing 2 m

M
SDS-
d25
at 298 K and pH 5.5. (A) In
the absence of paramagnetic agents. (B) In the presence of 5 m
M
12-doxylPtdCho (12-doxylPC). (C) In the presence of 0.2 m
M
Mn
2+
.(D)Inthe
presence of 0.1 m
M
Gd
3+
.
Fig. 7. pH-regulated location of the C-terminus in SDS micelles.
Residual relative intensities of H
a
-H
N
2D cross-peaks of DMT1-TM4
in SDS micelles in the presence of 0.2 m
M
Mn
2+
at pH 5.5 (j)and4.0
(d). Those calculated from H
a
-H

M
Mn
2+
(H. Li, F. Li, Z. M. Qian & H. Sun, unpublished
observation).
Titration of Gd
3+
(0.1, 0.2 and 0.4 m
M
)into2m
M
DMT1-TM4 containing 300 m
M
SDS-d
25
at pH 5.5 was
also made. It was noticed that the N-terminal residues
(Val2–Leu9) almost retained their intensities, whereas the
C-terminal residues and those involved in the formation of
helix completely disappeared from the TOCSY spectrum
(Fig. 6D) in the presence of 0.1 m
M
Gd
3+
at pH 5.5. This is
in agreement with the Mn
2+
titrations, but the paramag-
netic effects were more evident in the presence of Gd
3+

residual intensities for Leu19 and Tyr24 were calculated
from the cross-peaks of H
a
-H
b
, as the amide protons of
Tyr24 and Leu19 overlapped with Asp22 and Thr15,
respectively, in the presence of Mn
2+
at pH 4.0. As
illustrated in Fig. 7, residues Leu9–Phe20 almost completely
regained their intensities; while residues Leu21–Tyr24
regained only  40% of their intensities. This was still the
case even in the presence of 1.0 m
M
Mn
2+
, although the
spectra were considerably broader (data not shown). Simi-
larly, the intensities of the cross-peaks were also recovered in
the presence of Gd
3+
at pH 4.0, but the degree to which the
intensities recovered was lower in the presence of Gd
3+
,
particularly for the C-terminal residues, than in the presence
of the same amounts of Mn
2+
(H.Li,F.Li,Z.M.Qian&

Interestingly, the C-terminal part became well folded only
at low pH values (e.g. pH 4.0). This is probably due to the
protonation of Asp22 (pK
a
) which consequently has less
repulsion with the anionic head group of SDS molecules.
Whether the C-terminus has a regulative role in metal
transport remains to be investigated further.
It is of interest to investigate whether the peptide is
inserted into the micelles or lies along the micelle surface.
Relaxation probes have been widely used to determine
micelle-embedded [50,51] or water exposed fragments of
polypeptides [52]. In the present study, paramagnetic
broadening effects on the peptide resonances were used to
investigate the topology of the peptide relative to the SDS
micelle surface. This includes 12-doxylPtdCho and para-
magnetic metal ions (Mn
2+
and Gd
3+
). Although we have
previously used 5- and 16-doxyl-stearic acids to probe the
location of the peptide relative to the micellar surface [24],
the N-terminus was found to be affected by both spin-labels,
and it is therefore hard to draw a firm conclusion for its
location. In addition, we could not exclude the possibility
that the positively charged N-terminus interacts directly
with the negatively charged carboxyl-function from the
stearic acids, thus forcing the N-terminus into spatial
proximity with the spin-labels. In order to avoid this effect,

ing the peptide bonds into the hydrocarbon core [53]. The
H
a
-H
N
resonances of the C-terminal residues Phe18–Tyr24
were unobservable in the presence of paramagnetic metal
ions at pH 5.5, suggesting that these residues are situated at
the micelle surface or outside micelles at this pH value.
Surprisingly, a periodic residue broadening was observed
for the residues in the a-helical region at pH 5.5 (Fig. 7).
Asp14 completely disappeared, while the intensities of
Thr11, Ile12 and Thr15 significantly decreased and this
effect was propagated in the presence of Gd
3+
.This
phenomenon seems at odds with the results obtained from
spin-labeling experiments as the paramagnetic metal ions do
not enter the micelle and should only broaden the residues
located in aqueous phase and/or at the surface of SDS
micelles [49]. Previously, Mn
2+
has also been used as a
paramagnetic probe to explore solvent-exposed residues for
membrane peptides. Relative high concentrations ( 1–
5m
M
) were used to broaden the residues located outside the
micelles, and membrane-spanning regions were found to be
unaffected even at such high concentrations, while the

movement of the C-terminus relative to SDS micelles at
different pH values is also supported by the calculated
structures, in which the C-terminus was folded into helix
only at low pH values (e.g. pH 4.0). This phenomenon, that
the residues situated near micellar aqueous boundary can
move Ôup and downÕ at different pH values, has been noticed
previously [57]. Alternatively, at such a pH ( 4), a
relatively high concentration of protons may compete with
Mn
2+
and thus retard the entry of this metal ion at pH 4.0.
For the best explanation of our experimental data, we
hypothesize that a channel comprised of several peptide
monomers might be formed, which allows the permeation
of metal ions. Our CD study has shown the change of the
molar residue ellipiticity of the peptide at 222 nm on peptide
concentration (Fig. 8), indicating the presence of inter-
molecular interactions. Neither the oligomerization by SDS-
Tricine-PAGE [24], nor intermolecular NOE peaks in
NOESY spectra were observed. An attempt to test aggre-
gation behavior in SDS micelles by means of a similar
approach was not made, as the aggregation probably
depends on both SDS concentrations and the ratio of SDS
to the peptide. It is reasonable to assume that peptide may
have a similar aggregation behavior in SDS micelles. The
peptide helical monomers are probably orientated with the
polar face (e.g. Thr11, Asp14 and Thr15) pointing to each
other in the inner lumen of the oligomer, to reduce the
unfavorable free energy caused by immersing these residues
in the hydrophobic milieu. Interactions between the polar

and Gd
3+
bound water with the water and amino
protons in the channel. In contrast, no observable changes
in 2D NOESY were found upon addition of diamagnetic
metal ions (Zn
2+
) under similar conditions (H. Li, F. Li,
Z. M. Qian & H. Sun, unpublished observation). Interest-
ingly, when the pH value was lowed to 4.0, almost all the
resonances in NMR spectra recovered their intensities,
indicating the movement of the C-terminus towards SDS
micellar interior, thus making either metal ions or solvent
molecules inaccessible.
In conclusion, we have determined the solution structures
of a synthetic peptide, corresponding to the TM4 of DMT1
in membrane-mimetic TFE and SDS micelles. The peptide
adopts remarkably similar structures in both environments.
The structures consist of three regions: a well-defined helical
region Leu9–Phe20 in TFE, a highly disordered N-terminus
(Arg1–Gly7) and a pH-sensitive C-terminus, which folds
into a helix only at low pH. The paramagnetic broadening
studies in combination with H-D exchange experiments
indicate that the peptide is inserted into the interior of SDS
micelles. More work is needed in the future to clarify
whether DMT1 functions through a channel in vivo and
what role the TM4 plays in the function of its integral
protein.
Acknowledgements
We would like to thank the University of Hong Kong and the Area of

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Supplementary material
The following material is available from http://
blackwellpublishing.com/products/journals/suppmat/EJB/
EJB4104/EJB4104sm.htm
Table S1. Chemical shifts for DMT1-TM4 in TFE and SDS.
Ó FEBS 2004 Structure and topology of TM4 of DMT1 (Eur. J. Biochem. 271) 1951