Báo cáo khoa học: Structure and topology of the transmembrane domain 4 of the divalent metal transporter in membrane-mimetic environments docx

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

˚
for the backbone heavy atoms and
0.82 ± 0.17 A
˚
for all heavy atoms. In SDS micelles, the
length of the helix is dependent on pH values. In particular,
the C-terminus becomes well-structured at low pH (4.0),
whereas the N-terminal segment (Arg1–Gly7) is flexible and
poorly defined at all pH values studied. The effects of
12-doxylPtdCho spin-label and paramagnetic metal ions on
NMR signal intensities demonstrated that both the N-ter-
minus and helical region of the TM4 are embedded into the
interior of SDS micelles. Unexpectedly, we observed that
amide protons exchanged much faster in SDS than in 2,2,
2-trifluoroethanol, indicating that there is possible solvent
accessibility in the structure. The paramagnetic metal ions
broaden NMR signals from residues both situated in aque-
ous phase and in the helical region. From these results we
speculate that DMT1-TM4s may self-assemble to form a
channel through which metal ions are likely to be trans-
ported. These results might provide an insight into the
structure-function relationship for the integral DMT1.
Keywords:DMT1;membrane;NMR;structure.
The divalent metal transporter (DMT1) gene, also known as
Nramp2 (natural resistance-associated macrophage protein-
2) and DCT1, was identified recently [1,2]. It belongs to a
large family of integral membrane proteins highly conserved
throughout evolution, from bacteria to human beings [3–6].
It is the only known cellular iron importer, and is
responsible for importing iron from the gut into the entero-

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+

[1]. Studies in cultured
mammalian cells have also shown that both isoforms of
DMT1 are capable of transporting a variety of divalent
metal ions across the plasma membrane [12,13]. Transport
of these metal ions was shown to occur at pH 5.5, but not at
7.4 [1]. The His267/His272 located in the transmembrane
domain (TM) 6 has been thought to play an important role
in pH regulation of metal transport by DMT1 [14].
However, it is not yet clear how pH regulates DMT1 metal
transport. The biological importance of this transporter is
shown by its involvement in two naturally occurring animal
mutants of iron metabolism. A mutation (G185R) in TM4
of DMT1 is responsible for microcytic anemia of the mk
mice and Belgrade rats, which exhibit severe defects in
intestinal iron absorption and erythroid iron utilization [2,7].
This suggests that the TM4 of DMT1 may have a unique
and important biological function. The sequence of this
domain is characterized by a high degree of hydrophobicity
and is highly conserved among different species [1].
Correspondence to H. Sun, Department of Chemistry, The University
of Hong Kong, Pokfulam Road, Hong Kong.
Fax: + 852 2857 1586, Tel.: + 852 2859 8974,
E-mail:
Abbreviations: doxylPtdCho, palmitol(doxyl) stearoyl-phosphatidyl-
choline; 12-doxylPtdCho, doxylPtdCho lipids containing the nitroxide
label on C12; DMT1, divalent metal transporter; DMT1-TM4,
transmembrane domain 4 of DMT1; HFIP, 1,1,1,3,3,3,-hexafluoro-2-
propanol; TFE, 2,2,2-trifluoroethanol; TM, transmembrane domain.
Note: The coordinate for the 16 lowest energy conformers both in
SDS micelles at pH 6.0 and TFE has been deposited in the protein data

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

simulated annealing approach to characterize the 3D
structures of DMT1-TM4 in both TFE and SDS micelles
at different pH values. The topology of the peptide in SDS
micelles was probed by the effects of spin-labels, including
both palmitol(doxyl) stearoyl-phosphatidyl-choline (doxy-
lPtdCho) lipids containing the nitroxide label on C12
(12-doxylPtdCho) and paramagnetic metal ions (Mn
2+
and
Gd
3+
), on the intensities of NMR signals. The peptide was
found to embed into the interior of SDS micelles. The
possibility of formation of a divalent metal channel has been
discussed.
Experimental procedures
Materials
The sequence of the peptide (RVPLYGGVLITIADT
FVFLFLDKY) was taken from rat DMT1 and represents
the putative TM4 (residues 179–202). The peptide was
synthesized by a solid-phase method and was purified by
HPLC on a Zorbax SB Phenyl reverse phase column using
0.1% (v/v) trifluoroacetic acid/water and 0.1% (v/v)
trifluoroacetic acid/acetonitrile as solvents (Biopeptide
Co., LLC. San Diego, CA, USA). The purity was assessed
by both mass spectrometry and analytical HPLC to be
above 95%. 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) was
obtained from Sigma. Deuterated reagents for NMR
sample preparation, e.g. 2,2,2-trifluoroethanol-d
2

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

described previously [24]. Briefly,  3 mg of the peptide
dissolved in HFIP was mixed with an equal volume of
SDS-d
25
aqueous solution. The mixture was further diluted
with water, and was subject to lyophilization. The resulting
powder was then redissolved in 0.6 mL H
2
O containing
10% (v/v) D
2
O. The concentration of the peptide was
approximately 2.0 m
M
in SDS-d
25
(300 m
M
). Spectra for
assignments and structure calculation in the presence of
SDS were recorded at 298 K. In the presence of TFE, the
spectra were recorded at 298 and 305 K to resolve spectral
overlap.
All spectra were recorded on a Bruker AV600 spectro-
meter, operating at a proton frequency of 600.13 MHz.
Water suppression was carried out using a 3-9-19 watergate-
pulse sequence [25,26]. The sodium salt of trimethylsilyl-
propionate-d
4
solution was used to reference chemical shifts.

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

software [30] and served as an input for
Ó FEBS 2004 Structure and topology of TM4 of DMT1 (Eur. J. Biochem. 271) 1939
the program of
CYANA
(1.0) [31]. On the basis of these
distance constraints obtained using the macro
CALIBA
,a
systematic analysis of the local conformation around the C
a
atom of each residue, including the dihedral angles /, w, v
1
and v
2
, was performed using the macro
GRIDSEARCH
as
implemented in
CYANA
[31]. The final nonredundant upper-
limit constraints and the resulting angle constraints were
used in the structural calculations. No stereospecific assign-
ments were obtained in any case. The 200 randomized
starting structures were energy minimized during 4000 steps
under the NMR constraints, and the 30
CYANA
conformers
with the lowest target function values were selected for
further energy minimization under the force field of Cornell
et al. [32] using a generalized Born solvent model with a

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

2
O (v/v) were used in these
experiments. The pH was adjusted to either 5.5 or 7.4 by
addition of small aliquots of NaOH. Spin-labeled 12-
doxylPtdCho was solubilized in methonal-d
4
, and aliquots
of this solution were then added to the peptide at pH 5.5 to
yield a final concentration of the spin-label of 5 m
M
.This
corresponded to approximately one spin-label per micelle
on the assumption of about 60 molecules per micelle [37].
The TOCSY spectrum (mixing time of 50 ms) was
acquired with a spectral width of 6 p.p.m. in F1 dimension,
with 120 transients, and 256 increments in F1 dimension.
The pH of the sample was then raised to 7.4 and the
TOCSY spectrum was collected. The reference spectrum in
the absence of the spin-label was also recorded under
identical conditions. For Gd
3+
and Mn
2+
broadening
studies, either GdCl
3
or MnCl
2
were dissolved in H
2

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

O was added to lyophilized
samples containing 2 m
M
peptide and 300 m
M
SDS-d
25
.
The pD was adjusted to 5.5 by addition of aliquots of
NaOD. Similarly, the fast exchanging amide protons were
monitored by 1D proton NMR spectra recorded at different
time intervals from 10 to 120 min. The NOESY spectrum
(mixing time 200 ms) was then acquired in a total time of
8.5 h, and the protons that appeared in the spectrum were
regarded as relatively slow exchanging protons. All amide
protons were exchanged completely within 12 h.
Results
Resonance assignment and secondary structure
determination
The DMT1-TM4 peptide is highly hydrophobic, and insol-
uble in water and a range of organic solvents. We have chosen
TFE and SDS to solubilize the peptide and to mimic
biological membranes. The peptide is stable in these environ-
ments for at least a couple of months at room temperature.
TOCSY and NOESY spectra with a set of mixing times were
recorded for DMT1-TM4 in SDS-d
25
at different pH values,
and reasonably well-resolved spectra were found at a wide
range of pH values. The spectra recorded in SDS micelles at

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

N
region
of the TOCSY and NOESY spectra. Using this technique it
was possible to unambiguously assign almost all the proton
resonances including side chains, apart from a few aromatic
protons from phenylalanine residues due to spectral overlap
(H. Li, F. Li, Z. M. Qian & H. Sun, unpublished observation).
We noticed that chemical shift values in TFE and in SDS
micelles were similar with the expected exception of amide
protons, in particular, the amide protons of N-terminal
residues.
The chemical shifts of the H
a
protons provide informa-
tion about secondary structural elements of the peptides.
Generally, all residues experience a H
a
upfield shift relative
to the random-coil value when adopting a helical confor-
mation and a downfield shift when found in an extended or
b-strand structure [39]. The peptide was predicted to adopt a
helical conformation in the segment of Leu9–Phe20 in SDS
micelles and Gly6–Lys23 in TFE from the chemical shift
index method [40] (Fig. 2). Further investigation by exam-
ining the observed NOE connectivities produced similar
results. All H
N
resonances from Tyr5 to Phe20 were found
to be connected by (i, i+1) connectivities except for Phe16
1940 H. Li et al.(Eur. J. Biochem. 271) Ó FEBS 2004

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

2
.TheNOE
connectivities and numbers of NOEs per constraints for
DMT1-TM4 in both solvents are summarized in Fig. 2.
Except for unresolved cross-peaks between the residue pairs
Leu21/Asp22 in SDS, and Phe16/Val17 and Phe20/Leu21 in
TFE, almost all of the possible H
N
i
=H
N
iþ1
[38], and sequential
NOEs were observed in the segment of Tyr5–Phe20. In
addition, the presence of medium-range connectivities [38],
such as H
a
i
=H
N
iþ3
,H
a
i
=H
b
iþ3
and H
a
i

=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

M
DMT1-TM4 in
300 m
M
SDS-d
25
at pH 6.0, 298 K. (B) 150 ms
NOE spectrum of 2 m
M
peptide in TFE-d
2
,
305 K. The sequential assignment of all resi-
dues is indicated.
Ó FEBS 2004 Structure and topology of TM4 of DMT1 (Eur. J. Biochem. 271) 1941
Totals of 241 and 265 meaningful upper-limit distance
constraints were obtained based on totals of 358 and 378
assigned NOE cross-peaks for DMT1-TM4 in SDS at
pH 6.0 and in TFE, respectively. A total of 79 dihedral
angle constraints for 50 angles in SDS vs. 85 constraints for
55 angles in TFE, derived using the macro
GRIDSEARCH
as
implemented in
CYANA
[31], were also included in the
structure calculations. In no case could stereospecific
assignment be achieved. The structures were calculated by
molecular dynamics in torsion angle space using a simulated
annealing protocol as implemented in the program

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

residues, i.e. Leu9–Phe18 in SDS (pH 6.0) and Leu9–Phe20
in TFE, 99.4% and 91.7% occupy the most favored regions
of the Ramachandran space in SDS and TFE, respectively,
and none are found in the disallowed regions (Table 1).
The overall structure of DMT1-TM4 in SDS micelles is
similar to that in TFE. The mean structures obtained from
MOLMOL
showed that the peptide folded into an a-helical
conformation for Leu9–Phe18 in SDS and Leu9–Phe20 in
TFE. The pairwise rmsds between the minimized structures
and the mean structure in SDS at pH 6.0 were 0.18 ± 0.06
and 0.85 ± 0.17 A
˚
for the backbone and all heavy atoms,
respectively, in the segment Leu9–Phe18, vs. 0.20 ± 0.06
and 0.89 ± 0.16 A
˚
in the segment Leu9–Phe20 (Table 1).
The pairwise rmsds between the minimized structures
and mean structure in TFE were 0.23 ± 0.10 and
0.82 ± 0.17 A
˚
for the backbone and all heavy atoms,
respectively, in the segment Leu9–Phe20, vs. 0.26 ± 0.12
and 0.87 ± 0.17 A
˚
in the segment Leu9–Lys23. This
suggested that the structures of the peptide from the residue
Leu9 towards the C-terminal residues were well-defined by
NMR constraints, which is consistent with the observed

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

micelles at pH 6.0. The aromatic rings of Phe16 and Phe20
were oriented more or less in a plane parallel to each other in
TFE (Fig. 3B). In contrast, the rings of Phe16 and Phe20
were almost perpendicular to each other in SDS (Fig. 3A).
In most structures, H
N
iþ4
! CO
i
hydrogen bonds were
present in the helical region comprising residues Leu9–
Phe20 in SDS micelles and TFE, with the exception of the
missing H-bonds between Phe16 and Phe20 in both SDS
and TFE. Additionally, a H
N
iþ3
! CO
i
hydrogen bond was
present between Val17 and Phe20 in both SDS and TFE.
pH effects on the structures
The effects of pH on peptide conformation in SDS micelles
were investigated by acquiring a series of NOESY spectra
over the pH range of 4.0–7.5 at 298 K. Changes in
conformation over this range were assessed by analyzing
the differences of H
a
chemical shifts from random coil
values (data not shown). Generally, chemical shifts were
closer to random coil values at a higher pH, implying that

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

rmsds over residues Leu9–Val17 were 0.31 ± 0.11 and
0.89 ± 0.21 A
˚
for backbone atoms and all heavy atoms,
respectively.
To highlight the structural difference between the
conformations at both pH values, the structures were
superimposed over the backbone atoms of residues Ile10–
Val17 for a best fit (Fig. 5A). In general, the N-terminus of
the peptide was highly flexible and mostly unstructured at
both pH values, consistent with the lack of medium-range
NOEs,e.g.H
a
i
=H
N
iþ3
,H
a
i
=H
b
iþ3
and H
a
i
=H
N
iþ4
(Fig. 4).

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

Experimental NMR constraints
No. of distance constrains 241 265
Intraresidues 111 118
Sequential (i ¼ 1) 89 75
Medium range
(1 < 1/2i-j1/2 £ 4)
41 72
Long range (1/2i-j1/2 > 4) 0 0
NOE constraint violations (A
˚
)
Sum 0.30 ± 0.10 1.10 ± 0.20
Maximum 0.08 ± 0.02 0.18 ± 0.02
AMBER energy (kcalÆmol
)1
) )856.7 ± 6.2 )857.2 ± 5.1
rmsd from the mean structure (A
˚
)
Residues 1–24
Backbone atoms 4.07 ± 1.28 2.84 ± 0.90
All heavy atoms 5.37 ± 1.36 4.00 ± 1.05
Residues 9–20
Backbone atoms 0.20 ± 0.06 0.23 ± 0.10
All heavy atoms 0.89 ± 0.16 0.82 ± 0.17
Ramachandran statistics
(residues 9–20)
a
(%)
The most favored regions 99.4 91.7

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

access [44]. These experiments have also been used recently
to determine the residues which are involved in the binding
of peptides to membranes [37,45,46]. The exchange rate of
the amide protons of DMT1-TM4 in both TFE and SDS
micelles was monitored by both 1D and 2D
1
HNMR
spectroscopy (e.g. TOCSY and NOESY) and was analyzed
semiquantitatively in terms of either a rapid or slow
exchange of the various residues (Fig. 2). In the presence
of TFE-d
3
,bothN-terminal(Val2,Leu4,Tyr5,Gly6and
Gly7) and C-terminal residues (Asp22, Lys23 and Tyr24)
decreased their intensities rapidly and almost completely
disappeared within 2 h (except Gly6, which disappeared 6 h
later). However, the residues involved in the formation of
the helix comprising of Val8–Leu21 retained their intensities
in this period. A TOCSY spectrum was then recorded that
Fig. 3. Stereoview of NMR structures of
DMT1-TM4 in SDS micelles and TFE. (A) All
atoms of 16 final structures of DMT1-TM4 in
SDS micelles at pH 6.0 with superimposition
over the backbone atoms of residues Leu9–
Phe18. (B) All atoms of 16 final structures of
DMT1-TM4 in TFE overlaid over the back-
bone atoms of Leu9–Phe20.
1944 H. Li et al.(Eur. J. Biochem. 271) Ó FEBS 2004
showed that residues Val8–Leu21 were still observable after
24 h, and thus considered as slowly exchanging protons

(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

caused by the lipid-soluble spin-labeled compound 12-
doxylPtdCho was used to determine the position of the
peptide relative to the micelle surface. We used the
doxylPtdCho lipids containing doxyl groups at C12 atoms
of the stearoyl side-chain, which have been demonstrated
previously to be located near the center of the micelles
[47,48]. Therefore, peptide protons located in the center of
the micelles would be the most affected, whereas those
located at the micelle surface or outside of micelles would be
the least affected. The specific broadening of proton signals
was monitored using TOCSY spectra at the SDS/12-
doxylPtdCho molar ratio of 60 : 1, i.e. approximately one
spin-label per micelle. The presence of 12-doxylPtdCho
caused the complete disappearance of the cross-peaks of
Ala13 in the TOCSY spectrum (Fig. 6B), a significant
reduction in intensities of the cross-peaks of Leu9 and
Phe16, and a moderate reduction in intensities of the cross-
Fig. 4. Effects of pH on secondary structures of DMT1-TM4 in SDS
micelles. The NOE connectivities of DMT1-TM4 in SDS micelles at
pH 4.0 (top) and pH 7.4 (bottom) are summarized.
Fig. 5. Comparison of NMR structures of
DMT-TM4 at different pH values as well as in
different membrane-mimetic environments. (A)
Backbone atoms of 16 structures of DMT1 in
SDS micelles at pH 4.0 (green) are overlaid
with those at pH 7.5 (blue). The structures are
superimposed over the backbone atoms of
Ile10–Val17 for a best fit. (B) Backbone atoms
of 16 structures of DMT1 in SDS pH 4.0
(green) are superimposed with those in TFE

(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
DMT1-TM4 in 300 m
M
SDS-d
25
,and
some of the results are shown in Figs 6 and 7.
Addition of 0.1 m
M
Mn
2+
to DMT1 in SDS micelles at
pH 5.5 led to the complete disappearance of cross-peaks of
the C-terminal residues in the 2D TOCSY spectrum of
Phe18–Tyr24, indicating that these residues might locate
outside SDS micelles. A 3–4 periodic residue broadening
was noticed from residues of Ile10–Phe18. The intensities of
the H
a
-H
N
cross-peaks of Thr11, Ile12 and Thr15 were
drastically reduced, and Asp14 completely disappeared in
Fig. 6. Effects of paramagnetic agents on the TOCSY spectra (H
N-
H
a
region)of2m
M
DMT1-TM4 in 300 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

b
cross-peaks are represented as s.
1946 H. Li et al.(Eur. J. Biochem. 271) Ó FEBS 2004
the presence of 0.2 m
M
Mn
2+
(Figs 6 and 7). This suggests
that there is possible solvent accessibility in the structure,
presumably due to formation of a channel through self
association of peptide monomers. In contrast, the
N-terminal residues (Val2 and Leu4–Leu9) almost retained
their intensities, indicating that the N-terminus was embed-
ded in the SDS micelles and was solvent inaccessible. When
Mn
2+
concentration was increased to 1 m
M
, only the
N-terminal residues were observable in the TOCSY spec-
trum at pH 5.5 (H. Li, F. Li, Z. M. Qian & H. Sun,
unpublished observation). Similar effects of Mn
2+
on the
resonances of DMT1 in SDS micelles were observed at
pH 7.4. However, the effects were more pronounced at this
pH value than at pH 5.5, in particular for the residues of
Ile10, Ile12, Ala13, Phe16 and Val17, which either signifi-
cantly reduced the intensities or completely disappeared
from the TOCSY spectrum in the presence of 0.2 m

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+

.
Further addition of Gd
3+
(0.4 m
M
) led to only a slight
decrease in the intensities of the remaining cross-peaks
(H. Li, F. Li, Z. M. Qian & H. Sun, unpublished
observation).
Interestingly, when the pH was lowered from 5.5 to 4.0 in
the presence of paramagnetic metal ions (0.2 m
M
Mn
2+
or
Gd
3+
), we unexpectedly observed that the resonances not
observable at pH 5.5 regained their intensities at pH 4.0 in
both 1D and 2D NMR spectra. Figure 7 shows the
normalized H
a
-H
N
cross-peak intensities for the peptide
in the presence of 0.2 m
M
Mn
2+
at pH 4.0 and 5.5. The

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&

H. Sun, unpublished observation). This indicated that the
peptide is probably embedded completely inside the SDS
micelles by shrinking the C-terminus towards the micelles,
thus the entrance of the Mn
2+
is probably ÔblockedÕ.
Discussion
In the present study, we have characterized the 3D
structures of DMT1-TM4 peptide in membrane-mimetic
environments, e.g. TFE and SDS micelles at different pH
values by NMR spectroscopy and simulated annealing
calculations. The solution structures of DMT1-TM4 in both
environments are remarkably similar, consistent with our
previous CD studies [24]. The N-terminal segment is
unstructured and highly flexible, and consists of residues
Arg1–Gly7 in both environments. The best-defined helical
region in TFE involves residues Leu9–Phe20, with a rmsd
value of 0.23 A
˚
for the backbone atoms, which is further
supported by the slow amide proton exchange of these
residues (Fig. 2B); while the C-terminal segment (Leu21–
Tyr24) folded into a conformation which is extremely close
to helical folding, based upon both Ramachandran plot and
angular order parameters for backbone / and w dihedral
angles ( 1.0). Similarly, the peptide adopts an a-helical
conformation in SDS micelles. However, the folding of the
peptide is sensitive to the pH, and particularly for the
C-terminus. It forms a helix comprising residues Leu9–
Lys23 at pH 4.0 vs. Leu9–Val17 at pH 7.4 (Fig. 5).

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,

we therefore used 12-doxylPtdCho to study the topology of
the peptide in SDS micelles. The doxylPtdCho does not
perturb the peptide structures as a NOESY spectrum
recorded after addition of the spin label a week later, a
period during which the free-radicals are normally
quenched, shows almost identical NOE cross-peaks com-
pared with those in the absence of the spin label (H. Li,
F. Li, Z. M. Qian & H. Sun, unpublished observation). The
presence of 12-doxylPtdCho, similar to 16-doxyl-stearic
acids, caused complete disappearance of Ala13 and signi-
ficant reduction intensities of Ile12 and Phe16. However,
there were no changes on the C-terminal residues, which
clearly suggested that the peptide was inserted into the
interior of SDS micelles with the C-terminal exposed outside
the micelles. 12-doxylPtdCho again reduced dramatically
the intensities for the cross-peaks of the N-terminal (Val2,
Leu4, Tyr5) residues (Fig. 6).
Although spin-label broadening is an effective approach
to study the membrane location of peptides, it is difficult to
Ó FEBS 2004 Structure and topology of TM4 of DMT1 (Eur. J. Biochem. 271) 1947
estimate the exact position of peptides relative to micelle
surface because doxyl spin-labels broaden the proton
resonances from a large distance. Paramagnetic broadening
via addition of paramagnetic metal ions was therefore
applied as an additional probe to address the topology of
the peptide. Our results showed that few broadening effects
have been observed for the N-terminal residues e.g. Val2–
Leu9 (Figs 6 and 7) at both pH 5.5 and 4.0, implying that
the N-terminus was located inside the micelles. This is
unexpected considering the high energetic cost of partition-

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

intensities for solvent-exposed segments either completely
disappeared or were drastically reduced [54–56]. However,
in the present study, 1 m
M
Mn
2+
caused complete disap-
pearance of the cross-peaks of almost all residues at pH 5.5,
except for the N-terminal residues. A possible explanation
is that there is solvent accessibility in the peptide-micelle
aggregate, and this possibility is also supported by the fast
amide proton exchange for those residues situated in the
helical-core region in SDS micelles (Fig. 2). The amide
protons of Thr11, Asp14 and Thr15, which were buried
inside SDS micelles, were found to exchange more rapidly
compared with other residues in the helical region in SDS.
This was particularly the case for Asp14, which completely
exchanged with deuterium (D
2
O) within 30 min in SDS
micelles. Interestingly, the paramagnetic effect of Mn
2+
(0.2 m
M
) on NMR resonances of the peptide was barely
observable at pH 4.0, except for the residues of Leu21–
Tyr24, which lost  60% of their intensities. This suggests
that the C-terminus of the peptide is partially embedded into
SDS micelles at pH 4.0, which may therefore block the
entrance of either solvent molecules or metal ions. 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

residues may be mediated by water molecules. The oligo-
meric assembly in the membrane thus may be a loose
association between several monomers, which is probably
highly symmetric judging from unobservable intermolecule
NOEs. Twenty one out of 33 known structures possess a
helical bundle topology ( />Membrane_Proteins_xtal.html), suggesting that pore or
channel-forming assembly may be the common features
for the function of transmembrane proteins. It has been
Fig. 8. Self association of DMT1-TM4 in TFE. CD spectra of DMT1-
TM4 at different concentrations ranging from 6 l
M
to 750 l
M
(top)
and dependence of molar residue ellipiticity at 222 nm on peptide
concentration, indicative of intermolecular interactions (bottom).
1948 H. Li et al.(Eur. J. Biochem. 271) Ó FEBS 2004
reported previously that model peptides, corresponding to a
segment of integral proteins, have been found to be able to
resume functional assembly in lipids or even in detergent
micelles, such as formation of channels or pores, as their
integral proteins [15,58,59]. The significant broadening
effects of paramagnetic metal ions, e.g. Mn
2+
and Gd
3+
on the peptide NMR signals in the present study are
probably either due to permeation directly or exchange of
Mn
2+

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

Excellence Scheme of the University Grants Committee (Hong Kong)
for their support. We are grateful for the support from a Hong Kong
RGC grant (BQ-445) and Hong Kong Polytechnic University Research
Grants (G-YW 47 and A-PC 23). The 600 MHz NMR spectrometer
was purchased through an equipment grant from the University of
Hong Kong.
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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