From the Wadsworth Center, David Axelrod Institute for Public Health, New York State Department of Health, Albany, New York 12208
Received for publication, January 5, 2001
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ABSTRACT |
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It is generally held with respect to
heterotrimeric guanine nucleotide binding protein-coupled
receptors that binding of ligand stabilizes a conformation of receptor
that activates adenylyl cyclase. It is not formally appreciated if, in
the case of G-protein-coupled receptors with large extracellular
domains (ECDs), ECDs directly participate in the activation process.
The large ECD of the glycoprotein hormone receptors (GPHRs) is 350 amino acids in length, composed of seven leucine-rich repeat domains,
and necessary and sufficient for high affinity binding of the
glycoprotein hormones. Peptide challenge experiments to identify
regions in the follicle-stimulating hormone (FSH) receptor (FSHR) ECD
that could bind its cognate ligand identified only a single synthetic
peptide corresponding to residues 221-252, which replicated a
leucine-rich repeat domain of the FSHR ECD and which had intrinsic
activity. This peptide inhibited human FSH binding to the human FSHR
(hFSHR) and also inhibited human FSH-induced signal transduction in Y-1
cells expressing recombinant hFSHR. The hFSHR-(221-252) domain was not
accessible to anti-peptide antibody probes, suggesting that this domain
resides at an interface between the hFSHR ECD and transmembrane
domains. CD spectroscopy of the peptide in dodecyl phosphocholine
micelles showed an increase in the ordered structure of the peptide. CD and NMR spectroscopies of the peptide in trifluoroethanol confirmed that hFSHR-(221-252) has the propensity to form ordered secondary structure. Importantly and consistent with the foregoing results, dodecyl phosphocholine induced a significant increase in the ordered secondary structure of the purified hFSHR ECD as well. These data provide biophysical evidence of the influence of environment on GPHR
ECD subdomain secondary structure and identify a specific activation
domain that can autologously modify GPHR activity.
Follicle-stimulating hormone
(FSH),1 luteinizing hormone
(LH), and thyroid-stimulating hormone of pituitary origin and chorionic gonadotropin of placental origin belong to the family of glycoprotein hormones (1). These hormones are heterodimeric and composed of a common
Understanding the structure-function relationships of FSH and its
receptor has clinical importance. In humans, a mutation in the FSH
receptor (FSHR) gene was identified as the cause of hereditary
hypergonadotropic ovarian failure (8) and reduced sperm counts (9).
Because these genetic disorders of mouse and men do not completely
abrogate sperm production, these reports emphasize the essential role
of FSH for the absolute initiation/maintenance of spermatogenesis.
Indeed, in some species, immunization against FSH results in impairment
of spermatogenesis, rendering the animals infertile (10). A male
anti-fertility vaccine based on ovine FSH (11) or recombinant ovine
FSHR (12) has been developed. Discovery of receptor domains that bind
hormone has potential importance in contraceptive vaccine or fertility
drug development and will provide a better understanding of how this
family of receptors binds cognate ligand.
Leucine-rich repeat motifs found in the glycoprotein hormone receptor
large extracellular domain (ECD) (13) are similar to those found in the
structure of porcine ribonuclease inhibitor and have a parallel
Peptide Synthesis--
Peptides from the extracellular domain of
the hFSHR or the rat FSHR, where indicated (viz. sequences
9-30, rat; 15-44, rat; 45-72; 72-100; 101-125; 126-150; 150-183;
183-220; 221-252, rat; and 265-296), were selected for the study.
Peptides 9-30, 265-296, and 221-252 derived from FSHR amino and
carboxyl termini are encoded primarily within single exons. All other
peptides spanned the junctions of adjacent exons. Peptides
corresponding to the primary sequence of the FSHR ECD were synthesized
by solid-phase Fmoc (N-(9-fluorenyl)methoxycarbonyl)
chemistry using an Applied Biosystems 431A peptide synthesizer. After
cleavage from the solid support, peptides were purified by preparative
reversed-phase high performance liquid chromatography (C18,
Delta-Pak, 19 × 300 mm). The molecular ion mass of purified
peptides was determined by mass spectrometry at the Wadsworth Center
Biological Mass Spectrometry Facility. Tyrosine and cysteine were
incorporated into the C terminus of each peptide to enable iodination
or conjugation to carrier proteins, respectively.
Modification of Cysteine--
In some experiments, free thiols
in peptides were derivatized to assess the effects on peptide activity.
For derivatization, 10 mg of the hFSHR-(221-252) Tyr-Cys peptide was
dissolved in 2 ml of buffer (0.1 M potassium phosphate (pH
8.0), 1 mM EDTA, and 0.02% NaN3). Aliquots (5 µl) of freshly prepared 0.5 M iodoacetamide in water were
added at 5-min intervals. The reaction was monitored by the method of
Ellman (17) using 5,5'-dithiobis(2-nitrobenzoic acid). Addition of
iodoacetamide was continued until all the sulfhydryl groups were
modified. The solution was then diluted with 5 ml of 0.1%
trifluoroacetic acid and subjected to reversed-phase preparative HPLC.
Following purification, the modified peptide was characterized by amino
acid analysis and mass spectrometry.
Modification of Radioreceptor Assay (RRA)--
Peptides or anti-peptide
antibodies were tested for their ability to inhibit the binding of hFSH
to its receptor in a RRA. Deoxyuridine-resistant Chinese hamster ovary
cells (19) stably transfected with the hFSHR (CHO-hFSHR) and obtained
from Ares Advanced Technologies (Randolph, MA) were used as a source of hFSHR. CHO-hFSHR cells were washed twice and left for 15 min in EDTA/PBS buffer. Cells were dislodged, counted, and centrifuged at 3000 rpm for 5 min. The cell pellets were resuspended in RRA buffer (0.05 M Tris and 0.25 M MgCl2 (pH 7.5))
and stored frozen (10 × 107 cells/ml). For
competitive displacement, 125I-hFSH (150,000 cpm, 25 µCi/µg) in RRA buffer with 0.3% bovine serum albumin was used as
the tracer for binding to the hFSHR (2.5 × 105 cells)
(20). The RRA was carried out in a reaction volume of 400 µl.
Increasing concentrations of hFSH or receptor peptide (100µl) were
preincubated with 125I-hFSH (100 µl) for 1 h at room
temperature. CHO-hFSHR cells were then added and incubated overnight at
room temperature with shaking. When antibodies to hFSHR peptides were
used, they were preincubated with 2.5 × 105 CHO-hFSHR
cells for 4 h at 4 °C. Radiolabeled hFSH was then added, and
the tubes were shaken overnight at room temperature. At all concentrations of peptide or antibody tested, controls were included in
which pure hFSH (1 µg) was added to assess nonspecific binding. To
separate bound from free hFSH, the assay was terminated by adding 2 ml
of ice-cold 0.05 M Tris (pH 7.5) and pelleting cells at
2500 × g for 1 h at 4 °C. The supernatant from
each tube was aspirated, and the radioactivity in each pellet was
determined using a
In some experiments, membrane-bound or truncated receptors were
solubilized using the detergent Nonidet P-40. Truncated forms of the
hFSHR ECD were prepared as previously described (21). A CHO cell line
(D7) stably expressing the hFSH-(1-335) variant was utilized. CHO
cells expressing either the full-length hFSHR (1.5 × 106 cells/100 µl) or hFSHR ECD (clone D7, hFSHR-(1-335);
4 × 106 cells/100 µl) were solubilized using 0.1%
Nonidet P-40 (in 0.05 M Tris, 0.025 M
MgCl2, 0.3% bovine serum albumin, and 30% glycerol). Antibodies at different concentrations (100 µl) were incubated with
the solubilized receptor preparation (100 µl) at 4 °C for 4 h; then the tracer was added (150,000 cpm/100 µl); and the incubation was continued at 4 °C overnight. To separate bound from free hFSH, the assay was terminated by precipitation of the hFSH·hFSHR complexes using polyethylene glycol. To each tube were added 200 µl of
In Vitro FSH Bioassay--
The effect of hFSHR peptides on
signal transduction was evaluated by an in vitro FSH
bioassay (19). Y-1 cells stably expressing FSHRs (Ares Advanced
Technologies) were cultured in 48-well plates at 3 × 105 cells/500 µl/well in Eagle's minimum essential
medium supplemented with 5% fetal bovine serum and 80 µg/ml G418
(Geneticin, Life Technologies, Inc.). The medium was replaced with
fresh medium on the third day. On the fourth day, cells were rinsed
with Y-1 cell assay medium (Eagle's medium with 0.1% bovine serum
albumin and 1% glutamine) and treated with different concentrations of hFSH or peptide with or without 1 ng of hFSH (ID50) and
then incubated for 20-24 h. All the incubations were carried out at
37 °C in a 5% CO2 atmosphere. After the last
incubation, the medium from each well was transferred to a glass tube,
heated for 10 min at 100 °C, and spun at 2500 × g
for 30 min at 4 °C. Supernatants were decanted and assayed for
progesterone or stored frozen at
Progesterone secreted in the medium was measured by radioimmunoassay.
Samples were diluted with PBS-G (0.1% gelatin in PBS with 0.02%
sodium azide) and incubated with 100 µl (1:2500) of sheep
anti-progesterone antiserum (GDN 337, obtained from Dr. Gordon
Niswender, Colorado State University, Fort Collins, CO), 100 µl
(25,000 dpm) of [3H]progesterone (PerkinElmer Life
Sciences) in PBS-G, and 200 µl of PBS-G overnight at 4 °C.
Following incubation, 0.5 ml of dextran-coated charcoal was added to
the tubes and incubated for 10 min at 4 °C to separate bound from
free progesterone. The tubes were spun at 2500 × g for
10 min at 4 °C. Supernatants were decanted into 20-ml scintillation
vials, and 10 ml of Aquasol (PerkinElmer Life Sciences) was added.
Vials were vortexed and counted in a Development of Anti-receptor Peptide Antibodies--
The
hFSHR-(221-252) peptide was conjugated to ovalbumin through the
C-terminal -SH of cysteine, which was incorporated during the
synthesis for this purpose. Sulfo(succinimidyl
4-(N-maleimidomethyl))cyclohexane 1-carboxylate was used as
the cross-linking reagent (23). The conjugate was used to immunize two
Flemish giant female rabbits (X-180 and X-183). Immunoglobulins were
purified using protein A-Sepharose (Bio-Rad antibody purification kit)
following the manufacturer's instructions. IgG fractions were dialyzed
overnight against 0.05 M Tris (pH 7.5), lyophilized, and
stored at Characterization of Anti-peptide Antibodies--
Anti-peptide
antibody was screened using an ELISA to detect binding to corresponding
peptides. Microtiter wells (Nunc Maxisorp) were incubated overnight
with peptide (1 µg/100 µl) in carbonate buffer (pH 9.4). After the
unabsorbed peptide was removed, the wells were blocked with
PBS-G (200 µl) for 2 h at 37 °C. Dilutions of antisera
(100 µl) in PBS-G with 0.05% Tween 20 were added to the wells
and incubated for 90 min at 37 °C. This was followed by addition of
peroxidase-conjugated goat anti-rabbit immunoglobulin (100 µl,
1:4000; Pierce) and further incubation at 37 °C for 1 h.
Substrate (200 µl) containing 25 µl of hydrogen peroxide and 4 mg
of o-phenylenediamine in 10 ml of citrate/phosphate buffer (pH 5.5) was added and incubated in the dark for 10 min. The reaction was terminated by addition of 4 N
H2SO4 (100 µl). Absorbance was determined at
492 nm. For competition experiments, serial dilutions of the parent
peptide, its fragments, or an unrelated peptide were preincubated with
the antisera before addition to the microtiter wells.
Polyacrylamide Gel Electrophoresis and Western Blotting--
To
confirm that antibodies to peptide 221-252 bind to the hFSHR,
denaturing SDS-polyacrylamide gel electrophoresis in a discontinuous buffer system (46) was carried out. D7 cells (5 × 106) expressing the FSHR ECD were solubilized for 30 min at
room temperature in 500 µl of Laemmli sample buffer (0.25 M Tris (pH 6.8), 4% SDS, 20% glycerol, and 0.24%
bromphenol blue) containing 1× protease inhibitor mixture (100×
protease inhibitor mixture = 1.6 mg/ml benzamidine HCl, 1 mg/ml
phenanthroline, 1 mg/ml aprotinin, 1 mg/ml leupeptin, and 1 mg/ml
pepstatin A in 100% ethanol), 5% 2-mercaptoethanol, and 8 M urea. Electrophoresis chambers were cooled in ice baths.
The gels were 7.5% polyacrylamide and made 8 M with urea.
18 µl of cell suspension was added per lane. After electrophoretic
separation, proteins were transferred to nitrocellulose by
electroblotting (47) for 45 min at 250 mA. The nitrocellulose membranes
were blocked with Protein Images blocking solution (U. S. Biochemical
Corp.) for 2 h at room temperature. Primary antibodies were
diluted in Tris-buffered saline containing 1% Tween 20 (TBST), 2%
nonfat dry milk, 1% ovalbumin, and plain CHO cell extract (1 × 106 cells/10 ml) and incubated overnight at room
temperature. The blots were washed with TBST and incubated with
alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin
(BIOSOURCE International, Camarillo, TX) for
2 h at room temperature. The membranes were washed and developed
with Western blue-stabilized substrate for alkaline phosphatase
(Promega, Madison, WI).
Flow Cytometry--
To determine whether the hFSHR-(221-252)
domain was accessible to antibodies in native receptor present on
CHO-hFSHR cells, flow cytometric analysis was carried out.
Confluent cells were washed with EDTA/PBS and then incubated with the
same buffer for 10-15 min. The cells were dislodged from the culture
flasks and centrifuged for 5 min at 3000 rpm. After aspiration of the
supernatant, cells were suspended in PBSA (PBS with 0.02% sodium
azide). CHO-hFSHR cells (106 cells/100 µl) were
added to each tube. Human serum (50 µl) was incubated with the cells
on ice for 1 h to block nonspecific sites. Purified rabbit IgG (50 µg/50 µl) from control serum or antiserum against the
hFSHR-(221-252) or hFSHR-(265-296) peptide was added, and incubation
was continued for 1 h on ice. Cells were washed with 2 ml of PBSA,
pelleted, resuspended in fluorescein isothiocyanate-conjugated anti-rabbit IgG (200 µl, 1:40 dilution), and incubated for 1 h on ice. Cells were then pelleted, washed, and resuspended in 2 ml of
PBSA. Cell-surface immunofluorescence was measured using a flow
cytometer (Becton Dickinson FACScan).
Circular Dichroism Studies of the hFSHR-(221-254)
Peptide--
Circular dichroism analyses were performed to assess the
bulk solvent environment of the conformation of the hFSHR-(221-254) peptide. Experiments were carried out on a Jasco J-720
spectropolarimeter at 20 °C using a cell with a path length of 0.05 mm. In one series of experiments, peptides were dissolved in 25 mM phosphate buffer (pH 7.0), and spectra were obtained in
a series of solutions containing from 0 to 50% trifluoroethanol,
increasing in steps of 5%. In another set of experiments, peptides
were dissolved in water; phosphate buffer (pH 7.0) was added to 25 mM, and dodecyl phosphocholine was added to 5.0 or 22.0 mM; the solution was incubated overnight at room
temperature; and spectra were obtained at 20 and 37 °C at a peptide
concentration of 0.5 mM. All data were analyzed using the
computer program SELCON (25-28).
Nuclear Magnetic Resonance Spectroscopy--
All spectra of
hFSHR-(221-252) were taken of a sample at 1.75 mM in 90%
H2O, 25 mM CD3COOD, 100 mM KCl, and 0.01% NaN3 (pH 4.5) at 305 K. Data
were recorded at 500 MHz for protons. A variety of data sets were
taken. Several one-dimensional 1H spectra were obtained
with a spectral width of 6000 Hz, with 8192 complex data points
collected during acquisition. Spectra were processed with appropriate
exponential/gaussian multiplication. Two-dimensional 1H
TOCSY spectra with gradient excitation sculpting for water suppression were recorded (48). In each case, an isotropic mixing time of 35 ms was employed to emphasize correlations between amide and
Two-dimensional 1H NOESY spectra with gradient excitation
sculpting for water suppression were recorded (49). In each case, a
mixing time of 120 ms was employed for generation of nuclear Overhauser
effects. For the two-dimensional spectra, spectral widths were 6000 Hz
in both the direct and indirect dimensions. Data sets were 8192 × 512 complex points in the direct and indirect dimensions, respectively.
Spectra were processed with exponential/gaussian multiplication in the
direct dimension and a quadratic sine-bell in the indirect dimension.
Circular Dichroism Studies of the hFSHR ECD--
Experiments
were carried out on a Jasco J-720 spectropolarimeter at 6 °C using a
cell with a path length of 0.05 cm. The purified hFSHR ECD was dialyzed
against 0.01 M phosphate buffer (pH 7.2) overnight prior to
the measurements. Spectra were measured with the following settings:
1.0-nm bandwidth, speed of 20 nm/min, 0.2-nm resolution, and wavelength
between 260 and 180 nm with five accumulations per measurement to
improve noise-to-signal ratio. The buffer blank measured under the same
conditions was subtracted from the sample spectra. Protein
concentrations were determined by amino acid analysis. To analyze the
influence of dodecyl phosphocholine on the structure of the
hFSHR ECD, protein was incubated with either 20 mM dodecyl
phosphocholine or buffer overnight at 4 °C, and the spectra were
taken the following day.
All data were analyzed using the Jasco software and the computer
program SELCON3 (25-28). Data presented herein have been analyzed based on a data base containing 29 proteins within the wavelength range
from 178 to 260 nm (29).
Peptide Synthesis and Characterization--
Peptides were purified
by HPLC to homogeneity. The observed molecular mass of each purified
peptide as determined by mass spectrometry was, for the most part, as
expected. We did note that when a C-terminal cysteine was engineered
into the peptide, an additional mass of ~50 Da was always
detected. This was not the case if the cysteine was at the N terminus.
Peptides representing FSHR sequences 45-72 and 150-183 were not
soluble in RRA buffer. Sequence listing mis-transcription resulted in
synthesis errors in peptides representing FSHR sequences 105-125 and
126-150 of Gln to Gly and of Glu to Val, respectively. The hFSHR
sequence of Minegishi et al. (30) was used for peptides
representing hFSHR sequences 72-100 and 265-296.
Peptide Activity in RRA and in Vitro Bioassay--
Peptides
representing FSHR sequences 9-30, 15-44, 101-125, 126-150,
183-220, 221-252, and 265-296 were purified and then screened in the
hFSH RRA to determine whether they affected binding of hFSH to the
receptor. The remaining peptides were not soluble in RRA buffer
and could not be tested. The hFSHR-(221-252) peptide (Fig.
1) was found to inhibit binding of hFSH
to CHO-hFSHR cells in a dose-dependent manner (Fig.
2A). This effect was specific to the FSHR, as the peptide did not inhibit hCG binding to the human
LHR (Fig. 2B). The hFSHR-(221-252) peptide had a cysteine incorporated at the C terminus for the purpose of conjugation to a
carrier protein. To rule out the possibility of involvement of free
-SH in the observed inhibition of FSH binding to receptor, this group
was modified with iodoacetamide. The alkylated peptide still exhibited
binding inhibition (data not shown). There are five lysines in this
peptide as compared with two in the corresponding region of the human
LHR (Fig. 1). To determine whether these lysines participated in
electrostatic interactions that affect peptide activity and
specificity, Ligand Binding Isotherm--
To gain a better understanding of the
mechanism by which the hFSHR-(221-254) peptide inhibited FSH binding
to its receptor, ligand binding isotherms were conducted at three
different concentrations of peptide. Inhibition appeared mixed where an
apparent decrease in Bmax was observed along
with an increase in Kd only at higher levels of
peptide (Table I). These data do not
support a simple competitive mechanism of inhibition where peptide
binds to hormone. Nonspecific binding increased with increasing doses of peptide, as is often seen in peptide challenge experiments.
Characterization of Antibodies against
hFSHR-(221-252)--
Following immunization of rabbits with the
hFSHR-(221-252) peptide-ovalbumin conjugate, both rabbits (X-180 and
X-183) responded with good titers (Fig.
5A). The specificity of the
antibodies for binding to the immobilized peptide was shown by
competition ELISA, where the free immunizing peptide could inhibit
binding of anti-peptide antibodies, whereas an unrelated peptide could not (Fig. 5B). In addition, antisera to this peptide did not
bind to other hFSHR peptides coated on an ELISA plate (data not shown). Antibodies were purified using protein A and tested in an hFSH RRA.
Membrane-bound FSHR was used to determine whether this region of the
FSHR is accessible in situ. Minimal non-dose-related
20-25% inhibition was observed at all concentrations of antiserum
X-180 or X-183 tested (Fig.
6A). In contrast, antiserum
X-179 showed dose-dependent inhibition, as previously
described (24). However, when these hFSHR-containing cells were
detergent-solubilized, and the extract was used as a source of
receptor, anti-hFSHR-(221-252) antibody (X-180 and X-183) inhibited
the binding of hFSH to its receptor in a dose-related manner (Fig.
6B). Antisera could be shown to bind to full-length and
truncated receptors by Western blotting (Fig.
7). When truncated hFSHR was extracted
from D7 cells expressing the FSHR ECD, antibodies against the
hFSHR-(221-252) peptide inhibited binding of FSH to its receptor (data
not shown).
To further clarify the epitope recognized by antisera X-180 and X-183,
subregions of the hFSHR-(221-254) peptide (hFSHR-(221-237) and
hFSHR-(238-254)) were characterized in a competitive ELISA. Antiserum
X-180 recognized hFSHR-(238-254) only at very high concentrations of
the peptide (Fig. 8A), whereas
antiserum X-183 could not recognize peptides 221-237 and 238-254
(Fig. 8B). These results were interpreted to mean that the
hFSHR-(221-254) peptide has secondary structure that is essential for
the observed inhibitory effect on FSH binding to the FSHR.
Accessibility of peptide 221-252 was further assessed by flow
cytometry to determine whether antiserum to hFSHR-(221-252) could bind
to the FSHR in situ. Antiserum against hFSHR-(221-252) could not bind the FSHR in situ, further corroborating the
inaccessibility of this sequence on the cell surface. However, the
positive control (antiserum against hFSHR-(265-296)), as previously
described (24), bound with high intensity compared with the nonimmune
rabbit serum negative control (data not shown).
Circular Dichroism Analysis of hFSHR-(221-254)--
It is clear
from the CD studies (Fig. 9) of the
peptide in trifluoroethanol (TFE) that the conformational change is
very steep. The development of helix-like signature was essentially
complete at 25% (when compared with the 50% plot). As little
as 10% cosolvent addition resulted in visible changes in the CD
spectra. Analysis of the spectra of peptide in 10% TFE using the
SELCON program calculated a change from 13.6 to 36.6%
The conformation of a peptide portion of a protein in TFE is not a
useful predictor of that peptide structure in the protein (32).
Moreover, the ECD of the hFSHR is likely to be closely apposed to the
plasma membrane. Therefore, additional CD spectra of the active peptide
were collected in the presence or absence of 5 and 22 mM
dodecyl phosphocholine at 20 and 37 °C. Clearly seen for spectra
collected at 20 °C is how addition of 5.0 mM dodecyl
phosphocholine induced a marked change in the secondary structure of
the active peptide (Fig. 10). The
calculated helical content was slightly higher for spectra obtained at
37 °C (26.9%) than for spectra obtained at 20 °C (23.8%).
Roughly 50%
Although CD spectroscopy is a useful tool to monitor changes in
conformation, NMR can be used to verify if the interpretation of
secondary structure conformation is reasonable. Indeed, the one-dimensional 1H NMR spectrum of hFSHR-(221-252) in the
absence of TFE was characteristic of a peptide with a predominantly
random-coil structure (Fig. 11,
upper trace). There was little dispersion in the amide
proton region of the spectrum (Fig. 11, upper trace); these
resonances clustered around the random-coil shift of Circular Dichroism Analysis of the hFSHR ECD--
Recently, we
have achieved the purification of the biologically active hFSHR
ECD.2 Since the data to date
clearly showed that dodecyl phosphocholine increased the secondary
structure of the hFSHR-(221-252) synthetic peptide, we felt it
important to determine whether dodecyl phosphocholine exerted similar
effects upon the hFSH ECD protein. Since we succeeded in expressing and
purifying the entire ECD of the hFSHR in quantities sufficient for
circular dichroism studies, this advance provided an opportunity to
examine the influence of the bulk solvent environment of the secondary
structure of the purified ECD. These studies demonstrated that the
hFSHR ECD undergoes a remarkable change in secondary structure
following incubation with dodecyl phosphocholine (Fig.
13 and Table
II). This result is reminiscent of the
conformational change in the hFSHR-(221-252) synthetic peptide that
was observed when the peptide was incubated with dodecyl
phosphocholine. The data suggest further that the proximity of the ECD
to the plasma membrane may influence the final conformation of the
ECD.
Several homology models of glycoprotein hormone receptors have
been published (15, 16, 35). All homology models have been based on the
ribonuclease inhibitor three-dimensional structure and a leucine-rich
repeat motif. The model of the LHR reported by Jiang et al.
(15) is consistent with some of the available mutagenesis data for the
LHR/CGR. Charge inversion mutations of LHR Lys40,
Lys104, Glu132, and Asp135 carried
out by Puett and co-workers (35, 36) led to undetectable binding or
activation. An Arg114 mutant had no effect on either hCG
binding or signal transduction. Lys104, Glu132,
and Asp135 are predicted to be located at or near the
predicted In this work, the hFSHR-(221-252) peptide appears to be largely
disordered in physiologic buffers, but becomes ordered in the presence
of dodecyl phosphocholine or TFE. Compared with smaller peptides, the
steepness of transition may indicate a higher degree of cooperativity
in the transition. It would be expected that a relatively large peptide
such as hFSHR-(221-252) would exhibit a sharp transition in the
TFE-induced transition if it were to form a single helix along a
significant portion of its length.
TFE is not a helix-inducing solvent in the sense that it will induce
helix formation independent of the sequence (32). It is rather a
helix-enhancing cosolvent that stabilizes helices in regions with some
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit noncovalently bound to a specific
-subunit (2). They are
members of a cystine knot growth factor superfamily (3). FSH is
considered essential for folliculogenesis in females (4) and for
spermatogenesis in males (5). A naturally occurring mutation in the
human
-FSH gene was found to be the cause for primary amenorrhea and
infertility (6). In a mouse strain deficient in
-FSH gene
expression, FSH was shown to be essential for normal ovarian follicular
maturation in females and for normal sperm counts in males (7).
-sheet/
-helix motif (14). Homology modeling to the ribonuclease
inhibitor has provided several models of the three-dimensional
structure of glycoprotein hormone receptor extracellular domains (15,
16). As each leucine-rich repeat has 25-30 residues, we attempted to
identify the hormone-binding regions of the human FSHR (hFSHR) by
synthesizing peptides corresponding to hFSHR regions consisting of at
least 25-30 residues. We hypothesized that FSHR synthetic peptides
could replicate appropriate receptor domain conformation and bind FSH,
but were unable to demonstrate such activity. Unexpectedly, we
discovered an autologous acting domain of the FSHR. This finding
demonstrates, in principle, the possibility of identifying autologous
biological response modifiers of the G-protein-coupled receptor with
knowledge of only the primary structure of the cognate receptor. These
data also support the concepts that receptor-active structure is
transient, that autologous biological response modifiers can stabilize
the inactive state, and that hormone does not bind to the inactive state.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-NH2 Groups of Lysine--
To
determine the effect of charge on peptide activity,
-NH2
groups of lysine residues were modified using acetic anhydride (18).
Pure hFSHR-(221-254) peptide (7.0 mg) was dissolved in 1.0 ml of 0.05 M sodium acetate buffer. Acetic anhydride (25 µl) was
added over a period of 2 h (5 µl at a time) with stirring. The
pH was maintained at 8.0 throughout the reaction. After the last
addition, the peptide was dialyzed against 0.05 M ammonium acetate for mass spectrometry or 0.01 M sodium phosphate
buffer for CD spectroscopy.
-counter (Wallac 1470 Wizard). In the case of the
hCG RRA, CHO cells expressing the human LHR obtained from Ares Advanced Technologies were used as the receptor source, and 125I-hCG
was used as the tracer (CR127; 10 µCi/µg).
-globulin (2% in 0.05 M Tris (pH 7.5)), 500 µl of RRA
buffer, and 1 ml of 25% polyethylene glycol in PBS. Tubes were allowed
to stand on ice for 15 min and then were spun at 4 °C for 1 h
at 2500 × g. The supernatant was aspirated, and the
radioactivity in the pellets was counted in a
-counter. The data
obtained were processed using the computer program LIGAND (33).
20 °C until assayed.
-counter (Wallac Model 1409),
and data were processed using the NIHRIA program (22).
90 °C. Nonimmune rabbit serum and antiserum against the
hFSHR-(265-296) peptide (24) were also processed similarly to use as
negative and positive controls, respectively. Fab' fragments from
purified IgG were generated by digestion with papain (Pierce) following the manufacturer's instructions.
-protons.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-NH2 groups of lysines were modified with
acetic anhydride. Incorporation of acetyl groups was confirmed by mass
spectrometry. All five lysine
-NH2 groups were modified, and the modification eliminated the activity of the peptide (data not
shown). The peptide was also re-synthesized as hFSHR-(221-254) by
removing Tyr and Cys at the C terminus (used for iodination and
conjugations, respectively) and including two additional residues, Thr
and Tyr, as found in the native sequence. This peptide also retained
its activity (Fig. 3). To determine if
charge alone could account for the binding inhibition, two subregions
of the hFSHR-(221-254) peptide (hFSHR-(221-237) and hFSHR-(238-254))
were synthesized and characterized as described earlier. When tested in
the RRA peptide challenge test, peptide 221-237 had no activity, and
peptide 238-254 caused high nonspecific binding and had minimal
activity at the highest dose tested (Fig. 3). As this daughter peptide was found to be relatively insoluble, higher doses could not be tested.
To determine whether peptide 221-254 affected signal transduction, an
in vitro bioassay was carried out. It was found that peptide 221-254 inhibited progesterone production induced in Y-1 cells by 1 and 5 ng of highly purified hFSH (Fig.
4).
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Fig. 1.
Primary sequence comparison between
hFSHR-(221-254) and the analogous sequence of the human LHR
(hLHR)/CGR.
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Fig. 2.
Synthetic peptide corresponding to hFSHR
residues 221-252 exhibits dose-dependent inhibition of
125I-hFSH binding to the hFSHR (A), but
minimal inhibition of 125I-hCG binding to the human LHR/CGR
(B) (both receptors expressed in CHO cells).
Various concentrations of peptide (100 µl) were incubated with
labeled hormone (150,000 cpm/tube/100 µl) at room temperature for
1 h, followed by addition of CHO cells stably expressing either
receptor (250,000 cells/100 µl). Nonspecific binding was determined
in the presence of 1 µg of unlabeled pure hFSH (100 µl) at every
dose of peptide tested.
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Fig. 3.
Daughter peptide fragments hFSHR-(221-237)
and hFSHR-(238-254) were synthesized and tested to determine whether
either peptide possesses the same activity as the hFSHR-(221-254)
parent peptide. Neither peptide was active as compared with the
hFSHR-(221-254) parent peptide. NG, nanograms;
UG, micrograms.
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Fig. 4.
The hFSHR-(221-254) peptide inhibits
FSH-induced steroidogenesis. Y-1 cells expressing the hFSHR were
cultured in 48-well plates at 3 × 105 cells/500
µl/well. The cells were stimulated with 1 and 5 ng of hFSH in the
presence of different doses of the peptide (pep) or with
hFSH at various dose levels in the absence of peptide. Media
progesterone was measured using a radioimmunoassay.
Parameters determined in FSHR binding isotherms at varying
concentrations of the hFSHR-(221-252) peptide reveal a mixed
inhibition with a decrease in receptor concentration and an increase in
Kd
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Fig. 5.
A, development and titers of antisera
against hFSHR-(221-252) conjugated to ovalbumin. The antisera raised
in rabbits X-180 and X-183 were screened by ELISA using immobilized
peptide, peroxidase-conjugated goat anti-rabbit immunoglobulin, and
o-phenylenediamine. B, specificity of the binding
of anti-hFSHR-(221-252) antibody to the immobilized peptide
(hFSHR-(221-254)). Serial dilutions of the free or unrelated peptide
were preincubated with the antiserum (X-180 at 1:8000 or X-183 at
1:2000) before addition to the immobilized peptide in the microtiter
wells.
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Fig. 6.
FSH-blocking activity of
anti-hFSHR-(221-252) antibodies. A, IgG of antiserum
against the hFSHR-(221-252) peptide (X-180 or X-183), which was
prepared by protein A purification, did not inhibit
125I-hFSH binding to the membrane-bound hFSHR on CHO cells.
Nonimmune IgG (normal rabbit serum (NRS)) or immune IgG was
preincubated with CHO-hFSHR cells at 4 °C for 4-5 h. Radioligand
with or without 1 µg of hFSH was added and incubated at room
temperature overnight. B, antibodies at different
concentrations were incubated with solubilized hFSHR expressed in CHO
cells (1.5 × 106 cells) at 4 °C for 4-5 h prior
to addition of radiolabeled hFSH. Bound FSH was separated from free FSH
by precipitating in 12.5% polyethylene glycol. Antiserum to
hFSHR-(265-296) (X-179), which blocks FSH binding to receptor, was
similarly purified and used as a positive control and did inhibit
125I-hFSH binding to detergent-solubilized CHO-hFSHR
cells.
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Fig. 7.
Determination of specificity of protein-A
purified anti-FSHR-(221-252) antibody (X-180). Cells expressing
the extracellular domain of the hFSHR (residues 1-340; line D7) were
used. Cell lysates were subjected to 7.5% SDS-polyacrylamide gel
electrophoresis under reducing conditions, and the resolved proteins
were electrophoretically transferred to nitrocellulose. The blots were
incubated with a 1:500 dilution of nonimmune rabbit serum (lane
1), anti-hFSHR-(265-296) antibody (lanes 2 and
3), or anti-hFSHR-(221-252) antibody (lanes 4 and 5). The detection system used was alkaline
phosphatase/5-bromo-4-chloro-3-indolyl phosphate.
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Fig. 8.
Antisera against FSHR-(221-252) (X-180 and
X-183) recognize a specific peptide conformation. Competitive
inhibition studies of the binding of anti-hFSHR-(221-252) antibody to
the immobilized peptide were performed by incubation with the free
hFSHR-(221-254) peptide or its fragments representing sequences
221-237 and 238-254. Serial dilutions of different peptides were
preincubated with X-180 at 1:800 (A) or X-183 at 1:2000
(B) before addition to the peptide immobilized in the
microtiter plate wells.
-helix. At
25% TFE,
-helix content was calculated as 58.3%, and no further
remarkable change was observed at higher TFE concentrations. At 25%
TFE and above, the CD spectra more clearly show the double-negative
extrema at ~208 and 222 nm that is the
-helix
signature.
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Fig. 9.
Circular dichroism spectra of the
hFSHR-(221-254) peptide in various concentrations of
trifluoroethanol. The spectra were taken at 20 °C using a
0.005-cm path length cell. Peptides were buffered in 0.025 M sodium phosphate buffer (pH 7), and peptide
concentrations were identical in all cases. WL,
wavelength.
-helix was calculated for the peptide at either
temperature and at either concentration of detergent. Analysis of the
spectra of peptide in 5 mM dodecyl phosphocholine was not
greatly different from that in 22 mM dodecyl
phosphocholine. The critical micellar concentration of dodecyl
phosphocholine, as reported by the manufacturer, is 1.2 mM.
Finally, the CD spectra of acetylated, inactive hFSHR-(221-254) in 5 mM dodecyl phosphocholine also contained characteristic
signatures of
-helical secondary structure (data not shown).
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Fig. 10.
Circular dichroism spectra of the
hFSHR-(221-254) peptide in various concentrations of dodecyl
phosphocholine (DPC). The spectra were taken at
20 °C using a 0.005-cm path length cell. Peptides were buffered in
0.025 M sodium phosphate buffer (pH 7), and peptide
concentrations were identical in all cases.
8.3 ppm.
Additionally,
-protons were mostly unresolved with a cluster around
4.2 ppm (data not shown). Although a small amount of helix may have
been present, it was not ascertainable from this spectrum. In both cases, chemical shift degeneracy in the amide and
-proton regions suggested the peptide to be overwhelmingly unstructured (data not
shown). Addition of TFE stimulated a transition from unstructured to
some secondary structure in the peptide (Fig. 11, lower
trace). CD spectra identified the evolving structural element as a
helix. The helix developed fully at ~20-25% TFE and remained
unchanged as the level of TFE increased. NMR spectra recorded at 25 and 50% TFE both indicated an essentially identical level of helical content (data not shown). Fig. 11 (lower trace) shows the
increase in resonance dispersion in the amide proton region of the
peptide upon addition of TFE. This dispersion is indicative of
formation of structure. More evidence that the stimulated structure is
helical is provided by two-dimensional NMR. To probe more thoroughly
for signs of helical content, two-dimensional NOESY and TOCSY spectra were run and analyzed. The extra resolution afforded by the TOCSY spectra of peptide in TFE shows that there is extra chemical shift dispersion in the
-proton, again indicating formation of structure (Fig. 12). The
-protons are all
shifted upfield of the water resonance, implying an
-helix.
Corresponding NOESY spectra displayed dNN connectivities between amide protons, again suggestive of a helical structure.
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Fig. 11.
One-dimensional proton NMR spectra of the
hFSHR-(221-254) peptide. The upper trace represents
the peptide without added TFE, and the lower trace
represents the peptide in buffer made 50% with TFE.
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Fig. 12.
Two-dimensional proton NMR spectra of the
hFSHR-(221-254) peptide made 50% with TFE.
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Fig. 13.
CD spectra of the purified hFSHR ECD
incubated in the presence or absence of 20 mM dodecyl
phosphocholine. The spectra were taken at 6° C using a 0.05-cm
path length cell in 0.01 M potassium phosphate buffer after
incubation overnight with 20 mM dodecyl phosphocholine
(DPC) (- - -) or buffer (------). The spectra represent
the average of two independent experiments. deg,
degrees.
Percentages of secondary structure calculated from two independent
experiments by SELCON3 using a data base containing 29 proteins
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheet region; and hence, they are expected to be involved
in hormone binding. Lys40 and Arg114 are
predicted to be on the outer surface and are not expected to affect
hormone receptor binding. Site-directed mutagenesis of Asn residues has
shown that the carbohydrate moieties of the glycoprotein hormone
receptor are not involved in hormone recognition and high affinity
binding (37). The model (15) also predicts that Asn77,
Asn152, and Asn173 will be located at the outer
surface (helices or loops). Therefore, glycosylation of these residues
is not expected to interfere with hormone binding at the
-strand inner face. The region corresponding to the
hFSHR-(221-254) peptide was not included in this model because it did
not conform to the authors' leucine-rich repeat motif. Bhowmick
et al. (35) have also reported a model for the hCG receptor.
It differs from the model reported by Jiang et al. (15)
because it encompasses two leucine-rich repeats encoded by exon 9, including region 220-253 of the human LHR, represented as two
-strands and an
-helix, and is homologous to
hFSHR-(221-252). Analysis of the active peptide in TFE by NMR revealed
-helical structure that would be expected in a leucine-rich repeat domain.
-helical propensity (39). This said, it is also clear that solvent
can determine the secondary structure of a peptide in vitro
and can override its propensity for secondary structure due to sequence
(32). A conformational change in the presence of TFE does not prove
that the peptide is so ordered in the intact protein. For this reason,
we collected CD spectra in dodecyl phosphocholine of the
hFSHR-(221-252) peptide and found that this detergent, which mimics a
membrane environment, caused a conformational change in the peptide. It
is tempting to speculate that if the peptide is not ordered in the
intact protein, the peptide may undergo a conformational change upon
interaction with the receptor or as it nears the plasma membrane. The
formation of secondary structural elements by the peptide in the
presence of dodecyl phosphocholine suggests that the peptide can become ordered in an appropriate environment. There are examples in the literature of bioactive peptides that adopt a helical conformation upon
interaction with a hydrophobic environment (40) or lipid micelles (41)
or upon interaction with cognate ligand (42). Micelles and vesicles are
thought to induce and stabilize helix formation by binding to residues
that fall in a longitudinal, hydrophobic strip (43). A helical wheel
projection of the peptide is shown in Fig.
14, illustrating the preponderance of
hydrophobic residues and their spatial relationships to other residues
in the helix. For clarity, the helical wheel projection is depicted as
the first and second halves of the peptide since the presence of a
proline would produce a kink in the helix. Therefore, the peptide
helical wheel presentation is divided into two wheels at the proline
residue. Sequence 221-254 of the hFSHR is very likely in close
association with the plasma membrane or is involved in protein-protein
contacts that are disrupted upon detergent solubilization. These data
suggest that a helical conformation of hFSHR-(221-254) might be
stabilized by association of this part of the extracellular domain with
the plasma membrane.
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Fig. 14.
Helical wheel projection of the
hFSHR-(221-254) peptide. For clarity, two independent wheels
encompassing sequences 221-238 and 239-254 are shown. This analysis
was done using the GCG program of the Genetics Computer Group (Madison,
WI). Boxed residues are hydrophobic.
The present data also strongly suggest that region 221-252 interfaces with the transmembrane domain in part because it is inaccessible to immunochemical probes without detergent solubilization of membrane-bound receptor, providing an experimental basis for orientation of the hFSHR ECD in the models of the FSHR. Support for these conclusions is derived not only from data collected by immunochemical approaches, but also by combining synthetic peptide studies that identified region 221-254 of the hFSHR as having an effect on signal transduction.
We began these studies by analyzing a series of synthetic peptides of the hFSHR ECD for biological activity because synthetic peptides from the ECDs of the LHR/CGR and thyroid-stimulating hormone receptor were used by others to identify the hormone-binding regions of these receptors. Roche et al. (44) found that three different peptides, 21-38, 102-115, and 253-272, of the LHR/CGR inhibited the binding of labeled hCG to its receptor. Similar studies carried out with the thyroid-stimulating hormone receptor (45) identified four different inhibitory peptides, 16-35, 106-125, 226-245, and 256-275, of which only peptide 226-245 was specific, as other peptides also inhibited binding of hCG to its receptor. An FSHR peptide corresponding to region 9-30 has been shown to affect FSH binding and signal transduction (34). As observed in the LHR/CGR and thyroid-stimulating hormone receptor systems, there could have been other regions in the ECD of the FSHR capable of binding to FSH. Therefore, this study was undertaken to identify the regions of the hFSHR ECD involved in hormone binding. Peptide challenge studies revealed that the hFSHR-(221-254) peptide had remarkable properties of FSH binding inhibition. We selected the hFSHR-(221-254) peptide for further study, as it could provide a model peptide for a better biophysical understanding of the three-dimensional structure of the FSHR ECD. However, in contrast to previous studies, where an implicit assumption was that receptor peptides that block hormone binding do so by binding to hormone, there is no evidence that the activity of peptide 221-252 is due to its binding to hFSH.
A prediction of the models is that charges in the cusp of the receptor
ECD may play an important role in electrostatic interactions between
hormone and receptor. Four residues in the LHR (Lys158,
Lys183, Glu184, and Asp206) are
essential to gonadotropin binding (31). Asp206 is conserved
in all glycoprotein hormone receptors. Indeed, modification of
-NH2 groups decreased the activity of the peptide, but
did not disallow a conformational change in the presence of dodecyl phosphocholine. Concern that basic peptides might have large
nonspecific effects in the system used is assuaged by the observation
that the inactive daughter peptide we synthesized, which contains four out of the five lysine residues in the parent peptide, has no biological effect. Since the N-terminal fragment 221-237 had no effect
on hormone binding and the C-terminal fragment 238-254 caused minimal
inhibition at the highest dose tested, correct conformation may be
essential for its activity. Since the hFSHR-(221-254) peptide was able
to inhibit FSH-induced signal transduction, we reason that this region
in the FSHR may be in close contact with one of the extracellular
domains making direct contact with the extracellular loops. Since
peptide does not appear to bind hormone, but does inhibit binding and
signal transduction, the peptide may associate with receptor domains to
alter receptor conformation into a nonactive state. This previously
unrecognized, autologous acting domain demonstrates, in principle, that
discrete proteodomains can have regulating activity.
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ACKNOWLEDGEMENTS |
---|
James Seeger (Peptide Synthesis Facility) synthesized all peptides. Dr. Li-Ming Changchen (Biochemistry Core Facility) performed amino acid analyses. Dr. Charles Hauer (Biological Mass Spectrometry Facility) and Robert Stack determined the molecular masses of the synthetic peptides by mass spectrometry. We gratefully acknowledge Lynn McNaughton (NMR Structural Biology Core Facility), who assisted with NMR data processing. Gerry Kornatowski and the staff of the Cell Culture Facility maintained the CHO and Y-1 cell lines, and Leslie E. Eisele (Biochemistry Core Facility) helped in various aspects of peptide analysis. Renjie Song (Molecular Immunology Core Facility) assisted with the flow cytometry experiments. Michele Losavio provided technical help throughout the study. Dr. Gordon Niswender provided the anti-progesterone antiserum (GDN 337).
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant HD 18407 (to J. A. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by an overseas research associateship from the
Department of Biotechnology, Government of India. Present address: Inst. for Research in Reproduction (ICMR), J. M. St. Parel,
Mumbai 400 012, India.
§ Present address: Dept. of Biochemistry, 128 Polk Hall, Campus Box 7622, North Carolina State University, Raleigh, NC 27695-7622.
¶ To whom correspondence should be addressed: Wadsworth Center, New York State Department of Health, David Axelrod Inst. for Public Health, 120 New Scotland Ave., Albany, NY 12208. Tel.: 518-486-2569; Fax: 518-474-5978; E-mail: James.Dias@wadsworth.org.
Published, JBC Papers in Press, January 16, 2001, DOI 10.1074/jbc.M100115200
2 A. Schmidt, R. MacColl, B. Lindau-Shepard, D. R. Buckler, and J. A. Dias, submitted for publication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: FSH, follicle-stimulating hormone; hFSH, human follicle-stimulating hormone; FSHR, follicle-stimulating hormone receptor; hFSHR, human follicle-stimulating hormone receptor; LH, luteinizing hormone; LHR, luteinizing hormone receptor; ECD, extracellular domain; HPLC, high performance liquid chromatography; RRA, radioreceptor assay; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; hCG, human chorionic gonadotropin; CGR, chorionic gonadotropin receptor; ELISA, enzyme-linked immunosorbent assay; TOCSY, total correlation spectroscopy; NOESY, nuclear Overhauser effect correlation spectroscopy; TFE, trifluoroethanol.
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