The Mycobacterium tuberculosis membrane protein
Rv2560 ) biochemical and functional studies
David F. Plaza
1
, Hernando Curtidor
2,4
, Manuel A. Patarroyo
1,4
, Julie A. Chapeton-Montes
1
,
Claudia Reyes
3
, Jose Barreto
3
and Manuel E. Patarroyo
1,5
1 Molecular Biology Department, Fundacion Instituto de Inmunologia de Colombia, Bogota, Colombia
2 Receptor–Ligand Department, Fundacion Instituto de Inmunologia de Colombia, Bogota, Colombia
3 Chemical Synthesis Department, Fundacion Instituto de Inmunologia de Colombia, Bogota, Colombia
4 Universidad del Rosario, Bogota, Colombia
5 Universidad Nacional de Colombia, Bogota, Colombia
Tuberculosis (TB) is a major public health problem
throughout the world, affecting almost nine million
people [1] and causing more than three million deaths
per year. An increasing incidence of TB, related to the
high risk of developing the disease in immuno-
suppressed individuals and the increasing proportion
of Mycobacterium tuberculosis drug-resistant strains,
has contributed to this problem [2,3]. This makes the
Keywords

GG304) displayed high binding activity to A549 cells only. Cross-linking
assays showed the specific binding of peptide 11024 to a 54 kDa membrane
protein on U937. Invasion inhibition assays, in the presence of shared
high-activity binding peptide identified for U937 and A549 cells, presented
maximum inhibition percentages of 50.53% and 58.27%, respectively. Our
work highlights the relevance of the Rv2560 protein in the M. tuberculosis
invasion process of monocytes and epithelial cells, and represents a funda-
mental step in the rational selection of new antigens to be included as
components in a multiepitope, subunit-based, chemically synthesized, anti-
tuberculosis vaccine.
Abbreviations
GRAVY, grand average of hydropathicity; HABP, high-activity binding peptide; SPf66, synthetic Plasmodium falciparum 66; SSP, simple
sequence protein; TB, tuberculosis.
6352 FEBS Journal 274 (2007) 6352–6364 ª 2007 The Authors Journal compilation ª 2007 FEBS
development of new therapies and ⁄ or vaccines, or the
improvement of existing ones, necessary to control this
disease.
The identification and characterization of the pro-
teins involved in the interaction of M. tuberculosis with
its target cells and the mechanisms for evading an
immune response produced against this pathogen are
thus of crucial interest. Of special interest are those
proteins found on the cell membrane and in the
secreted fraction, as they represent the ‘face’ shown by
the bacterium to its host, thereby leading to its identifi-
cation and elimination.
Although TB is mainly a pulmonary disease, it can
also affect other organs. Few microorganisms can sur-
vive within macrophages because of the abundance of
hydrolytic enzymes contained in acid phagocytic com-

protein sequence in different organisms, and for pre-
dicting (with some degree of confidence) the existence
of membrane anchoring or signal export regions within
them, making it a fundamental tool when screening
and ⁄ or selecting for a vaccine candidate [14,15]. Fur-
thermore, work carried out on the proteomics of the
bacterium has led to the identification of a large
number of membrane proteins with an as yet undeter-
mined function [11–13].
A previous study has shown the importance of the
melA gene (encoding an Rv2560 homologous protein)
in Mycobacterium marinum invasion of THP-1 cells,
indicating that its homologous gene might be relevant
in M. tuberculosis–host cell interaction [16].
This work has led to the identification of the pres-
ence of the Rv2560 proline- and glycine-rich trans-
membrane protein encoding gene and its transcripts in
the M. tuberculosis complex and clinical isolate strains,
as well as the characterization of the high-activity
binding peptide (HABP) involved in the binding to
and invasion of monocytes (U937) and type II alveolar
epithelial cells (A549), using synthetic peptides. The
protein encoded by the Rv2560 gene was synthesized
in 20-mer-long, nonoverlapping peptides, which were
analysed by a highly specific and robust methodology
(widely used in Plasmodium falciparum research) for
the identification of the HABPs interacting with U937
and A549 cells (in this case), their binding dissociation
constants, their critical binding residues and their role
in cell invasion inhibition, with the specific purpose of

bacterium microti (Fig. 1A, lanes 2–7). tblastn analy-
sis showed similar (although not identical) genes in
M. marinum and Mycobacterium ulcerans.
DNA sequencing
The amplified fragments in the different clinical iso-
lates studied were purified, and their DNA was
sequenced to determine this region’s polymorphism. It
was observed that this region was completely con-
served in all M. tuberculosis clinical isolates analysed,
independent of geographical origin. This result was
confirmed by sequencing each fragment’s complemen-
tary chain (data not shown).
RT-PCR assay
Two specific primers were designed and synthesized to
determine the transcriptional pattern of the Rv2560
gene in M. tuberculosis complex strains. A single
308 bp amplification band was observed in some
M. tuberculosis complex strains (Fig. 1B, lanes 2–7).
This gene’s transcription was exclusive to M. tuberculo-
sis H37Rv, M. tuberculosis H37Ra, M. bovis BCG and
M. africanum strains, but not M. bovis and M. microti,
suggesting that it was not transcribed, in spite of this
gene being present as assessed by PCR. Figure 1C
shows a 360 bp conserved fragment from a mycobacte-
rial genus constitutive gene (the rpoB gene encoding
the RNA polymerase b-subunit) used as transcription
control.
Western blot
Polymerized synthetic peptides corresponding to the
Rv2560 protein peptide 11027 (181ADGKPVTIATFF

was only amplified on DNA from M. tuberculosis complex strains
(lanes 2–7). Lane 1, 1 kb molecular weight marker (Gibco); lane 2,
M. tuberculosis H37Rv; lane 3, M. tuberculosis H37Ra; lane 4,
M. bovis; lane 5, M. bovis BCG; lane 6, M. africanum; lane 7,
M. microti; lane 8, PCR negative control. (B) M. tuberculosis
Rv2560 protein H37Rv 308 bp RT-PCR product obtained from
M. tuberculosis complex strain cDNA. Lane 1, 1 kb molecular
weight marker (Gibco); lane 2, M. tuberculosis H37Rv; lane 3,
M. tuberculosis H37Ra; lane 4, M. bovis; lane 5, M. bovis BCG;
lane 6, M. africanum; lane 7, M. microti; lane 8, negative control
[M. tuberculosis H37Rv DNA treated with DNAse Q (Promega)];
lane 9, PCR positive control (M. tuberculosis H37Rv DNA); lane 10,
PCR negative control. A single 308 bp amplification band was
observed in some M. tuberculosis complex strains. (C) PCR 360 bp
product from the same strains as (B) but with amplification of the
Mycobacterium rpoB gene as positive control for mRNA expression
and cDNA synthesis.
M. tuberculosis Rv2560 protein characterization D. F. Plaza et al.
6354 FEBS Journal 274 (2007) 6352–6364 ª 2007 The Authors Journal compilation ª 2007 FEBS
Immunoelectron microscopy
Rabbit serum raised against peptide 11027 was used
for immunolabelling. Immunoelectron microscopy
showed that colloidal gold particles (5 nm) were
mainly located on the surface of M. tuberculosis
H37Rv (Fig. 3), supporting the concept that this
expressed gene is located on the surface and is recog-
nized by serum directed against one of its forming pep-
tides. No labelling was observed in negative controls
with preimmune serum (data not shown).
High specific binding peptides

PVAALIHVYTYRKLSGG304) showed high binding
activity to the epithelial A549 cell line only.
Affinity constants
Saturation assays and Hill analysis [24,25] were carried
out for shared 11024 HABPs based on initial screening
results, using a greater
125
I-radiolabelled peptide
Fig. 2. Immunoblotting assay. Western blotting was performed
with rabbit preimmune and post-third inoculation sera against a
M. tuberculosis sonicate (lanes 1, 2 and 5) and a membrane frac-
tion (lanes 3 and 4). Lanes 1 and 3, absence of recognition of
M. tuberculosis proteins by rabbit preimmune sera; lane 2, serum
from rabbit inoculated with polymerized peptide 11031; lane 4,
serum from rabbit inoculated with polymerized peptide 11027;
lane 5, serum from rabbit inoculated with M. tuberculosis total son-
icate. Molecular weight markers are shown on the left-hand side.
Fig. 3. Immunoelectron microscopy. Immunogold location of
Rv2560 protein on M. tuberculosis H37Rv membrane, as assessed
by 5 nm gold-labelled anti-rabbit IgG particles. Intact cells were
incubated with a 1 : 10 dilution (1 and 2) or pure (3) rabbit sera
directed against peptide 11027, and prepared for immunoelectron
microscopy by negative contrast, as described. No labelling of the
surface was obtained in control experiments from which these anti-
bodies had been omitted, or with preimmune serum (data not
shown). The arrows indicate the location of the Rv2560 protein on
the mycobacterial surface.
D. F. Plaza et al. M. tuberculosis Rv2560 protein characterization
FEBS Journal 274 (2007) 6352–6364 ª 2007 The Authors Journal compilation ª 2007 FEBS 6355
concentration range (500–2000 nm) (Fig. 5A). Scat-

cell membrane, specifically recognizing a 54 kDa hypo-
thetical receptor. Radiolabelled peptide binding to this
receptor protein was inhibited in the presence of non-
radiolabelled peptide; this was clearly shown by the
band intensities (Fig. 6). Peptide 11024 binding was
target cell specific, as it did not show any binding to
HepG2 cells or erythrocytes (data not shown).
Invasion inhibition assay using HABPs
A549 and U937 cell lines were preincubated in the
presence of HABP 11024 in two independent experi-
ments to evaluate the effect of HABP on bacterial
invasion of target lung cells. It was found that invasion
was inhibited in both cell types. The inhibitory power
of HABP 11024 was greater in assays carried out using
the A549 cell line, where it reached close to 60% inhi-
bition when such cells were preincubated in the pres-
ence of 1 lm peptide. Monocyte invasion assays
revealed around 50% maximum inhibition only when
higher peptide concentrations were used than those
employed for A549 cell assays (Fig. 7). It was also
Fig. 4. Cell binding activity of Rv2560 peptides. Amino acid sequence and specific U937 and A549 cell binding activity for 20-mer long chem-
ically synthesized Rv2560 peptides from the M. tuberculosis H37Rv strain. The peptides are given on the left-hand side, with numbers indi-
cating their position within the native protein; in peptides that did not contain tyrosine, it was added to the carboxy-terminal end. To the
right, the black bar represents each peptide binding activity, determined as the specific binding ⁄ total added peptide ratio. The dotted line
separates peptides having ‡ 1% binding activity. HABP 11095 was used as a binding activity control. Rv2560 transmembrane topology is
presented on the left-hand side. Grey shows the transmembrane helices present in the sequence according to the
TMHMM prediction server.
nd, not done because of solubilization problems.
M. tuberculosis Rv2560 protein characterization D. F. Plaza et al.
6356 FEBS Journal 274 (2007) 6352–6364 ª 2007 The Authors Journal compilation ª 2007 FEBS

The saturation curves resulted from plotting the specifically bound
125
I-HABP concentration versus free
125
I-HABP. The affinity constants and
maximum number of sites per cell were obtained from these curves. Inset: the abscissa is log F in the Hill plot and the ordinate is
log [B ⁄ B
m
) B], where B
m
is the maximum bound peptide, B is the bound peptide and F is free peptide. (B) Competition binding assay with
analogous peptides. Specific original radiolabelled peptide binding inhibited by analogous peptide (at 16 and 400 n
M) is shown. Amino acids
underlined in bold represent the critical binding residues, as their binding activity decreased by 50% or more when modified and assayed at
the two concentrations used.
Fig. 6. Cross-linking assay for peptide 11024. Autoradiographs for
U937 proteins specifically cross-linked with radiolabelled peptide
11024. Lanes 1 and 2, U937 cells; lanes 3 and 4, HepG2 cells;
lanes 1 and 3, total radiolabelled peptide binding; lanes 2 and 4,
inhibited binding. The cross-linking assays show that peptide 11024
specifically binds to a 54 kDa membrane protein on U937 cells.
D. F. Plaza et al. M. tuberculosis Rv2560 protein characterization
FEBS Journal 274 (2007) 6352–6364 ª 2007 The Authors Journal compilation ª 2007 FEBS 6357
Moreover, it has been observed that M. tuberculosis
can promote monocyte differentiation into dendritic
cells that lack CD1, have low CD80 and produce inter-
leukin-10; this altered phenotype is unable to prime
effector T cells, thus becoming an escape mechanism
[28]. The identification of proteins specifically binding
to receptors on monocytes or other M. tuberculosis

enriched fraction and the mycobacterial surface, and is
involved in the mycobacterial invasion of host cells.
On PCR amplification of a 631 bp fragment, includ-
ing the sequence of nucleotides encoding a high specific
binding Rv2560 protein region (amino acids 80–290)
(Fig. 1), it was found that this fragment was amplified
in M. tuberculosis complex strains. The presence of this
gene was also determined in clinical M. tuberculosis
isolates obtained from patients having different types
of TB (pulmonary, pleural, osseous, meningeal,
abdominal, genitourinary and renal), with the 631 bp
fragment being amplified in all clinical isolates. All of
the foregoing suggests that the Rv2560 gene is present
in different M. tuberculosis complex strains, including
clinical isolates, and has no genetic variability, as
shown by DNA sequencing (data not shown).
The transcription of this gene was also confirmed
after the presence of the highly conserved gene had
been established in different M. tuberculosis complex
strains. cDNA was therefore obtained from M. tuber-
culosis complex strains and amplified by PCR, using
specific primers for the 308 bp fragment (using more
internal primers to amplify smaller fragments in order
to improve RT-PCR efficiency). The results (Fig. 1B)
led to the conclusion that the Rv2560 gene was
transcribed in M. tuberculosis H37Rv, M. tuberculosis
H37Ra, M. bovis BCG and M. africanum strains, but
not in M. bovis or M. microti.
The two polymerized peptides used in rabbit immu-
nization produced an antibody response (differing in

displaying the VVALSDRATTAYTNTSGVSS
sequence (where the amino acids shown in bold italic
correspond to those identified as being critical in 11024
binding to U937 cells). These residues may be involved
directly in target cell binding or may be important for
the structural characteristics of the peptide, thereby
allowing specific binding. BLAST analysis of this small
region led to the identification of 78% homology with
Burkholderia cenocepacia-dependent AMP synthase,
which causes respiratory tract infection and is cyto-
toxic for alveolar epithelial cells [30].
A 54 kDa protein was found to be a receptor on
U937 cells; its binding by peptide 11024 was inhibited
by an excess of nonradiolabelled peptide. This receptor
was specific for the U937 cell line, but was not seen in
hepatocytes or erythrocytes. A receptor having a simi-
lar molecular mass (51 kDa) has been described for
the Rv1510c protein HABP 11095; however, further
studies are required to determine whether the same
membrane protein is involved.
Peptide 11024 (for which high monocyte and alveo-
lar epithelial cell binding has already been determined)
induced a 36.96–50.53% decrease in M. tuberculosis
invasion of U937 cells and a 30.98–58.27% decrease in
M. tuberculosis invasion of A549 cells (Fig. 7). This
indicates that this HABP is important in the recogni-
tion and invasion of monocytes and type II alveolar
epithelial cells by the mycobacterium.
It was observed in preliminary assays that HABP
concentrations of less than 100 lm had a greater

calculated using the protparam tool (asy.
org ⁄ tools⁄ protparam.html), for which a score of greater
than ) 0.4 (mean score for the cytosolic proteins) suggests
membrane association probability; the higher the score, the
greater the probability [13]. Transmembrane regions were
predicted by tmhmm ( />TMHMM) and tmpred ( />software/TMPRED_form.html). Cell localization, lipid
attachment sites and signal sequences were predicted using
psort ().
Mycobacterial species and strains
The following M. tuberculosis complex species and strains
were used: M. tuberculosis H37Rv (ATCC27294), M. tuber-
culosis H37Ra (ATCC25177), M. bovis (ATCC19210),
M. bovis BCG (ATCC27291, Pasteur sub strain), M. africa-
num (ATCC25420) and M. microti (kindly donated by
F. Portaels, Prince Leopold Institute of Tropical Medicine).
D. F. Plaza et al. M. tuberculosis Rv2560 protein characterization
FEBS Journal 274 (2007) 6352–6364 ª 2007 The Authors Journal compilation ª 2007 FEBS 6359
Ten M. tuberculosis clinical strains were isolated from speci-
mens obtained from different patients attending the TB pro-
gramme at either the San Juan de Dios Hospital or Santa
Clara Hospital, both in Bogota, Colombia. Patients came
from several geographical areas of Colombia. All mycobacte-
rial strains were grown for 5–15 days in 7H9 Middlebrook
broth (Difco Laboratory, Detroit, MI, USA) with 0.04%
Tween 80 and in Middlebrook agar 7H10 (Difco), both sup-
plemented with oleic acid, albumin, dextrose and catalase
(BBL, Becton Dickinson, Mountain View, CA, USA).
Genomic DNA extraction
Cells were harvested in Tris–EDTA buffer (TE, pH 8.0)
and suspended in 25% sucrose in 10 · TE. Lysozyme

with ethidium bromide and photographed.
DNA sequencing
The dideoxy chain termination method was used for
sequencing reactions with a Taq FS DyeDeoxy Terminator
cycle sequencing kit (Applied Biosystems, Foster City, CA,
USA) on a GeneAmp PCR System 9600 (Perkin-Elmer Life
Sciences Inc.), and run on a 373 DNA sequencer model
(Applied Biosystems). The sequencing strategy involved for-
ward and reverse specific primers for the 631 bp fragment
of the Rv2560 gene.
RNA isolation
The bacilli were harvested by spinning at 12 000 g for
15 min at 4 °C. Sodium azide (10 mm) was added to the
culture just before harvesting. The cell pellet was suspended
in 2 mL of cold lysis buffer for each 200 mg wet weight of
cells [33], and sonicated twice for 15 min. Two volumes of
Trizol (Gibco BRL, Gaithersburg, MD, USA) were then
added and extracted according to the manufacturer’s
instructions. The pellet was suspended in 100 lL of distilled
water and stored in aliquots at ) 80 °C.
RT-PCR
Total RNA was quantified by a GeneQuant spectropho-
tometer (Pharmacia Biotech, Piscataway, NJ, USA), treated
with RNAse-free DNAse RQ1 at 37 °C for 3 h, precipi-
tated with isopropanol, washed with 70% ethanol and sus-
pended in distilled water. M. tuberculosis H37Rv DNA was
included as DNAse Q activity control (1 UÆlg
)1
DNA).
Target RNA (500 lg) was reverse transcribed in a tube

gia’s Bioethics Committee recommendations. The Rv2560
protein peptide sequences chosen for the immunization of
rabbits were obtained using T-epitope prediction software
M. tuberculosis Rv2560 protein characterization D. F. Plaza et al.
6360 FEBS Journal 274 (2007) 6352–6364 ª 2007 The Authors Journal compilation ª 2007 FEBS
syfpeithi (15-mer T-epitope for major histocompatibility
complex class II only) downloaded from http://www.
syfpeithi.de/Scripts/MHCServer.dLl/EpitopePrediction.htm.
SDS-PAGE and immunoblotting
M. tuberculosis sonicate or membrane fraction (1 mg) was
loaded in a discontinuous SDS-PAGE system, using a 10–
20% (w ⁄ v) acrylamide gradient, and then transferred to
nitrocellulose paper using the semidry blotting technique
[34]. The filters were incubated with a 1 : 100 (v ⁄ v) dilution
of the sera obtained from rabbits immunized with polymer-
ized 11027 or 11031 peptides. Sera were diluted in NaCl ⁄
Tris ⁄ Tween 20 (0.02 m Tris-HCl, pH 7.5, 0.05 m NaCl, 1%
Tween 20) and 5% skimmed milk. Incubation for 1 h with
1 : 3000 (v ⁄ v) alkaline phosphatase-conjugated anti-rabbit
IgG (ICN Biomedicals, Costa Mesa, CA, USA) followed
five NaCl ⁄ Tris ⁄ Tween 20 washes. The reaction was devel-
oped with Nitro Blue tetrazolium ⁄ 5-bromo-4-chloroindol-
2-yl phosphate (KPL, Gaithersburg, MD, USA).
Immunoelectron microscopy
Immunoelectron microscopy studies were carried out on a
Philips CM 10 transmission electron microscope (Philips,
Suresne, Hauts-de-Seine, France). Briefly, a wet pellet
(50 lL) of M. tuberculosis H37Rv was fixed with a 4%
paraformaldehyde)0.5% glutaraldehyde solution for 2 h at
4 °C. After fixation, the pellet was dehydrated in graded

1640.
Peptide synthesis
Sixteen sequential 20-mer peptides, corresponding to the
Rv2560 amino acid sequence [9], were synthesized in this
study by a solid-phase multiple peptide system [36,37].
4-Methylbenzhydrylamine resin (0.7 meqÆg
)1
), t-Boc amino
acids and low–high cleavage techniques were used [38]. Pep-
tide identity and purity were analysed by MALDI-TOF MS
and analytical reverse phase HPLC. An extra tyrosine resi-
due was added to any peptide C-terminus that did not
contain it to enable radiolabelling.
Peptide radiolabelling
Radiolabelling with
125
I was performed according to previ-
ously described techniques [24,25,39,40], in which chlor-
amine-T (2.25 mgÆmL
)1
) and 3.2 lLNa
125
I (100 mCiÆmL
)1
)
were added to 5 lL peptide solution (1 lgÆlL
)1
); 15 lL
sodium bisulfite (2.75 mgÆmL
)1

) were incubated with increas-
ing concentrations (100–2000 nm) of radiolabelled peptide
at 120 lL total volume for 90 min at 4 °C, in the presence
or absence of 40 lm unlabelled peptide, to determine the
binding specificity. After incubation, unbound peptide was
D. F. Plaza et al. M. tuberculosis Rv2560 protein characterization
FEBS Journal 274 (2007) 6352–6364 ª 2007 The Authors Journal compilation ª 2007 FEBS 6361
removed from the cells by sedimentation through a dioctyl-
phthalate–dibutylphthalate cushion (d ¼ 1.015 gÆmL
)1
) and
centrifuged at 9000 g for 2 min. As before, each assay was
performed in triplicate; bound and free peptides were deter-
mined by measuring the cell-associated radioactivity on a
gamma counter. Triplicate assay data were averaged. The
curves obtained were analysed and the dissociation con-
stants were determined by the Hill equation [18,19].
Analogue peptide competition binding assay
HABP 11024 glycine scanning analogues were synthesized
to identify critical residues for binding to U937 cells. For
the competition binding assays, 1 · 10
6
U937 cells were
incubated with increasing quantities (16 and 400 nm)of
each unlabelled analogue peptide or original unlabelled
peptide in the presence of native
125
I-labelled peptide. After
90 min of incubation at 4 °C, the mixture was centrifuged
at 9000 g for 2 min on a dioctylphthalate–dibutylphthalate

i
. The sus-
pension was briefly sonicated (30 W for 5 s) on a Branson
sonifier (VWR Scientific, Boston, MA, USA); the suspen-
sion turbidity was adjusted to 3 · 10
8
colony-forming
unitsÆmL
)1
according to McFarland’s pattern. M. tuber-
culosis was labelled with SYBR Safe (Invitrogen, Eugene,
OR, USA) at 20 · final concentration at 37 °C for 20 min
in the dark with constant shaking, washed twice and centri-
fuged at 12 000 g for 20 min; excess dye was removed.
Bacteria were then suspended in RPMI 1640, and labelling
was further verified by flow cytometry and fluorescence
microscopy.
Invasion inhibition assay with HABPs
The possible biological relevance of the HABPs identified
in Rv2560 was evaluated by an assay developed by our
group [41]. U937 or A549 cells (1 · 10
6
) suspended in com-
plete RPMI 1640 medium without antibiotics were preincu-
bated for 1 h in the presence of 50, 100 or 200 lm
HABP 11024 for U937 cells and 10
0
)10
5
nm or 200 lm

Samples were analysed on a FACScan (Becton Dickinson)
flow cytometer. CellQuest software (Becton Dickinson) was
used for flow cytometry capture and analysis. NaCl ⁄ P
i
,
which had been filtered with a 0.2 lm membrane, was used
as sheath fluid. Uninfected cells were discriminated from
infected cells on the basis of light characteristics on fluores-
cence channel one. Samples were run at 2000 events per sec-
ond; 40 000 events were collected. The percentage of positive
SYBR safe events in the gated region was determined.
CD spectroscopy
CD was carried out for HABP 11024 and control peptide
11021 to determine whether there were any conformational
changes when the peptide concentration in the solution
M. tuberculosis Rv2560 protein characterization D. F. Plaza et al.
6362 FEBS Journal 274 (2007) 6352–6364 ª 2007 The Authors Journal compilation ª 2007 FEBS
was increased. CD spectra were recorded at 20 °Cona
Jasco J-810 spectropolarimeter at wavelengths ranging
from 260 to 190 nm in 1 cm cuvettes (Jasco, Tokyo,
Japan). The peptides were dissolved at 5, 25, 50, 100 and
200 lm concentration in 30% trifluoroethanol aqueous
solution. Each spectrum was obtained from the average of
three scans taken at a scan rate of 20 nmÆ min
)1
with a
spectral bandwidth of 1 nm, corrected for baseline. The
results were expressed as the mean residue ellipticity [Q].
Acknowledgements
We wish to thank Dr Ramses Lopez, Rosalba Alfonso,

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