From the Gustaf H. Carlson School of Chemistry, Clark
University, Worcester, Massachusetts 01610, the § Division
of Bone and Mineral Metabolism, Harvard-Thorndike & Charles A. Dana
Laboratories, Beth Israel Deaconess Medical Center, Harvard Medical
School, Boston, Massachusetts 02215, and the ¶ Department of
Pharmacology and Molecular Toxicology, University of Massachusetts,
Medical Center, Worcester, Massachusetts 01655
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ABSTRACT |
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Parathyroid hormone (PTH) regulates mineral
metabolism and bone turnover by activating specific receptors located
on osteoblastic and renal tubular cells and is fully functional as the
N-terminal 1-34 fragment, PTH-(1-34). Previously, a "U-shaped"
conformation with N- and C-terminal helices brought in close proximity
by a turn has been postulated. The general acceptance of this
hypothesis, despite limited experimental evidence, has altered the
direction of the design of PTH-analogs. Examining the structure of
human PTH-(1-34) under conditions that encompass the different
environments the hormone may experience in the approach to and
interaction with the G-protein-coupled receptor (including benign
aqueous and saline solutions and in the presence of
dodecylphosphocholine), we observe no evidence for a U-shape
conformation or any tertiary structure. Instead, the N- and C-terminal
helical domains, which vary in length and stability depending on the
conditions, are separated by a highly flexible region of undefined
conformation. These observations are in complete accord with recent
conformational studies of PTH-related protein analogs containing
lactams (Mierke, D. F., Maretto, S., Schievano, E., DeLuca, D.,
Bisello, A., Mammi, S., Rosenblatt, M., Peggion, E., and Chorev, M. (1997) Biochemistry 36, 10372-10383) or a model
amphiphilic -helix (Pellegrini, M., Bisello, A., Rosenblatt, M.,
Chorev, M., and Mierke, D. F. (1997) J. Med.
Chem. 40, 3025-3031). Reliable structural data from different environmental conditions are absolutely requisite for the next step in
the design of non-peptide PTH analogs.
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INTRODUCTION |
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Human parathyroid hormone (hPTH)1 has been the object of numerous studies for many years now, owing to its crucial role in the regulation of extracellular calcium homeostasis (1). More recently, we evidence a growing interest in PTH and PTH-derived agonists as anabolic agents in bone remodeling and their promise as new drugs for the treatment of osteoporosis. It has been established that the 34 residues at the N terminus of the 84-amino acid protein, hPTH-(1-34), are sufficient for high affinity binding to the PTH receptor (a G-protein-coupled receptor) and retain PTH-like bone-related activities (2, 3). Biological activity studies on hybrid peptides of PTH and the parathyroid hormone-related protein (PTHrP), which binds to and activates the same receptor, indicate a functional interaction between the 1-14 and 15-34 domains (4).
On the basis of theoretical methods for secondary structure prediction,
both the N and C termini of PTH-(1-34) are expected to be helical.
Furthermore, the amphipathic character of the two putative helices has
suggested helix-helix interactions resulting in a "U-shaped"
tertiary structure with a hydrophobic core and outwardly facing
hydrophilic residues (5). Notably, only a low -helical content could
be identified for hPTH-(1-34) in aqueous solution
([
]r222 =
6800; average of literature values
as reported by Willis (see Ref. 6) corresponding to approximately 8 residues in
-helix (see Ref. 7)). Only upon addition of the
structure inducing solvent trifluoroethanol (TFE), a helix content
consistent with the two predicted helical segments was observed.
Unfortunately, the majority of the NMR-derived structures of
hPTH-(1-34) in TFE (8-10) do not provide any evidence of long range
interactions between the two helices. This has been attributed to the
low dielectric constant (
= 26.7) of TFE, which could weaken
hydrophobic interactions and therefore destabilize the alleged tertiary
structure (11).
Recently, new evidence emerging from the NMR study of hPTH-(1-37) in aqueous solution in the presence of salts was interpreted in favor of the U-shape hypothesis (11). However, the reported long range NOEs are limited only to the turn portion of the U-shape and do not include residues further toward the termini. Therefore, the experimental data do not define the relative topological arrangement of the helices; any helix-loop-helix conformation is consistent with the data. In addition, the conformational consequences of the experimental conditions employed, namely the pH and the high salt concentration, were not investigated. Highly questionable U-shape conformations have been reported for PTHrP analogs in the presence of TFE (12-14).
Here, we report high resolution structures of hPTH-(1-34) in aqueous solution, investigating the effect of pH and salt concentration on secondary and tertiary structure (hPTH-(1-34) is equipotent to hPTH-(1-37) and displays identical affinity to the PTH/PTHrP receptor). In addition, we examined the peptide in the presence of dodecylphosphocholine (DPC) micelles. Micellar systems have often been used as an NMR-compatible model system for mimicking biological membranes (15-24). A membrane-ligand interaction and membrane-induced conformation have been proposed as an operational pathway for the interaction between peptide hormones and their receptors (25-28).
The structural study utilizes experimental data both from circular
dichroism (CD) and NMR spectroscopies, providing, respectively, an
estimate of the helix content and the location of the -helix, possibly including long range interactions determining a tertiary fold.
The NMR study was coupled with a protocol of structure calculation that
could assure adequate sampling of conformational space (metric matrix
distance geometry, DG), followed by further refinement with extensive
molecular dynamics (MD) simulations incorporating explicit
solvent. The high resolution conformational preferences of
hPTH-(1-34) in aqueous and saline solutions and in the presence of a
membrane mimetic is required to advance the structure-activity relationship of the hormone and provide a reliable template for rational design of non-peptidic PTH-mimetic agonists.
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EXPERIMENTAL PROCEDURES |
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The synthesis and purification of the peptide followed published procedures (29).
Circular Dichroism---
Spectra were recorded using an Aviv CD
spectrometer (model 62 DS), interfaced with a personal computer.
Spectra were acquired on 30 µM solutions of the peptide
in H2O, at different pH, buffer composition, and
temperature and in the presence of DPC micelles. Each spectrum is the
average of four scans from 255 to 195 nm. The ellipticity is reported
as mean residue molar ellipticity ([]r), in degrees
cm2 dmol
1. Estimates of the helical fraction
of the peptide were calculated using the value of
[
]obs at 222 nm and the formula for percent helix = {([
]obs222
[
]coil222)}
{([
]helix222
[
]coil222)} × 100 (7). Estimations of
the error associated with this formula has been discussed extensively
in the literature (30, 31). Data manipulation and graphic
representation were achieved with the program Kaleidograph.
NMR Methods-- hPTH-(1-34) was examined in benign water solution (1.4 mM, 9:1 H2O/2H2O, pH = 4.3, not corrected for deuterium), in the presence of buffer and salts (2 mM, 270 mM NaCl, 50 mM phosphate buffer, in 9:1 H2O/2H2O, pH = 6.0) and in the presence of micelles (1.4 mM, 9:1 H2O/2H2O, pH = 4.3, 200 mM DPC-d38 from CIL). All the experiments were recorded on a Varian Unity500 spectrometer (500 MHz) at temperatures varying between 285 and 318 K. Data processing utilized Varian (VNMR) or Felix (Biosym Technologies Inc., San Diego) software. Chemical shifts were referenced to the signal of tetramethyl silane (0.0 ppm).
The complete amino acid spin systems were identified from double quantum filtered correlation spectroscopy (32) and TOCSY (33, 34) spectra. NOESY (35, 36) and ROESY (37) experiments with mixing times of 125-350 ms were employed for the sequential assignment. All the experiments were recorded in the phase-sensitive mode according to the method detailed by States et al. (38). The TOCSY utilized a MLEV-17 sequence to realize mixing times of 40-70 ms with a spin-lock field of 10,000 Hz. In the ROESY experiment, a spin-lock field of 2500 Hz was realized by a continuous wave pulse. Suppression of the solvent signal was achieved by continuous wave presaturation at low power level during the relaxation delay (1.5-2 s), and for NOESY experiments also during the mixing time. The typical spectral width was 6500 Hz in both dimensions, with 4096 data points in t2 and 512-640 data points in t1 and with 32-128 scans at each increment. Forward linear prediction to 1024 points was applied to the incremented dimension; both dimensions were multiplied by Gaussian or shifted squared sine bell apodization functions, prior to Fourier transformation. Back-calculation of NOESY spectra utilized the matrix doubling method within the Felix program. The cutoff distance was set at 8 Å, the correlation time to 1 ns, and the smallest back-calculated intensity to be saved was set to 0.001, representing a 0.1% NOE enhancement.Distance Geometry--
NOESY spectra acquired at 285 K with a
mixing time of 200 ms were utilized to measure cross-peak volumes for
the two water samples, while a mixing time of 150 ms at 308 K was
employed for the DPC sample. The volumes were converted to distances
using the two-spin approximation and the cross-peaks between couples of
aromatic protons of Trp23 and between the two protons
of Trp23 (Ser1 for the DPC sample) as a
reference (respectively: Trp23 4H/5H = 2.4 Å;
Trp23 7H/6H = 2.4 Å;
1/
2 = 1.78 Å). Addition and subtraction
of 10% to the calculated distances yielded upper and lower bounds
utilized in the distance geometry calculations. A home-written program, based on the random metrization algorithm of Havel (39), was utilized
to calculate an ensemble of structures fulfilling holonomic (constitutional) and experimental (proton-proton distances) restraints. The 100 starting structures were obtained by random sampling of the
distances between the upper and lower bounds. They were first embedded
in four dimensions and optimized versus the initial distance restraints (conjugate gradients) (40). This step was followed by
distance-driven dynamics (DDD) (41, 42), carried out at 500 K for 100 ps, and by slow cooling to 1 K. A new metrization reduced the resulting
structures to three dimensions for the next optimization and DDD. The
DDD procedure applies a simple square well potential on holonomic and
experimental distances and an additional penalty term to maintain
chirality and planarity, to generate the violation "energy" and
calculate the forces.
Molecular Dynamics--
MD simulations were performed with
GROMACS (45), and interactive modeling was performed using
Insight II (Biosym Technologies Inc., San Diego). Only the
structures resulting from the study in water and in the presence of
salts were examined. The starting structures for the simulations were
chosen from the low energy structures obtained from DG as described
under "Results." All atoms and the solvent were simulated
explicitly. The peptide structure was placed in a rectangular box of
H2O (46, 47), allowing at least 10 Å between the walls and
the peptide and using three-dimensional periodic boundary condition.
The charges of ionizable groups correspond to the pH of the NMR
solution; no counter ions were included. A time step of 2.0 fs was
employed. Neighbor lists for calculation of nonbonded interactions were
updated every 20 steps within a radius of 10 Å. No switching function
was utilized. The system was energy-minimized for 100 steps using
steepest descent. Following this, 10 ps of MD at 300 K (48) with the
peptide restrained to its original position with a force constant of
1000 kJ mol1 nm
1 were carried out.
Experimental distance restraints were then introduced with a force
constant of 6000 kJ mol
1 nm
1 for 20 ps, and
subsequently raised to 10,000 kJ mol
1 nm
1
for the rest of the simulation (200-400 ps). Structures were sampled
every 0.5 ps. One iteration took approximately 2.7 s on one
processor of a SGI Challenger (R8000, 150 MHz). The resulting trajectories were examined with analysis programs in the GROMACS package and home-written Fortran programs.
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RESULTS |
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The helix content of hPTH-(1-34) based on CD spectra (given in
Fig. 1) increases in the presence of
phosphate buffer at pH 6 (from 24% in benign water to 28% in
buffer) and reaches a maximum of 54% in the presence of DPC micelles.
Spectra acquired in the presence of phosphate or acetate buffer but at
acidic pH are almost identical to the spectrum in benign
H2O (data not shown). The solution containing phosphate
buffer at pH = 6 displays a small temperature dependence. A small
decrease of helix content (indicated by the ellipticity at 222 nm)
occurs upon changing the temperature from 5 to 45 °C.
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NMR in Benign H2O--
The NMR study confirmed the
presence of helical structure and localized the secondary structure
elements to segments in the N- and C-terminal part of the peptide
sequence. The analysis of the chemical shift values of the H
protons, compared with values for amino acids in a random-coil peptide
(49), chemical shift index (CSI), provides a first estimate of
secondary structure (50, 51). The results, shown in Fig.
2A, indicate a helical region
(CSI
0.1) extending from residue 17 to 30, separated by a
zero point from a second "helical" domain with a small deviation from random coil values, centered at Leu7.
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NMR in H2O and Salts--
The CSI values for protons in the presence of phosphate buffer and salts are similar to
that observed in benign water solution (Fig. 2B). The extent
of the negative deviations in this case is significantly larger for
both the C- and N-terminal helix. This qualitatively indicates a larger
content/population of
-helix, as do the greater dispersion of
chemical shifts and the larger number of medium range NOEs (Fig.
4). The
HN(i)-HN(i+1) NOEs are present along the entire
peptide sequence. An N-terminal
-helix from residue 5 to 12 is
indicated by 4 H
(i)-HN(i+3) (i = 5, 7, 9, 10) and 3 HN(i)-HN(i+2)
(i = 3, 8, 12) cross-peaks. NOEs between Gly12HN and Ser14HN and
Gly12H
-Leu15HN are
of weak intensity and may result from a small population in which the
helix extends to residue 15. In addition, H
(i)-H
,
(i+3) (i = 2, 3, 5, 6, 7) and
H
(i)-H
,
(i+4) (i = 3, 6)
NOEs are observed.
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NMR in H2O/DPC Micelles--
The pattern of two
helices located at the N and C terminus of the molecule, very well
defined by CSI and NOEs, is also observed in the membrane mimetic. The
CSI, reported in Fig. 2C, show remarkably higher values in
the two helical regions, compared with the values observed in the
aqueous and saline conditions. The C-terminal helix extends less toward
the N terminus, with residues 17-19 assuming values typical for a
random coil structure. This is confirmed by NOEs; cross-peaks of type
H(i)-HN(i+3) (i = 2, 4, 5, 6, 10, 11, 12, 18, 20, 21, 22, 23, 24, 25, 26, 28, 29) and
H
(i)-HN(i+4) (i = 5, 21, 22, 27) locate the two helices approximately from residues 2-4 to 15 and
from 18-20 to 32. No long range NOEs were detected, even after
increasing the mixing time of the NOESY experiments; no interactions
between Trp23 and Leu15 were observed. A total
of 249 informative distance restraints (144 are inter-residue) were
utilized in the DG calculations. The 90 low energy structures fulfill
the experimental distance restraints (the largest violation is 0.16 Å). The large number of NOEs typical of
-helix produces a
converging ensemble of conformations, with values of the dihedral
angles
and
characteristic for
-helix in the region 21-33
(pairwise r.m.s. deviation = 1.72 Å, average over 90 structures,
backbone atoms). An irregular helix at the N terminus starts at residue
4 and extends to residue 15 and possibly 17, with strong deviations
about Asp10 and Leu11 (r.m.s. deviation value
of 2.85 Å for residues 4-14). The well ordered structure is reflected
by high order parameters for the
and
dihedral angles (Fig. 3).
Low values at residues 18 and 19 indicate flexibility in the central
part of the molecule (importantly, there was no resonance overlap
preventing the detection of NOEs in this region). A flexible domain in
the center of the molecule allows a range of different orientations of
the two well defined helices relative to each other. Consequently, the
r.m.s. deviation value for the 34 amino acids of the peptide is very
large (6.0 Å). The family of structures and the consequences of the
flexible domain are indicated in Fig. 6.
The average angle between the two helical domains is 115 ± 30°.
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DISCUSSION |
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The NMR studies and relative DG/MD calculations presented in this work indicate that the conformation of hPTH-(1-34) in aqueous solution depends largely on the experimental conditions. In addition, the presence of conformational averaging fast on the NMR time scale must be considered to produce accurate and high resolution structures consistent with the experimental observations.
In benign aqueous solution, the peptide is mainly disordered. The CD
data account for about eight helical residues, in agreement with much
of the literature data (6). The only NMR evidence for the presence of
ordered structure (indeed, an equilibrium between ordered and random
conformations) is provided by a few weak NOEs, typical of -helix.
These NOEs are in general agreement with the structure of Lee and
Russell (53) and with our CD data, and provide additional evidence for
one loop of helix at the N terminus. Our results are in stark contrast
with the structure presented by Barden and Kemp (54). Despite very
similar experimental conditions, their proposed conformation consists
of two helical domains with a tertiary fold determined by a large
number of long range interactions. These contrasting structures cannot
be attributed to the difference at the C terminus (i.e. a
free carboxylic acid used in this study versus a carboxamide
used in their study). Importantly, the structure proposed by these
authors is not consistent with the CD data. It is important to note
that a significant overlap of NMR signals, particularly of the side
chain resonances, are reported by these authors (54) that would make an
unambiguous assignment of all of the NOEs listed in this publication
particularly difficult. Additionally, the collapsing of the two helical
domains to adopt a U-shape may arise from the utilization of in
vacuo calculations for structure refinement. The observation of
collapsed structures, particularly peptides, from the overemphasis of
the non-bonded terms (i.e. Coulombic and van der Waals
attraction) in the absence of explicit solvent has been addressed in
the literature (55, 56).
Concerning hPTH-(1-34) in high salt concentration, our NMR data
provide no evidence of tertiary structure or any long range interaction
deriving from a hydrophobic collapse of residues 15 and 23, despite
careful examination of NOESY spectra collected with differing mixing
times and temperatures. The molecule is far too flexible to prefer any
definite secondary or tertiary fold. Two regions of the sequence appear
to have a tendency to fold into a -helix and account for the CD
spectra (namely residues 6-10 in the N terminus and residues 18-29 in
the C terminus), but they do not constitute stable, long-lived
-helices.
The difference between the conformation in H2O at pH 4.3 and in saline solution does not arise exclusively from the different ionic strength of the solution or the salt utilized. CD spectra in
aqueous solutions containing 50 mM phosphate buffer at pH
4.6 or 50 mM acetate buffer at pH 4.9 (results not shown)
give similar values of []r222 to that observed
for hPTH-(1-34) in benign H2O solution (
6800 versus
6300). The average ellipticity values measured for
solutions containing 50 mM phosphate buffer at pH 6 or 9, in the absence or presence of NaCl, are [
]r222
of
8000, accounting for about 10 residues in
-helix.
The apparent discrepancy between the differences in the NMR spectra of
the two solutions and the similarity of the CD spectra can be explained
by averaging of the various conformational states in solution. Indeed,
a good agreement between the NMR data and calculated structures for
hPTH-(1-34) in water/salts was achieved only upon considering an
equilibrium between helical and extended conformations. The ensemble of
two structures utilized in the ensemble calculations produced two
conformations, each of them representative of 50% of the population
and displaying shorter -helical segments, located in different,
alternating regions of the peptide. If the combination of the helical
segments observed in the two conformations is considered, it is
consistent with the distribution of NOEs, indicative of 17-24 residues
in an
-helix (residues 3-5 to 12 and 16-18 to 28-31), while the
population of 50% of each conformation explains the 10 residues in
-helix calculated from the CD spectra.
Despite the similar medium range interactions observed here for hPTH-(1-34) and those reported for hPTH-(1-37) (11), the resulting conformations are drastically different. Indeed, the C-terminal helix is more stable in the presence of salt than in benign H2O and extends to include residues 16-18, placing the hydrophobic side chains of amino acids Trp23 and Leu15 at a short distance, separated by only two loops of helix. The close proximity may induce a hydrophobic interaction that will contribute to stabilize the helix. The interaction between amino acids containing aromatic rings with hydrophobic residues has been reported to significantly stabilize helices, although usually spaced i, i+4 residues apart (57). From the computational searching of conformational space, we find that the medium range NOEs between Leu15 and Trp23 are compatible with a large number of tertiary folds or topological arrangements of the two helical domains. This is a consequence of the flexibility of the central portion of the molecule producing a large variability of interhelical angles between the two helical domains. Two representative conformations, a linear and a U-shaped arrangement, representing the conformational extremes, were further examined by extensive MD simulations. During the MD simulations of the U-shaped conformation, the two helices (N- and C-terminal) are too far apart for interhelical contacts or justify a hydrophobic interaction as a stabilizing factor of the tertiary fold.
To further examine the putative U-shaped conformation, theoretical
NOESY spectra were calculated using coordinates of a U-shaped conformation (as previously reported in Ref. 11) generated by molecular
modeling. The back-calculation of NOEs from a structure is a powerful
method for identification of conformations not consistent with the
experimentally observed NOEs. One of the theoretical NOESY spectra is
illustrated in Fig. 7; the experimental
NOESY spectrum is shown for comparison in black. The
agreement between the theoretical and experimental spectra is generally
good. The higher regularity of the helices in the MD structure,
discussed in the previous session, is reflected by an additional
cross-peak, Ser3H-Gln6HN, and by
an increased intensity in the
Ser3H
-Ile5HN and
Asn10H
-Lys13HN
cross-peaks. There are many additional theoretical NOEs produced by the
U-shaped conformation; those giving rise to resolved, unambiguous cross-peaks are labeled in the figure. Clearly visible are NOEs between
the aromatic protons of Trp23, which is buried in between
the two helices, and backbone protons of Leu7 and
Met8 belonging to the N-terminal helix. Additional
correlations involve Leu24-Met8 and
Arg20-Met8. The absence of these cross-peaks in
the experimental NOESY indicates that under these experimental
conditions, either such a conformation is not present or is not
populated to an extent to generate observable NOEs.
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The NMR study of hPTH-(1-34) in the presence of DPC micelles indicates
that indeed the membrane environment can induce a higher degree of
conformational order, extending and stabilizing both the N-terminal and
C-terminal -helix. If the two helices, and the consequent
amphipathic exposure of the amino acids, are relevant elements in the
bioactive conformation, the interaction with the cellular membrane
could present to the receptor a ligand folded into a preferred
conformation (25, 58). Similar to the results obtained in water and
saline solution the two helical domains are separated by a region of
flexibility, in this case centered about residues 18 and 19 (i.e. shifted more toward the C terminus than was observed
in the other two environments). This allows the two "halves" of the
molecule to adopt a range of relative orientations. However, no
evidence for helix-helix interaction in a tertiary structure was
observed in the presence of DPC. Likewise, we could not find any
support for a turn conformation in the connecting loop separating the
two helices.
In conclusion, under the experimental conditions examined in this
study, the formation of a hydrophobic core between the two amphiphilic
helices, resulting in a U-shaped conformation is not detectable either
in aqueous solution or in the presence of micelles. A conformation in
which the N and C termini of hPTH are in close proximity may indeed be
preferred upon binding to the receptor, as suggested by biological
activities and receptor binding affinities of chimeric PTH/PTHrP (4).
However, our results clearly indicate that, even if the U-shape fold is
energetically accessible, the peptide does not assume this conformation
in solution. Instead it is possible that the flexibility of the
molecule, allowing different relative spatial arrangements of the two
helical domains, is a requirement to adopt the proper conformation for
interacting with the receptor. Results from a series of lactam
containing PTHrP-derived analogs have shown that flexibility around
residue 19 is required for high biological activity of these analogs
(59). The PTHrP analog, RS-66271, containing a model amphiphilic
-helix, likewise displayed a flexible domain immediately N-terminal
of the C-terminal helix (60). The magnitude of the conformational changes induced by the receptor will only be obtained from analysis of
the receptor bound hormone. We are approaching this by photoaffinity cross-linking and mapping out contact points between the hormone and
receptor (61). These investigations, coupled with high resolution structures of the hormone in the different environments that the hormone may experience upon interacting with the receptor, will provide
the structural insight required for the rational design of PTH-derived
agonists.
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ACKNOWLEDGEMENT |
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We gratefully acknowledge the assistance of Dr. Robert Talanian of BASF (Worcester, MA) with the CD measurements.
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FOOTNOTES |
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* This work was supported in part by Grants R01-DK47940 (to M. R.) and GM54082 (to D. F. M.) from the National Institutes of Health.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.
To whom correspondence should be addressed. Present address:
Dept. of Molecular Pharmacology, Box G-B4, Brown University, Providence, RI 02912. Tel.: 401-863-2139; Fax: 401-863-1595; E-mail: dale_mierke{at}brown.edu.
1 The abbreviations used are: hPTH, human parathyroid hormone; CSI, chemical shift index; DDD, distance-driven dynamics; DG, distance geometry; DPC, dodecylphosphocholine; G-protein, guanine nucleotide-binding regulatory protein; MD, molecular dynamics; NOE, nuclear Overhauser enhancements; NOESY, nuclear Overhauser enhancement spectroscopy; PTH, parathyroid hormone; PTHrP, parathyroid hormone-related protein; r.m.s., root-mean-square; ROESY, rotational-Overhauser enhancement spectroscopy; TFE, trifluoroethanol; TOCSY, total-correlation spectroscopy.
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REFERENCES |
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