From the Lehrstuhl für Biopolymere,
Universität Bayreuth, D-95440 Bayreuth and the
§ Niedersächsisches Institut für
Peptid-Forschung, Feodor-Lynen-Straße 31, D-30625 Hannover, Federal Republic of Germany
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ABSTRACT |
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Human parathyroid hormone (hPTH) is involved in the regulation of the calcium level in blood. This hormone function is located in the NH2-terminal 34 amino acids of the 84-amino acid peptide hormone and is transduced via the adenylate cyclase and the phosphatidylinositol signaling pathways. It is well known that truncation of the two NH2-terminal amino acids of the hormone leads to complete loss of in vivo normocalcemic function. To correlate loss of calcium level regulatory activity after stepwise NH2-terminal truncation and solution structure, we studied the conformations of fragments hPTH-(2-37), hPTH-(3-37), and hPTH-(4-37) in comparison to hPTH-(1-37) in aqueous buffer solution under near physiological conditions by circular dichroism spectroscopy, two-dimensional nuclear magnetic resonance spectroscopy, and restrained molecular dynamics calculations. All peptides show helical structures and hydrophobic interactions between Leu-15 and Trp-23 that lead to a defined loop region from His-14 to Ser-17. A COOH-terminal helix from Met-18 to at least Leu-28 was found for all peptides. The helical structure in the NH2-terminal part of the peptides was lost in parallel with the NH2-terminal truncation and can be correlated with the loss of calcium regulatory activity.
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INTRODUCTION |
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All known extracellular biological activity of human parathyroid
hormone (hPTH)1 is located in
the NH2 terminus of this 84-amino acid peptide
hormone (1). hPTH-(1-37) is the naturally occurring bioactive hormone
extractable from human blood (2, 3), and hPTH-(1-34) is known to
maintain normocalcemia in blood via adenylate cyclase activation. To
increase calcium flow into blood, the hormone acts directly on bone and kidney and indirectly on the intestine (1). In addition to the cyclic
adenosine monophosphate (cAMP) pathway, involvement of the
phosphatidylinositol hydrolysis signaling pathway is postulated for
these functions (4). The receptor binding region mediating the calcium
regulatory activity is located within sequence His-14 to Phe-34 (5, 6).
The complete NH2-terminal part of hPTH-(1-34) is required
for stimulation of the cAMP-dependent pathway (4), and the
minimum sequence affecting bone and kidney comprises amino acids 2-27
(1, 7). Adenylate cyclase activity is lost on deletion of the first
NH2-terminal amino acid, whereas receptor binding capacity
is not influenced, indicating that the activation region for cAMP
production and the receptor binding region are located in two distinct
domains (4, 8). Adenylate cyclase activity measured in vitro
does, however, not reflect the sequence-activity relationship indicated
by various in vivo assays (4). hPTH-(2-34) is nearly
inactive in an in vitro bioassay of cAMP stimulation, but
in vivo the calcium level in blood is regulated with
identical efficiency by hPTH-(2-34) and hPTH-(1-34) (Ref. 4 and
references therein). This indicates that hPTH utilizes other second
messengers in addition to cAMP for signal transduction and possibly
additional receptors in vivo (9). Furthermore, hPTH is
stimulating cell proliferation in skeletal derived cell cultures (10,
11) as well as DNA synthesis in chondrocytes (12). Different sequence regions of the peptide are responsible for these functions; for stimulation of DNA synthesis, amino acids Asp-30 to Phe-34 are postulated as an indispensable region, but flanking residues seem to be
required in addition for this function (12).
hPTH stimulates an increase of bone formation and axial bone mass after periodic administration of the hormone (13). Thus, hPTH is useful in the treatment of patients with hypoparathyroidism and, moreover, in the treatment of osteoporotic patients. Therefore, it would be highly desirable to construct a stable mimetic of this peptide hormone. Thus, recent studies focused on the determination of the three-dimensional structure of NH2-terminal peptides in solution by nuclear magnetic resonance (NMR) spectroscopy. In particular, hPTH-(1-34) is an intensely studied hormone fragment as it contains all functional domains (14-17). From most experiments it was concluded that hPTH-(1-34) does not form secondary structure elements in the absence of TFE (14, 16, 18), but helix formation in TFE-free solution is nevertheless observed for hPTH-(1-34), residues 4-13 and 21-29 (19), and for hPTH-(1-37), residues 5-10 and 17-28 (20). In TFE-containing solution hPTH-(1-34) displays helical regions from Ser-3 to Gly-12 and from Ser-17 to Lys-26 (16, 18), but no tertiary interactions for hPTH-(1-34) are found under these conditions. It is commonly known that TFE stabilizes secondary structures, in particular helices (21-26), but bears the risk of weakening hydrophobically stabilized tertiary structure domains (24), an effect also observed for hPTH-(1-34).2
Since hPTH is of considerable medical importance, drugs mimicking this structure could be useful as therapeutics. In a first step in this direction, we determine here the structures of the NH2-terminally truncated fragments hPTH-(2-37), hPTH-(3-37), and hPTH-(4-37) in comparison with the biologically active fragment hPTH-(1-37) (20) under near physiological conditions to elucidate a possible correlation between the loss of calcium regulatory activity after stepwise truncation of NH2-terminal amino acids and structural features of the peptides.
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MATERIALS AND METHODS |
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Peptide Synthesis-- Synthesis of hPTH fragments was carried out using a PerSeptive 9050 automated peptide synthesizer on preloaded Fmoc-L-Leu-PEG-PS or Fmoc-L-Leu-TentaGelS PHB resin (loading 0.2 mmol/g, PerSeptive, Wiesbaden, Rapp Polymere, Tübingen, Germany) (20, 27, 28). Acylations with a 4-fold excess of Fmoc amino acids in N,N-dimethylformamide were performed in the presence of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate/N,N-diisopropylethylamine/1-hydroxybenzotriazole for 30 min. The following protective groups were used: Ser-(tert-butyl), Glu-(O-tert-butyl), Gln-(triphenylmethyl), His-(triphenylmethyl), Asn-(triphenylmethyl), Lys-(tert-butyloxycarbonyl), Arg-(2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl), and Trp-(tert-butyloxycarbonyl). Fmoc groups were cleaved in 10 min with 20% piperidine in N,N-dimethylformamide. The peptides were deprotected and cleaved from the resin with trifluoroacetic acid/ethanedithiol/water, 94:3:3, for 120 min. After filtration and precipitation of the crude peptide by addition of cold tert-butyl methyl ether, the peptide was lyophilized from 10% acetic acid and purified by preparative reversed phase-high performance liquid chromatography (Vydac C18, 300A, 10 mm, 25 × 250 mm, flow rate 10 ml/min; buffer A, 0.6% trifluoroacetic acid in water; buffer B, 0.5% trifluoroacetic acid in acetonitrile/water, 4:1, detection at 230 nm). Pure fractions were pooled, and the final product was checked by reversed phase-high performance liquid chromatography (Vydac C18) and capillary zone electrophoresis (Biofocus 3000, Bio-Rad, München, Germany). Electrospray mass spectrometry (Sciex API III, Perkin-Elmer, Langen, Germany), gas phase sequencing (473A Protein Sequencer, Applied Biosystems/Perkin-Elmer, Weiterstadt, Germany), and amino acid analysis (Aminoquant 1090L, Hewlett Packard, Waldbronn) showed correct mass, amino acid sequence, and composition.
Biological Activity--
In vitro biological activity
of the synthetic hPTH-(1-37), hPTH-(2-37), hPTH-(3-37), and
hPTH-(4-37) fragments was tested by observation of the stimulation of
the cAMP generation in osteogenic cells (rat osteosarcoma cells)
compared with synthetic hPTH-(1-34) fragment. ROS 17/2.8 cells were
grown in 25-cm2 plastic flasks at 37 °C in a humidified
atmosphere of air/CO2 in Ham's F12/Dulbecco's modified
Eagle's medium supplemented with 5% fetal calf serum, 50 mg of
streptomycin/ml, and 50 units of penicillin/ml. The medium was changed
on alternate days. The cells reached confluence within 3-4 days and
were plated into 24-well dishes for experiments. Assays were performed
on confluent cultures 1-2 days after change in medium. cAMP
measurements were as follows. The cells were preincubated with 1 mM 3-isobutyl-1-methylxanthine for 15 min. The cells were
then incubated for an additional 5 min in the presence of the agonists
(hPTH-(1-34), hPTH-(1-37), hPTH-(2-37), hPTH-(3-37), and
hPTH-(4-37)). Incubation with forskolin was used as positive control.
Supernatant was aspirated, and cAMP was extracted after addition of
70% chilled ethanol, evaporation, and redilution of the cells in cAMP
buffer. The samples were kept at 20 °C until cAMP levels were
determined by a specific radioimmunoassay (29).
CD Spectroscopy-- CD spectra were recorded at 25 °C in 0.1-mm cells from 250 to 190 nm at 20 nm/min on a Jasco J 600A CD spectropolarimeter. Peptide concentrations ranged from 270 to 310 µM in 50 mM phosphate buffer, pH 6.0, with 270 mM sodium chloride in 30 µl volume. The reference sample contained buffer without peptide. Eight scans were accumulated from samples and reference, respectively.
NMR Spectroscopy-- Two-dimensional NMR spectra were obtained on a commercial Bruker AMX600 spectrometer at 298 K with standard methods (31, 32). For hPTH-(4-37) an additional set of spectra was measured at 288 K to resolve frequency degeneracy. The measurements were carried out in 50 mM phosphate buffer with 270 mM sodium chloride. Peptide concentrations were 1.6 mM, pH 6.0 (hPTH-(2-37)), 2.1 mM, pH 6.0 (hPTH-(3-37)), and 1.9 mM, pH 5.8 (hPTH-(4-37)).
The H2O resonance was presaturated by continuous coherent irradiation at the H2O resonance frequency prior to the reading pulse. The sweep widths inRestrained Molecular Dynamics Calculations-- Distance geometry and molecular dynamics (MD) calculations were performed with the XPLOR 3.1 program package (33) on an HP735 computer. The number of nontrivial interresidual NOESY cross-peaks used for structure calculation was 171 for hPTH-(2-37), 210 for hPTH-(3-37), and 159 hPTH-(4-37) (Table I). These cross-peaks were divided into three groups according to their following relative intensities: strong, 0.2 to 0.3 nm, medium, 0.2 to 0.4 nm, and weak, 0.2 to 0.5 nm. 0.05 nm was added to the upper distance limit for distances involving unresolved methyl or methylene proton resonances (pseudoatom approach).
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Structure Analysis-- The final structures were analyzed with respect to stable idealized elements of regular secondary structure using the DSSP (definition of secondary structure of proteins) program package (35). To elucidate the stability of the structures, we calculated the local root mean square deviations with a five-amino acid window (36). For visualization of structure data the SYBYL 6.0 (TRIPOS Association), the RASMOL V 2.6 (37), and the MOLSCRIPT program packages (38) were used.
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RESULTS AND DISCUSSION |
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Activity Tests-- The biological activities of hPTH-(1-37), hPTH-(2-37), and hPTH-(1-34) are virtually identical in the in vivo activity test of calcium homeostasis in blood using Parsons' Chicken Assay (30). Fragment hPTH-(3-37) shows less than 10% of this activity, and hPTH-(4-37) is inactive (Fig. 1a). In the in vitro activity test measuring only the cAMP production in cultured rat osteosarcoma cells, hPTH-(2-37) is much less active than hPTH-(1-37). hPTH-(3-37) and hPTH-(4-37) do not stimulate the adenylate cyclase (Fig. 1b).
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CD Spectroscopy--
To compare the overall content of helical
structure of the different peptides, far UV CD spectroscopy was used
(Fig. 2) with peptide concentrations
ranging from 270 to 310 µM. The overall shape of the
spectra of the different peptides indicates the presence of both
-helical and random coil structural elements (40, 41). With the
stepwise truncation of the NH2-terminal amino acids the ellipticity at 222 nm changes to less negative values. The evaluation of the helix content of the different peptides by standard methods (42)
shows the following approximate fractional helix contents: hPTH-(1-37), 29%; hPTH-(2-37), 24%; hPTH-(3-37), 23%; and
hPTH-(4-37), 22%.
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Analysis of C- Proton Chemical Shifts--
To allow an initial
mutual comparison of the truncated fragments and hPTH-(1-37), we used
the chemical shift data available from our experiments to perform a
secondary structure estimation based on the chemical shift index
strategy (44, 45). The procedure depends on a direct correlation
between the chemical shifts of C-
proton resonances of consecutive
amino acids and the local secondary structure: an upfield shift of the
C-
proton resonances relative to the corresponding "random coil"
values indicates local
-helical structure (negative value in Fig.
3), and a downfield shift of C-
proton
resonances compared with the corresponding random coil values indicates
a local
-sheet structure (positive value in Fig. 3). Only deviations
from the random coil values by more than 0.1 ppm are taken into account
for secondary structure estimation. For hPTH-(1-37) and hPTH-(2-37),
the chemical shifts of C-
proton resonances suggest two helical
regions extending from Ser-17 to at least Gln-29 and around Glu-4 to
His-9. In contrast, no indication of an NH2-terminal helix
is found for hPTH-(3-37) and hPTH-(4-37), although the helical region
in the COOH-terminal part can clearly be derived (Fig. 3). No other
elements of regular secondary structure were evidenced by this
procedure.
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Analysis of Medium Range NOEs--
The NOEs observed for the
various hPTH fragments were determined from the 200-ms NOESY spectra at
298 K (Table I and Fig. 4). The
dN(i,i + 3) and
d
(i,i + 3) NOESY cross-peaks fully
corroborate the existence of two helical regions for hPTH-(1-37) and
hPTH-(2-37). Indications for an NH2-terminal helix for
hPTH-(3-37), however, are weak and are entirely missing for
hPTH-(4-37), thus confirming the results from the chemical shift index
procedure. In particular, helix typical (i,i + 3) NOEs are
clustered from Ile-5 to Leu-11 and Ser-17 to Phe-34 for hPTH-(1-37)
and hPTH-(2-37), respectively. For hPTH-(1-37), two helical regions
were found earlier, a short one from Ile-5 to Asn-10 and a longer one
from Ser-17 through at least Leu-28 (20). For hPTH-(3-37) two weak
helix typical NOEs are found in the NH2-terminal region,
and for hPTH-(4-37) no helix typical NOE could be found in the
NH2-terminal region, and frequency degenerations of
possible (i,i + 3) NOEs were not present. In contrast, clear
evidence of the COOH-terminal helix in these two fragments is found
from Ser-17 to Phe-34 and His-32, respectively. To investigate whether
the missing (i,i + 3) NOEs in the NH2-terminal
region of hPTH-(4-37) can be accounted for by the lower concentration
of this peptide (1.9 mM), two-dimensional NMR spectra of a
sample of hPTH-(1-37) with 1.8 mM concentration were
measured with the same buffer, temperature, and spectrometer
conditions. From the 200-ms NOESY spectrum of this sample, two
d
N(i,i + 3) and five d
(i,i + 3) NOEs in the
NH2-terminal region of hPTH-(1-37) could be assigned.
Thus, the lower concentration cannot account for the missing
(i,i + 3) NOEs in the NH2-terminal region of
hPTH-(4-37). The loss of the NH2-terminal helix on removal
of the first two amino acids was fully confirmed by the NOESY
cross-peak patterns (Fig. 4).
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Relative NOE Intensities--
Relative intensities of sequential
and medium range NOEs may be used to estimate the perfection and
stability of helical structures, in addition to the upfield shift of
the -proton resonances. For an ideal
-helix the
d
N(i,i + 1) and
d
N(i,i + 3) distances should be nearly
identical, whereas the dNN(i,i + 1)
distances should be shorter, yielding higher intensity NOEs (47-49).
For all PTH fragments employed in our experiments, most of the
sequential d
N(i,i + 1) NOEs are of
higher intensity than the corresponding
dNN(i,i + 1) and
d
N(i,i + 3) NOEs (Fig. 4), indicating
that the helices are not ideal but are in an equilibrium with a more
extended conformation, possibly a 310-helix. The helices of
the PTH fragments are clearly more stable than nascent helices that do
not show (i,i + 3) NOEs (48, 50). Simultaneous observation
of d
N(i,i + 2) and d
N(i,i + 4) NOEs may arise from a
mixture of 310- and
-helix type structures (50). This
effect was observed for the COOH-terminal region of the different PTH
fragments. Karle and Balaram (51, 52) suggest that six-residue
sequences are equally likely to form 310- or
-helices.
Both helices in the hPTH fragments we studied seem to represent an
equilibrium between an
-helix and 310-helical
conformation, the NH2-terminal helix having a higher
tendency to a more extended 310-helical conformation. This
phenomenon is also reflected by values of the upfield shift of the
-proton resonances (Fig. 3).
Other NOEs--
For each fragment, four to six long range
(|i j| > 5) NOEs could be assigned
(Table I). All fragments show several long range NOEs between Leu-15
and Trp-23. For hPTH-(1-37), five NOEs were found between Leu-15 and
Trp-23 (20), four NOEs for hPTH-(2-37) and hPTH-(3-37), and five NOEs
for hPTH-(4-37). Additionally, two NOEs between Leu-15 and Val-21 were
observed in hPTH-(3-37). These NOEs indicate a spatial proximity
between Leu-15 and Trp-23, probably due to hydrophobic interactions
between these two residues. The observed NOEs are responsible for a
clear restriction of the conformational space of the calculated
structures and lead to a defined loop region around His-14 to Ser-17.
Furthermore, due to the ring current field of the spatial neighboring
aromatic ring system of Trp-23 the
proton resonances of Leu-15 are
shifted upfield in comparison to the analogous resonances of other
leucines for all four fragments.
Structure Calculation and Analysis--
159-210 interresidual
NOEs per fragment were collected from 200-ms NOESY spectra at 298 K and
used in restrained MD calculations (Table I). For structure calculation
of the NH2-terminally truncated fragments, the combined
distance geometry/simulated annealing protocol described earlier (20,
33) was used. For each fragment a family of 30 structures was
calculated, and the 10 structures with lowest energy values and lowest
number of NOE violations were selected from each group. To resolve
frequency degenerations of proton resonances in the spectra of
hPTH-(4-37) an additional set of spectra was obtained at 288 K. From
this NOESY spectrum, 175 unambiguous interresidual NOEs could
be assigned. Only NOEs were taken into account for
structure calculation that were also observed, albeit ambiguously, in
the NOESY spectrum at 298 K. For each of the four fragments the
COOH-terminal helix extending from Met-18 to at least Leu-28 is found
by DSSP analysis. For hPTH-(1-37) an NH2-terminal helix
from Gln-6 to His-9 exists. For hPTH-(2-37), five structures show an
NH2-terminal -helix around Leu-7; the others show turns
or 310-helix in this region. None of the 10 calculated
structures of hPTH-(3-37) displays an NH2-terminal
-helix, and only two structures exhibit a 310-helix from
Glu-4 to Gln-6. No structure of hPTH-(4-37) shows an
NH2-terminal helix, and only in one case a turn is
indicated by DSSP in this region. The extension of the COOH-terminal
helix of hPTH-(4-37) is virtually identical to that of the
corresponding helix in the other fragments. The loss of the
NH2-terminal helix after truncation of the first two amino
acids is corroborated by the structure calculations.
Local RMSD Values-- To elucidate the stability of the structures in the helical regions and the defined loop, we calculated the local root mean square deviations (RMSD) using a five-amino acid window (36) (Fig. 5). The upper trace represents the local RMSD values for all heavy atoms, and the lower trace represents the values for the peptide backbone. The regions with defined structure show substantially reduced local RMSD values compared with the flexible regions at the termini and around Gly-12. For hPTH-(1-37) and hPTH-(2-37) two regions with local backbone RMSD values lower than 0.07 nm were found from Gln-6 to His-9 and Asn-16 to Lys-26 for hPTH-(1-37) and from Leu-7 to His-9 and Asn-16 to Asp-30 for hPTH-(2-37), respectively. Comparatively high RMSD values for the amino acids Leu-11 to Lys-13 for fragments hPTH-(1-37) and hPTH-(2-37) indicate a flexible hinge region between the NH2-terminal helix and the loop region followed by the COOH-terminal helix. For hPTH-(3-37) and hPTH-(4-37) a decrease of the RMSD values is found in the region of the COOH-terminal helix from Ser-17 to Gln-29. Compared with the fragments hPTH-(1-37) and hPTH-(2-37) the NH2-terminal region is structurally less well defined for the fragments hPTH-(3-37) and hPTH-(4-37) (Fig. 5).
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Helix Content--
From NMR and structure calculation data the
following helix contents for the different peptides were estimated. The
secondary structure analysis using the DSSP program (35) result in a
helix content of 43% for hPTH-(1-37), 44% for hPTH-(2-37), 37% for
hPTH-(3-37), and 32% for hPTH-(4-37). For this calculation only
amino acids that reside in a helical conformation in more than 50% of
the calculated structures were taken into account. Under the assumption that residues which contribute to medium and strong (i,i + 3) NOEs are part of helical structures (Fig. 4), the helix content is
59% for hPTH-(1-37), 55% for hPTH-(2-37), 43% for hPTH-(3-37), and 38% for hPTH-(4-37). The helix content according to the chemical shift indexing procedure is 46 and 50% for hPTH-(1-37) and
hPTH-(2-37), respectively, 31% for hPTH-(3-37), and 32% for
hPTH-(4-37) (Fig. 3). From the NMR results a clear decrease in the
helix content is derived between hPTH-(2-37) and hPTH-(3-37).
Assuming that the length of helical regions is reflected correctly by
the combined NMR results, there is a significant underestimation of the
helix content from CD spectra (22-29%), which is also reported for
other peptides (54, 55). One explanation for the apparent lower helix
content estimated from CD spectra is that the helical sequences are in
helical conformation in 50-70% on time average in the case of PTH.
Other explanations are the absolute length of the helices and the
associated end group effects (56-60) as well as a possible contribution of the aromatic side chain of Trp-23 to the far UV CD
signal (59, 61). Additionally, the shape and intensity of the CD signal
depends on the geometry of a peptide helix. An ideal -helix has a
stronger CD signal than a 310-helix (59) with a different
shape (50, 58). These phenomena lead to a lower percentage of helicity
estimated from the [
]222 value. Thus, changes in the
short NH2-terminal helix could not be detected on the basis
of the CD signal at 222 nm alone. The possibility of an equilibrium
with a 310-helix is also reflected by the values of the
upfield shifts of the C-
proton resonances (Fig. 3).
Conclusion-- After deletion of the NH2-terminal two or three amino acids, PTH's biological activity is lost, but its receptor binding ability remains unimpaired (8, 53). Table II summarizes the results of the activity tests and the structure calculations. All fragments show the loop region and the COOH-terminal helix. His-14 to Leu-28 (loop and COOH-terminal helix) comprises the major part of the receptor binding region that is known to reside within His-14 to Phe-34 (5, 6, 68). The NH2-terminal helix, however, is present only in the in vivo bioactive fragments hPTH-(1-37) and hPTH-(2-37), but not in the inactive fragments hPTH-(3-37) and hPTH-(4-37) (Table II). This may indicate that the NH2-terminal helix is correlated with the in vivo bioactivity of the PTH fragments concerning the calcium level in blood. Existence of the NH2-terminal helix, however, cannot be connected to the ability to stimulate adenylate cyclase, as hPTH-(2-37) is nearly inactive in the cAMP assay. This result may imply different structural requirements for triggering the different signal transduction pathways (4), and may thus indicate the occurrence of different PTH receptors as discussed in the literature (69). To decide whether or not the in vivo biological activity is determined on a structural level by the NH2-terminal helix or depends on a direct functional role of the first two amino acids, structure calculations and activity tests of stabilized PTH fragments are currently under investigation.
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FOOTNOTES |
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* 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.
¶ Present address: MPI für Molekulare Physiologie, Abteilung Physik. Biochemie, Rheinlanddamm 201, D-44139 Dortmund, Germany.
To whom correspondence should be addressed: Lehrstuhl
für Struktur und Chemie der Biopolymere, Universität
Bayreuth, D-95440 Bayreuth, Germany. Tel.: 49 921 553540; Fax: 49 921 553544; E-mail: paul.roesch{at}uni-bayreuth.de.
1 The abbreviations used are: hPTH, human parathyroid hormone; Clean-TOCSY, TOCSY with suppression of NOESY-type cross-peaks; COSY, correlated spectroscopy; DSSP, definition of secondary structure of proteins; Fmoc, 9-fluorenylmethoxycarbonyl; MD, molecular dynamics; NOE, nuclear Overhauser effect, also used for NOESY cross-peak; NOESY, NOE spectroscopy; PTH, parathyroid hormone; RMSD, root mean square deviation; TOCSY, total correlation spectroscopy; TFE, trifluoroethanol.
2 U. C. Marx, K. Adermann, W.-G. Forssmann and P. Rösch, unpublished data.
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REFERENCES |
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