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• W2061465147 abstract "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 vivonormocalcemic 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. 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 vivonormocalcemic 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. All known extracellular biological activity of human parathyroid hormone (hPTH) 1The 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. is located in the NH2− terminus of this 84-amino acid peptide hormone (1Potts Jr., J.T. Kronenberg H.M. Rosenblatt M. Adv. Prot. Chem. 1982; 35: 323-396Crossref PubMed Scopus (120) Google Scholar). hPTH-(1–37) is the naturally occurring bioactive hormone extractable from human blood (2Forssmann W.G. Schulz-Knappe P. Meyer M. Adermann K. Forssmann K. Hock D. Aoki A. Yanaihara N. Peptide Chemistry. ESCOM, Leiden1993: 553-557Google Scholar, 3Hock D. Mägerlein M. Heine G. Ochlich P.P. Forssmann W.G. FEBS Lett. 1997; 400: 221-225Crossref PubMed Scopus (22) Google Scholar), 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 (1Potts Jr., J.T. Kronenberg H.M. Rosenblatt M. Adv. Prot. Chem. 1982; 35: 323-396Crossref PubMed Scopus (120) Google Scholar). In addition to the cyclic adenosine monophosphate (cAMP) pathway, involvement of the phosphatidylinositol hydrolysis signaling pathway is postulated for these functions (4Coleman D.T. Fitzpatrick A. Bilezikian J.P. Bilezikian J.P. Levine M.A. Marcus R. The Parathyroids. Raven Press, New York1994: 239-258Google Scholar). The receptor binding region mediating the calcium regulatory activity is located within sequence His-14 to Phe-34 (5Caulfield M.P. McKee R.L. Goldmann M.E. Duong L.T. Fisher J.E. Gay C.T. DeHaven P.A. Levy J.J. Roubini E. Nutt R.F. Chorev M. Rosenblatt M. Endocrinology. 1990; 127: 83-87Crossref PubMed Scopus (90) Google Scholar, 6Lopez-Hilker S. Martin K.J. Sugimoto T. Slatopolsky E. J. Lab. Clin. Med. 1992; 119: 738-743PubMed Google Scholar). The complete NH2-terminal part of hPTH-(1–34) is required for stimulation of the cAMP-dependent pathway (4Coleman D.T. Fitzpatrick A. Bilezikian J.P. Bilezikian J.P. Levine M.A. Marcus R. The Parathyroids. Raven Press, New York1994: 239-258Google Scholar), and the minimum sequence affecting bone and kidney comprises amino acids 2–27 (1Potts Jr., J.T. Kronenberg H.M. Rosenblatt M. Adv. Prot. Chem. 1982; 35: 323-396Crossref PubMed Scopus (120) Google Scholar, 7Tregear G.W. van Rietschoten J. Greene E. Keutmann H.T. Niall H.D. Reit B. Parsons J.A. Potts Jr., J.T. Endocrinology. 1973; 93: 1349-1353Crossref PubMed Scopus (275) Google Scholar). 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 (4Coleman D.T. Fitzpatrick A. Bilezikian J.P. Bilezikian J.P. Levine M.A. Marcus R. The Parathyroids. Raven Press, New York1994: 239-258Google Scholar, 8Segre G.V. Rosenblatt M. Reiner B.L. Mahaffey J.E. Potts Jr., J.T. J. Biol. Chem. 1979; 254: 6980-6986Abstract Full Text PDF PubMed Google Scholar). Adenylate cyclase activity measured in vitrodoes, however, not reflect the sequence-activity relationship indicated by various in vivo assays (4Coleman D.T. Fitzpatrick A. Bilezikian J.P. Bilezikian J.P. Levine M.A. Marcus R. The Parathyroids. Raven Press, New York1994: 239-258Google Scholar). hPTH-(2–34) is nearly inactive in an in vitro bioassay of cAMP stimulation, butin vivo the calcium level in blood is regulated with identical efficiency by hPTH-(2–34) and hPTH-(1–34) (Ref. 4Coleman D.T. Fitzpatrick A. Bilezikian J.P. Bilezikian J.P. Levine M.A. Marcus R. The Parathyroids. Raven Press, New York1994: 239-258Google Scholar and references therein). This indicates that hPTH utilizes other second messengers in addition to cAMP for signal transduction and possibly additional receptors in vivo (9Jouishomme H. Whitfield J.F. Chakravarthy B. Durkin J.P. Gagnon L. Isaacs R.J. MacLean S. Neugebauer W. Willick G. Rixon R.H. Endocrinology. 1992; 130: 53-60Crossref PubMed Scopus (85) Google Scholar). Furthermore, hPTH is stimulating cell proliferation in skeletal derived cell cultures (10Sömjen D. Bindermann I. Schlüter K.-D. Wingender E. Mayer H. Kaye A.M. Biochem. J. 1990; 272: 781-785Crossref PubMed Scopus (60) Google Scholar,11Sömjen D. Schlüter K.-D. Wingender E. Mayer H. Kaye A.M. Biochem. J. 1991; 277: 863-868Crossref PubMed Scopus (37) Google Scholar) as well as DNA synthesis in chondrocytes (12Schlüter K.-D. Hellstern H. Wigender E. Mayer H. J. Biol. Chem. 1989; 264: 11087-11092Abstract Full Text PDF PubMed Google Scholar). 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 (12Schlüter K.-D. Hellstern H. Wigender E. Mayer H. J. Biol. Chem. 1989; 264: 11087-11092Abstract Full Text PDF PubMed Google Scholar). hPTH stimulates an increase of bone formation and axial bone mass after periodic administration of the hormone (13Reeve J. Meunier P.J. Parsons J.A. Bernat M. Bijovoet O.L.M. Coupron P. Edouard C. Klenerman L. Neer R.M. Renier J.C. Slovik D. Vismans F.J. Potts J.T. Br. Med. J. 1980; 280: 1340-1344Crossref PubMed Scopus (523) Google Scholar). 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 (14Klaus W. Dieckmann T. Wray V. Schomburg D. Wingender E. Mayer H. Biochemistry. 1991; 30: 6936-6942Crossref PubMed Scopus (82) Google Scholar, 15Barden J.A. Cuthbertson R.M. Eur. J. Biochem. 1993; 215: 315-321Crossref PubMed Scopus (50) Google Scholar, 16Strickland L.A. Bozzato R.P. Kronis K.A. Biochemistry. 1993; 32: 6050-6057Crossref PubMed Scopus (50) Google Scholar, 17Chorev M. Rosenblatt M. Bilezikian J.P. Levine M.A. Marcus R. The Parathyroids. Raven Press, New York1994: 139-156Google Scholar). From most experiments it was concluded that hPTH-(1–34) does not form secondary structure elements in the absence of TFE (14Klaus W. Dieckmann T. Wray V. Schomburg D. Wingender E. Mayer H. Biochemistry. 1991; 30: 6936-6942Crossref PubMed Scopus (82) Google Scholar, 16Strickland L.A. Bozzato R.P. Kronis K.A. Biochemistry. 1993; 32: 6050-6057Crossref PubMed Scopus (50) Google Scholar, 18Wray V. Federau T. Gronwald W. Mayer H. Schomburg D. Tegge W. Wingender E. Biochemistry. 1994; 33: 1684-1693Crossref PubMed Scopus (67) Google Scholar), but helix formation in TFE-free solution is nevertheless observed for hPTH-(1–34), residues 4–13 and 21–29 (19Barden J.A. Kemp B.E. Biochemistry. 1993; 32: 7126-7132Crossref PubMed Scopus (63) Google Scholar), and for hPTH-(1–37), residues 5–10 and 17–28 (20Marx U.C. Austermann S. Bayer P. Adermann K. Ejchart A. Sticht H. Walter S. Schmid F.-X. Jaenicke R. Forssmann W.-G. Rösch P. J. Biol. Chem. 1995; 270: 15194-15202Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). In TFE-containing solution hPTH-(1–34) displays helical regions from Ser-3 to Gly-12 and from Ser-17 to Lys-26 (16Strickland L.A. Bozzato R.P. Kronis K.A. Biochemistry. 1993; 32: 6050-6057Crossref PubMed Scopus (50) Google Scholar, 18Wray V. Federau T. Gronwald W. Mayer H. Schomburg D. Tegge W. Wingender E. Biochemistry. 1994; 33: 1684-1693Crossref PubMed Scopus (67) Google Scholar), 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 (21Nelson J.W. Kallenbach N.R. Proteins. 1986; 1: 211-217Crossref PubMed Scopus (407) Google Scholar, 22Dyson H.J. Merutka G. Waltho J.P. Lerner R.A. Wright P.E. J. Mol. Biol. 1992; 226: 795-817Crossref PubMed Scopus (368) Google Scholar, 23Sönnichsen F.D. van Eyk J.E. Hodges R.S. Sykes B.D. Biochemistry. 1992; 31: 8790-8798Crossref PubMed Scopus (619) Google Scholar, 24Sticht H. Willbold D. Bayer P. Ejchart A. Herrmann F. Rosin-Arbesfeld R. Gazit A. Yaniv A. Frank R. Rösch P. Eur. J. Biochem. 1993; 218: 973-976Crossref PubMed Scopus (16) Google Scholar, 25Lancelin J.M. Bally I. Arlaud G.J. Blackledge M. Gans P. Stein M. Jacquot J.P. FEBS Lett. 1994; 343: 261-266Crossref PubMed Scopus (35) Google Scholar, 26Morton C.J. Simpson R.J. Norton R.S. Eur. J. Biochem. 1994; 219: 97-107Crossref PubMed Scopus (13) Google Scholar), but bears the risk of weakening hydrophobically stabilized tertiary structure domains (24Sticht H. Willbold D. Bayer P. Ejchart A. Herrmann F. Rosin-Arbesfeld R. Gazit A. Yaniv A. Frank R. Rösch P. Eur. J. Biochem. 1993; 218: 973-976Crossref PubMed Scopus (16) Google Scholar), an effect also observed for hPTH-(1–34). 2U. C. Marx, K. Adermann, W.-G. Forssmann and P. Rösch, unpublished data. 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) (20Marx U.C. Austermann S. Bayer P. Adermann K. Ejchart A. Sticht H. Walter S. Schmid F.-X. Jaenicke R. Forssmann W.-G. Rösch P. J. Biol. Chem. 1995; 270: 15194-15202Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) 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. 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) (20Marx U.C. Austermann S. Bayer P. Adermann K. Ejchart A. Sticht H. Walter S. Schmid F.-X. Jaenicke R. Forssmann W.-G. Rösch P. J. Biol. Chem. 1995; 270: 15194-15202Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 27Atherton E. Sheppard R.C. Solid Phase Peptide Synthesis: A Practical Approach. IRL Press, Oxford1989Google Scholar, 28Forssmann W.G. Marx U. Bayer P. Adermann K. Hock D. Rösch P. Kaumaya P.T.P. Hodges R.S. Peptides: Chemistry and Structure. Mayflower Worldwide Ltd., Birmingham, UK1995: 225-226Crossref Google Scholar). 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 inN,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 coldtert-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. 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 (29Abou-Samra A.B. Harwood J.P. Manganiello V.C. Catt K.J. Aguilera G. J. Biol. Chem. 1987; 262: 1129-1136Abstract Full Text PDF PubMed Google Scholar). In vivo biological activity of these hPTH fragments was tested using Parsons' Chicken Assay (30Parsons J.A. Reit B. Robinson C.J. Endocrinology. 1973; 92: 454-462Crossref PubMed Scopus (82) Google Scholar) which is indicative of the Ca2+ level homeostasis in blood. 6.25 μg of hPTH fragment together with 20 μmol of CaCl2 was injected intravenously into 10–14-day-old male chickens. After 60 min the chickens were anesthetized and then decapitated, and the blood was collected. The serum was diluted in a 1:50 ratio with 1% lanthanum nitrate solution. Atomic absorption spectroscopy was used for determination of serum calcium concentration. A hPTH-(1–34) sample served as a standard. Pure solvent without PTH was used as control. 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. Two-dimensional NMR spectra were obtained on a commercial Bruker AMX600 spectrometer at 298 K with standard methods (31Ernst R.R. Angew. Chem. Int. Ed. Engl. 1992; 104: 817-852Crossref Google Scholar, 32Wüthrich K. NMR of Proteins and Nucleic Acids. John Wiley & Sons, New York1986Crossref Google Scholar). 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 in ω1 and ω2 were 7042.3 Hz. Quadrature detection was used in both dimensions with the time proportional phase incrementation technique in ω1. 4 K data points were collected in ω2and 512 data points in ω1. Zero filling to 1 K data points was used in ω1. All two-dimensional NMR spectra were multiplied with a squared sine bell function phase shifted by π/4, π/3, and π/2, respectively, for the NOESY spectra, by π/6 or π/4 for the Clean-TOCSY spectra, and π/8 or π/4 for the double quantum filtered COSY spectra. Base-line and phase correction of the 6th order was used. Data were evaluated on X-Window work stations with the NDee program package (Software Symbiose GmbH, Bayreuth, Germany). Data from the following 600 MHz spectra were employed for the sequence-specific assignment of spin systems and the evaluation of the NOESY distance constraints for the different PTH fragments: double quantum filtered COSY spectra, Clean-TOCSY spectra with mixing times of 80 ms, and NOESY spectra with mixing times of 200 ms. For the structure calculations only NOEs visible in the NOESY spectra at 298 K were taken into account. Distance geometry and molecular dynamics (MD) calculations were performed with the XPLOR 3.1 program package (33Brünger A.T. X-PLOR, Version 3.1. Howard Hughes Medical Institute and Yale University, New Haven1993Google Scholar) 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).Table IEnergy contributions to the structures and deviations from standard geometry NOE and X-PLOR statisticshPTH(1–37) 1831-aTotal NOE number.hPTH(2–37) 1711-aTotal NOE number.hPTH(3–37) 2101-aTotal NOE number.hPTH(4–37) 1591-aTotal NOE number.‖i − j‖ = 1106101126100‖i − j‖ = 266 105‖i −j‖ = 3 43 43 53 38‖i− j‖ = 4 18 11 17 10‖i − j‖ = 52401‖i −j‖ > 55645Average energies (kJ/mol)Etotal−1579.36 −522.05−1083.16−1401.76Ebonds268.77300.77 268.54 259.53Eangles535.08715.08 584.73 481.28Eimpr109.99148.24 101.9175.26EVDW−2411.21−2280.64−2130.27−2123.47ENOE553.99 1273.86 772.71 558.95Deviations from standard geometryBonds0.0010 nm0.0011 nm0.0010 nm0.0010 nmAngles0.8452 deg0.9956 deg0.9120 deg0.8342 degImpr0.6973 deg0.8432 deg0.7006 deg0.6140 degNOEs0.0118 nm0.0119 nm0.0134 nm0.0130 nmNOE violations > 0.05 nm≤5 (φ = 1.7)≤8 (φ = 5.0)≤5 (φ = 2.3)≤6 (φ = 3.1)Whole peptide1-bRMSD among backbone structures (nm).0.5260.4910.4530.479Met-18–Leu-281-bRMSD among backbone structures (nm).0.0520.0560.0600.074His-14–Leu-281-bRMSD among backbone structures (nm).0.0690.0860.0840.0961-a Total NOE number.1-b RMSD among backbone structures (nm). Open table in a new tab The structure calculations followed standard procedures employing a hybrid distance geometry-restrained MD approach with simulated annealing refinement and subsequent energy minimization (protocol distance geometry simulated annealing (33Brünger A.T. X-PLOR, Version 3.1. Howard Hughes Medical Institute and Yale University, New Haven1993Google Scholar)). For the refinement the dielectric constant was changed to ε = 4. Structure parameters were extracted from the standard parallhdg.pro and topallhdg.pro files (34Brooks B.R. Bruccoleri R.E. Olafson B.D. States D.J. Swaminathan S. Karplus M. J. Comput. Chem. 1983; 4: 187-217Crossref Scopus (14017) Google Scholar). For each fragment 30 structures were calculated. Ten structures for every fragment were selected on the criteria of smallest number of NOE violations over 0.05 nm and lowest overall energy. 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 (35Kabsch W. Sander C. Biopolymers. 1983; 22: 2577-2637Crossref PubMed Scopus (12414) Google Scholar). To elucidate the stability of the structures, we calculated the local root mean square deviations with a five-amino acid window (36Wagner G. Braun W. Havel T.F. Schaumann T. Go N. Wüthrich K. J. Mol. Biol. 1987; 196: 611-639Crossref PubMed Scopus (635) Google Scholar). For visualization of structure data the SYBYL 6.0 (TRIPOS Association), the RASMOL V 2.6 (37Sayle R. RASMOL, Version 2.6, Molecular Visualisation Program. Glaxo Wellcome Research and Development, Stevenage, Hertfordshire, UK1995Google Scholar), and the MOLSCRIPT program packages (38Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 976-980Crossref Google Scholar) were used. 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 (30Parsons J.A. Reit B. Robinson C.J. Endocrinology. 1973; 92: 454-462Crossref PubMed Scopus (82) Google Scholar). Fragment hPTH-(3–37) shows less than 10% of this activity, and hPTH-(4–37) is inactive (Fig. 1 a). 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. 1 b). It is generally accepted that PTH initiates multiple intracellular signals, for example cAMP formation, phosphatidylinositol hydrolysis, and release of intracellular calcium by activating G protein-linked receptors in bone and kidney (4Coleman D.T. Fitzpatrick A. Bilezikian J.P. Bilezikian J.P. Levine M.A. Marcus R. The Parathyroids. Raven Press, New York1994: 239-258Google Scholar). A single receptor was shown to stimulate intracellular accumulation of both cAMP and inositol triphosphates (39Abou-Samra A.B. Jüppner H. Force T. Freeman M.W. Kong X.-F. Schipane E. Urena P. Richards J. Bonventre J.V. Potts Jr., J.T. Kronenberg H.M. Segre G.V. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2732-2736Crossref PubMed Scopus (1007) Google Scholar). PTH has the concentration-dependent ability to stimulate two separate signal pathways (9Jouishomme H. Whitfield J.F. Chakravarthy B. Durkin J.P. Gagnon L. Isaacs R.J. MacLean S. Neugebauer W. Willick G. Rixon R.H. Endocrinology. 1992; 130: 53-60Crossref PubMed Scopus (85) Google Scholar), and different sequential regions of the hormone may be responsible for initiation of the adenylate cyclase and the phospholipase C activating pathway. The existence of these multiple pathways is possibly reflected by the fact that hPTH-(2–37) is virtually inactive in the adenylate cyclase assay but can induce substantial hypercalcemia in the in vivomodel (Fig. 1, a and b). 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 α-helical and random coil structural elements (40Greenfield N. Fasman G. Biochemistry. 1969; 8: 4108-4116Crossref PubMed Scopus (3331) Google Scholar, 41Schmid F.-X. Creighton T.E. Protein Structure: A Practical Approach. IRL Press, Oxford, UK1989: 251-285Google Scholar). 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 (42Morrisett J.D. David J.S.K. Pownall H.J. Gotto A.M. Biochemistry. 1973; 12: 1290-1299Crossref PubMed Scopus (250) Google Scholar) shows the following approximate fractional helix contents: hPTH-(1–37), 29%; hPTH-(2–37), 24%; hPTH-(3–37), 23%; and hPTH-(4–37), 22%. After truncation of the first two amino acids resulting in hPTH-(3–37), the wavelength corresponding to zero ellipticity and the minimum between 200 and 210 nm are shifted to lower wavelength. For hPTH-(1–37) and hPTH-(2–37) the ellipticity vanishes at 197 nm, and for hPTH-(3–37) and hPTH-(4–37) the ellipticity vanishes at 194 nm. The CD spectrum shows a minimum at 205 nm for hPTH-(1–37) and hPTH-(2–37), whereas the minimum for hPTH-(3–37) and hPTH-(4–37) is at 203 nm, indicating a structural transition between hPTH-(2–37) and hPTH-(3–37). These changes in the shape of the spectra may be interpreted as relative increase of random coil structure upon truncation of the first two amino acids (40Greenfield N. Fasman G. Biochemistry. 1969; 8: 4108-4116Crossref PubMed Scopus (3331) Google Scholar, 41Schmid F.-X. Creighton T.E. Protein Structure: A Practical Approach. IRL Press, Oxford, UK1989: 251-285Google Scholar, 43Neugebauer W. Barbier J.-R. Sung W.L. Whitfield J.F. Willick G.E. Biochemistry. 1995; 34: 8835-8842Crossref PubMed Scopus (50) Google Scholar). 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 (44Wishart D.S. Sykes B.D. Richards F.M. Biochemistry. 1992; 31: 1647-1651Crossref PubMed Scopus (2024) Google Scholar, 45Wishart D.S. Bigam C.G. Holm A. Hodges R.S. Sykes B.D. J. Biomol. NMR. 1995; 5: 67-81Crossref PubMed Scopus (1426) Google Scholar). The procedure depends on a direct correlation between the chemical shifts of C-α proton resonances of consecutive amino acids and the local secondary structure: an upfield shift of the C-α proton resonances relative to the corresponding “random coil” values indicates local α-helical structure (negative value in Fig. 3), and a downfield shift of C-α proton resonances compared with the corresponding random coil values indicates a local β-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-α 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. From the difference values of the observed C-α proton chemical shifts relative to the random coil values (45Wishart D.S. Bigam C.G. Holm A. Hodges R.S. Sykes B.D. J. Biomol. NMR. 1995; 5: 67-81Crossref PubMed Scopus (1426) Google Scholar), one can estimate the stability of structural elements, as there is not only a correlation between the existence of secondary structure elements and chemical shifts of C-α proton resonances but also a correlation between the inherent main chain flexibility of these structure elements and the chemical shift data (46Wishart D.S. Sykes B.D. Richards F.M. J. Mol. Biol. 1991; 222: 311-333Crossref Pub" @default.
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• W2061465147 title "Structure-Activity Relation of NH2-terminal Human Parathyroid Hormone Fragments" @default.
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