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W2000147308 abstract "We have investigated the prefibrillar state of salmon (s) and human (h) calcitonin (CT). Size exclusion chromatography at pH 3.3 and 7.4 indicates that sCT is present in solution as a dimer, whereas hCT elutes as a monomer at pH 3.3 and as monomer-dimer at pH 7.4. Guanidine hydrochloride unfolding experiments show that dimerization is stabilized by hydrophobic interactions. We investigated the dimeric structure by multidimensional nuclear magnetic resonance spectroscopy and calculations by using an sCT mutant (LAsCT) in which Pro23 and Arg24 were substituted for Leu23 and Ala24. As indicated by the Leu9–Tyr27 and Leu12–Leu19 contacts, the mutated hormone forms a head-to-tail dimer whose basic unit is an α-helix in the region Leu12–Tyr22. The solution behavior of LAsCT is identical to that of sCT, so the dimeric structure can safely be extended to sCT: we believe that such a structure inhibits fibril maturation in sCT. No stable dimer was observed for hCT, which we attributed to the absence of a defined helical structure. However, we suggest that intermolecular collisions of short ordered regions (for example, a sequence of turns) in hCT favors intermolecular contacts, and specific orientation can be obtained through hydrogen bond formation involving Tyr12, Phe16, and Phe19, with the aromatic ring acting as an acceptor. Taken together, our results indicate that hCT fibrillation can be reduced by favoring a helical dimer, obtainable by replacing the three aromatic amino acids with leucines. We have investigated the prefibrillar state of salmon (s) and human (h) calcitonin (CT). Size exclusion chromatography at pH 3.3 and 7.4 indicates that sCT is present in solution as a dimer, whereas hCT elutes as a monomer at pH 3.3 and as monomer-dimer at pH 7.4. Guanidine hydrochloride unfolding experiments show that dimerization is stabilized by hydrophobic interactions. We investigated the dimeric structure by multidimensional nuclear magnetic resonance spectroscopy and calculations by using an sCT mutant (LAsCT) in which Pro23 and Arg24 were substituted for Leu23 and Ala24. As indicated by the Leu9–Tyr27 and Leu12–Leu19 contacts, the mutated hormone forms a head-to-tail dimer whose basic unit is an α-helix in the region Leu12–Tyr22. The solution behavior of LAsCT is identical to that of sCT, so the dimeric structure can safely be extended to sCT: we believe that such a structure inhibits fibril maturation in sCT. No stable dimer was observed for hCT, which we attributed to the absence of a defined helical structure. However, we suggest that intermolecular collisions of short ordered regions (for example, a sequence of turns) in hCT favors intermolecular contacts, and specific orientation can be obtained through hydrogen bond formation involving Tyr12, Phe16, and Phe19, with the aromatic ring acting as an acceptor. Taken together, our results indicate that hCT fibrillation can be reduced by favoring a helical dimer, obtainable by replacing the three aromatic amino acids with leucines. Protein amyloid fibrils are found in about 20 diseases of unrelated origin, including Alzheimer's disease, diabetes mellitus (type II diabetes), familial amyloidosis, light chain amyloidosis, transmissible spongiform encephalopathies, and Parkinson's disease (1Rochet J.C. Lansbury Jr., P.T. Curr. Opin. Struct. Biol. 2000; 10: 60-68Crossref PubMed Scopus (999) Google Scholar). It is not known which structural features cause specific proteins to form amyloid fibrils in vivo; however, evidence is accumulating that aggregation is initiated from specific regions within a polypeptide chain (2Taddei N. Capanni C. Chiti F. Stefani M. Dobson C.M. Ramponi G. J. Biol. Chem. 2001; 276: 37149-37154Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 3Azriel R. Gazit E. J. Biol. Chem. 2001; 276: 34156-34161Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar, 4Tjernberg L.O. Callaway D.J.E. Tjernberg A. Hahne S. Lilliehöök C. Terenius L. Thyberg J. Nordstedt C. J. Biol. Chem. 1999; 274: 12619-12625Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar, 5Reches M. Porat Y. Gazit E. J. Biol. Chem. 2002; 277: 35475-35480Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 6Tjernberg L. Hosia W. Bark N. Thyberg J. Johansson J. J. Biol. Chem. 2002; 277: 43243-43246Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). Human calcitonin (hCT), 1The abbreviations used are: CT, calcitonin; hCT, human CT; sCT, salmon CT; LAsCT, sCT with Pro23–Arg24 substituted by Leu-Ala; BPTI, bovine pancreatic trypsin inhibitor; MD, molecular dynamics; NOE, nuclear Overhauser enhancement; NOESY, nuclear Overhauser spectroscopy; r.m.s., root mean square; TOCSY, total correlation spectroscopy. a 32-amino-acid polypeptide hormone of ∼3.4 kDa (Scheme 1) produced by thyroidal C-cells, forms amyloid fibrils associated with medullary carcinoma of the thyroid (7Benvegna S. Trimarchi F. Facchiano A. J. Endocrinol. Invest. 1994; 17: 119-122Crossref PubMed Scopus (9) Google Scholar). Synthetic hCT also forms amyloid fibrils in vitro, with a morphology similar to the thyroid deposits (8Arvinte T. Cudd A. Drake A.F. J. Biol. Chem. 1993; 268: 6415-6422Abstract Full Text PDF PubMed Google Scholar). Calcitonin (CT) can be used to treat various diseases including Paget's disease and osteoporosis (9Zaidi M. Inzerillo A.M. Moonga B.S. Bevis P.J. Huang C.L. Bone (NY). 2002; 30: 655-663Crossref PubMed Scopus (132) Google Scholar), but the tendency of hCT to associate into amyloid fibrils at physiological pH limits its efficacy as a drug. Salmon CT (sCT) (Scheme 1), the clinically used alternative to hCT, causes immunogenic reactions (10Levy F. Muff R. Dotti-Sigrist S. Dambacher M.A. Fisher J.A. J. Clin. Endocrinol. Metab. 1988; 67: 541-548Crossref PubMed Scopus (61) Google Scholar). Therefore, understanding the mechanism of amyloid formation by hCT and controlling this process are important not only in the context of amyloid formation but also as a step toward improved therapeutic use of CT. The detailed molecular mechanism of CT fibril formation is not yet well understood. It has been proposed that the first step is a homogeneous association of α-helices to form the nucleus of a fibril followed by an autocatalytic heterogeneous fibrillation of β-sheets to form a mature fibril (11Kamihira M. Naito A. Tuzi S. Nosaka Y.A. Saitô H. Protein Sci. 2000; 9: 867-877Crossref PubMed Scopus (101) Google Scholar). It has been suggested that hydrophobic interactions favor the formation of α-helical bundles, whereas charged amino acids regulate the β-sheet association (12Kanaori K. Nosaka A.Y. Biochemistry. 1995; 34: 12138-12143Crossref PubMed Scopus (43) Google Scholar). The fibrillation of sCT is much slower than hCT, requiring more than 8 months as compared with 21 min for hCT at 1 mg/ml and pH 7.4 (8Arvinte T. Cudd A. Drake A.F. J. Biol. Chem. 1993; 268: 6415-6422Abstract Full Text PDF PubMed Google Scholar); furthermore, the fibrillation of hCT at low pH is slower than that at physiological pH (8Arvinte T. Cudd A. Drake A.F. J. Biol. Chem. 1993; 268: 6415-6422Abstract Full Text PDF PubMed Google Scholar). Considering that the proposed fibrillation mechanism involves a homogeneous aggregation of α-helices, it is surprising that sCT does not fibrillate, despite having a helical propensity higher than hCT (13Siligardi G. Samorì B. Melandri S. Visconti M. Drake A.F. Eur. J. Biochem. 1994; 221: 1117-1125Crossref PubMed Scopus (52) Google Scholar, 14Amodeo P. Motta A. Strazzullo G. Castiglione Morelli M.A. J. Biomol. NMR. 1999; 13: 161-174Crossref PubMed Scopus (33) Google Scholar). To understand the structural determinants that cause the differing fibrillation mechanism of hCT and sCT, we investigated the prefibrillar state of both hormones. Size exclusion chromatography at pH 3.3 and 7.4 indicate that sCT is present in solution as a dimer. hCT is eluted as a monomer at pH 3.3 but is in equilibrium between dimer and monomer at pH 7.4. The structure of dimeric sCT was investigated by NMR spectroscopy and calculations by resorting to an sCT mutant (referred to as LAsCT) in which Pro23 and Arg24 were substituted for Leu23 and Ala24 (Scheme 1). LAsCT showed properties identical to those of sCT, the only difference being the stability and the length of the amphipathic α-helix in membrane-like environment. 2G. Andreotti, B. López Méndez, P. Amodeo, M. A. Castiglione Morelli, H. Nakamuta, and A. Motta, submitted. We report here that, in water, LAsCT forms an α-helical head-to-tail dimer in the region Leu12–Tyr22. Guanidine HCl unfolding experiments at physiological pH indicate that hydrophobic interactions are responsible for the association of all three calcitonins. The striking similarity between LAsCT and sCT strongly suggests that sCT dimerizes via the hydrophobic face of the helix using the leucines at sites 12, 16, and 19. On the contrary, hCT, in which the key leucines are substituted by aromatic residues (Scheme 1), takes up a sequence of turns in the central region. An important issue is whether aromatic amino acids in hCT have a driving role in fibril formation (5Reches M. Porat Y. Gazit E. J. Biol. Chem. 2002; 277: 35475-35480Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). Our results support this idea. The absence of a defined helix in the central region of hCT corroborates the requirement for fibrillation of specific chemical properties in a definite region of the hormone (5Reches M. Porat Y. Gazit E. J. Biol. Chem. 2002; 277: 35475-35480Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). Intermolecular collision of short ordered regions is a possible mechanism to bring together two molecules to allow chemical interaction. In particular, the presence of aromatic residues at sites 12, 16, and 19 in hCT would drive a specific orientation through hydrogen bond formation, the benzene ring acting as hydrogen bond acceptor. Such a mechanism could well explain fibril formation by short hCT-based peptides. Furthermore, leucines (but not aromatic residues) in the central region of sCT and LAsCT favor a stable prefibrillar helical dimer that prevents amyloid formation. We therefore propose that hCT fibrillation can be prevented by stabilizing a leucine-based helical dimer, which can be achieved by substituting aromatic amino acids for leucine residues at sites 12, 16, and 19. Biological support for our conclusion is given by a report showing a 20-fold increase of hCT hypocalcaemic potency, obtained by replacing aromatic amino acids by Leu residues (15Maier R. Kamber B. Riniker B. Rittel W. Clin. Endocrinol. 1976; 5 (suppl.): 327S-332SCrossref PubMed Scopus (27) Google Scholar). Finally, our finding strongly supports the hypothesis (16Kallberg Y. Gustafsson M. Persson B. Thyberg J. Johansson J. J. Biol. Chem. 2001; 276: 12945-12950Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar) that stabilization of an α-helix/β-strand-discordant stretch (i.e. an α-helix in a polypeptide segment that should form a β-strand according to secondary structure predictions) into α-helix avoids amyloid fibril formation. Peptide Synthesis—sCT, LAsCT, and hCT (a gift from Dr. Nagana A. Goud, Bachem, Torrance, CA) were prepared by standard methods. Size Exclusion Chromatography—Size exclusion chromatography was carried out at room temperature, using a 1.5 × 50-cm Sephadex G-50 Fine column at a flow rate of 0.3 ml/min. Chromatography was performed at pH 7.4, using 20 mm phosphate containing 100 mm NaCl, and at pH 3.3, using 20 mm acetate containing 100 mm NaCl. The concentrations used for hCT were as follows: 0.15, 0.36, and 1.33 mm at pH 3.3 and 0.081 and 0.31 mm at pH 7.4. The concentrations used for sCT and LAsCT were as follows: 0.40 and 0.45 mm at pH 3.3, and 0.58 mm at pH 7.4. Insulin B-chain (3.5 kDa) and bovine pancreatic tripsin inhibitor (BPTI, 6.6 kDa), both purchased from Sigma, were used as molecular weight standards. Peptide concentrations were determined by ultraviolet absorption spectroscopy using coefficients at 275 nm of 1531 and 1515 cm-1m-1 for hCT, sCT, and LAsCT, respectively (17Epand R.M. Epand R.F. Orlowski R.C. Schleuter R.J. Boni L.T. Hui S.W. Biochemistry. 1983; 22: 5074-5084Crossref PubMed Scopus (121) Google Scholar). Circular Dichroism Spectroscopy—Measurements were performed on a Jasco-J710 spectropolarimeter connected to a water bath used to control the temperature of the cell. LAsCT CD spectra were recorded in the far UV region (200–240 nm) at 295 K and pH 3.3 (20 mm acetate, 100 mm NaCl) and 7.4 (20 mm phosphate, 100 mm NaCl) with a peptide concentration of 0.072 mm in a 1.0-cm path length cell. Spectra in the presence of 0.4 m SDS, at both pHs, were acquired with a peptide concentration of 0.41 mm in a 0.1-cm path length cell. A spectral bandwidth of 2.0 nm and a scan speed of 10 nm/min were used. The precision of the data was improved by averaging five scans, and the results are reported as mean residue ellipticity (θ). Prediction of percentages of secondary structure from CD spectra was obtained using the k2d software, a Kohonen neural network with a two-dimensional output layer (18Andrade M.A. Chacón P. Morán F. Protein Eng. 1993; 6: 383-390Crossref PubMed Scopus (952) Google Scholar, 19Merelo J.J. Andrade M.A. Prieto A. Morán F. Neurocomputing. 1994; 6: 443-454Crossref Scopus (124) Google Scholar) (www.embl-heidelberg.de/∼andrade/k2d). Possible fibril formation of each peptide was monitored at 293 K with time course experiments for 24 h by following the ellipticity at 205 nm. Freshly dissolved peptide was used at different concentrations (0.030, 0.060, and 0.18 mm at pH 3.3 and 7.4, in 20 mm acetate and phosphate buffers, respectively, both containing 100 mm NaCl). CD spectra of LAsCT and sCT samples corresponding to the above concentrations, as obtained from a sample of 0.59 mm kept at room temperature, were examined after 8 months. Guanidine Denaturation—The guanidine hydrochloride denaturation studies of hCT, sCT, and LAsCT were carried out at 295 K in 100 mm phosphate buffer at pH 7.4. Mixtures of freshly prepared stock solutions of peptide, phosphate buffer, and guanidine hydrochloride were made to obtain denaturant concentration ranging from 0 to 4 m. The final peptide concentration was 0.1 mg/ml. Preliminary experiments showed that incubation of 30 min was sufficient to reach the equilibrium. Guanidine-induced unfolding was monitored by measuring ellipticity at 220 nm as a function of denaturant concentration. The CD spectrum of each sample was recorded between 210 and 230 nm in a 0.1-cm path length cell. A spectral bandwidth of 1.0 nm and a scan speed of 50 nm/min were used, and each spectrum was accumulated five times. All the spectra were corrected for the contribution of the buffer. The results are reported as mean residue ellipticity. NMR Data Collection—All CT samples were prepared by dissolving the appropriate amounts of the peptide in 0.5 ml of 1H2O, 2H2O (90/10 v/v) to yield concentrations of 0.59 mm. Deuterated water was obtained from CortecNet (Paris, France). 1H NMR spectra, recorded at 293 K and pH 3.3 and 7.4, were acquired on a Bruker DRX-500 spectrometer operating at 500 MHz using an inverse multinuclear probehead fitted with gradients along the x, y, and z axes. Spectra were referenced to sodium 3-(trimethylsilyl)-(2,2,3,3-2H4)propionate. Homonuclear two-dimensional clean TOCSY (20Griesinger C. Otting G. Wüthrich K. Ernst R.R. J. Am. Chem. Soc. 1988; 110: 7870-7872Crossref Scopus (1199) Google Scholar) and NOESY (21Jeener J. Meier B.H. Bachmann P. Ernst R.R. J. Chem. Phys. 1979; 71: 4546-4553Crossref Scopus (4857) Google Scholar) spectra were recorded by incorporating the excitation sculpting sequence (22Hwang T.-L. Shaka A.J. J. Magn. Reson. 1995; 112: 275-279Crossref Scopus (1590) Google Scholar) for water suppression. We used a pulsed-field gradient double echo with a soft square pulse of 4 ms at the water resonance frequency, with the gradient pulses of 1 ms each in duration. 512 equally spaced evolution time period t1 values were acquired, averaging 16 transients of 2048 points, with 6024 Hz of spectral width. Time domain data matrices were all zero-filled to 4096 in both dimensions, yielding a digital resolution of 2.94 Hz/point. Prior to Fourier transformation, resolution enhancement was applied with a Lorentz-Gauss window to both t1 and t2 dimensions for all the experiments. NOESY spectra were obtained with different mixing times (100, 200, 300, and 400 ms), TOCSY experiments were recorded with a spin-lock period of 64 and 96 ms, achieved with the MLEV-17 pulse sequence Structure Calculations—Distance restraints for structure calculations were obtained by integrating the volumes of NOE peaks at different mixing times and representing the buildup of the NOEs by a second-order polynomial. The corresponding interproton distances were calculated using an r-6 dependence of the initial slope. An upper bound was set for all distance restraints to 10% above the calculated distance, whereas the lower bound was set conservatively to 0.25 nm, representing a value close to van der Waals contact. For the methyl protons, a correction of 0.03 nm was made (23Koning T.M.G. Boelens R. Kaptein R. J. Magn. Reson. 1990; 90: 111-123Google Scholar). ϕ and Ψ dihedral angle restraints were derived from 3JNHα coupling constants. The structure of the monomer was calculated with 230 NOEs (109 intraresidue, 85 sequential (αCHi–NHi+1, βCHi–NHi+1, and NHi–NHi+1), and 36 medium range (αCHi–NHi+n, n ≥ 2, and αCHi–βCHi+3)). For the dimer, we used a total of 464 NOEs, which include four intermolecular NOEs defining the mutual orientation of single chains. Model building and preliminary calculations were performed with the united atom model (24Weiner S.J. Kollman P.A. Case D.A. Singh U.C. Ghio C. Alagona G. Profeta S. Weiner P. J. Am. Chem. Soc. 1984; 106: 765-784Crossref Scopus (4904) Google Scholar) with the SYBYL 6.2 package (Tripos Inc., St. Louis, MO). The solvation effects were approximated by using a distance-dependent dielectric constant ϵ = r. A cutoff radius of 0.8 nm for non-bonded interactions, with a residue-based pair list generation routine, was used for all calculations. In the united atom model, distance restraints were included as C–C or C–N distance, increasing the upper limits calculated for interproton distances by 0.1 nm. Semiparabolic penalty functions were used with force constants in energy minimization and room temperature molecular dynamics (MD) of 83.33 kJ mol-1 radians-2 for dihedral restraints and 8.33 kJ mol-1 nm-2 for distance restraints. In simulated annealing and preliminary MD simulations, a time step of 1 fs was used, with no restraints on bond length. Simulated annealing runs were performed with different lengths (from 10 to 250 ps), temperatures (maximum values from 500 to 800 K, applied for 25–75% of the total time, cooling rates from 1 to 5 K ps-1), and restraint force constant time profiles. Energy minimization and MD simulations with solvent were performed with the SANDER module of the AMBER 4.1 package. A time step of 2 fs, with rigid restraint of all bond lengths (SHAKE algorithm) (25Ryckaert J.P. Ciccotti G. Berendsen H.J.C. J. Comp. Phys. 1977; 23: 327-341Crossref Scopus (17344) Google Scholar, 26van Gunsteren W.F. Berendsen H.J.C. Mol. Physiol. 1977; 34: 1311-1327Crossref Scopus (1595) Google Scholar), and periodic boundary conditions were applied. All MD simulations were performed in the isothermal-isobaric ensemble (300 K, 1 atm), with a solvent box that initially extended 0.8 nm from the most external solute atom on each side of the box. Molecular structures were drawn and analyzed with the graphics program MOLMOL (27Koradi R. Billeter M. Wüthrich K. J. Mol. Graph. 1996; 14: 51-55Crossref PubMed Scopus (6503) Google Scholar). Size Exclusion Chromatography—The apparent molecular weights of hCT and sCT at non-fibrillating concentration were measured by gel filtration on a Sephadex G-50 column using as eluents 20 mm phosphate containing 100 mm NaCl (pH 7.4) and 20 mm acetate containing 100 mm NaCl (pH 3.3). sCT (Fig. 1A, open circles) eluted at pH 3.3 with an apparent molecular mass of 6.6 kDa (compare with BPTI, 6.6 kDa, stars) instead of 3.4 kDa as indicated by its amino acid composition. On the contrary, hCT (open squares) eluted with an apparent molecular mass corresponding to that of the insulin B-chain (3.5 kDa, crosses). Therefore, whereas sCT elutes as a dimer, hCT appears as a monomer. Both elution patterns are symmetric, as are those of the two standards, and this implies the presence of a single species in solution. At pH 7.4 (Fig. 1B), sCT shows an elution profile (filled circles) with a predominant component corresponding to the molecular weight of a dimer, with a slight tail due to the presence of a second minor component corresponding to sCT monomer. Such an asymmetry of the eluted peak is characteristic of a dissociating system (28Cunningham B.C. Mulkerrin M.G. Wells J.A. Science. 1991; 253: 545-548Crossref PubMed Scopus (212) Google Scholar), suggesting an equilibrium between the two forms strongly favoring the dimer. The elution pattern of hCT (Fig. 1B, filled squares) is asymmetric with a sharper front edge between monomer and dimer and a tail region showing a distinct second component corresponding to hCT monomer, suggestive of a slow equilibrium between the two forms. Denaturation with Guanidine Hydrochloride—Fig. 2 depicts the denaturation curves at pH 7.4 and 295 K, obtained by monitoring the hCT (filled squares) and sCT (filled circles) ellipticities at 220 nm as a function of guanidine HCl concentration. Upon addition of guanidine, we observed a reduction of ellipticity for both hormones. This is consistent with the presence of hydrophobicity-induced association for both hCT and sCT. Identical behavior was observed for sCT at pH 3.3 (not shown), supporting the relevance of hydrophobic association in the formation of the dimer at both acidic and physiological pH. LAsCT, an sCT Mutant Used to Characterize the Dimer—The possibility of characterizing the sCT dimer relies on its stability in solution. In water, at physiological conditions, CD spectra indicate that neither hCT nor sCT have significant ordered structure (29Arvinte T. Drake A.F. J. Biol. Chem. 1993; 268: 6408-6414Abstract Full Text PDF PubMed Google Scholar). The reported α-helix percentages are ∼8% for hCT and ∼14% for sCT, with ∼18% of turns. NMR NOESY experiments of hCT and sCT in water at pH 7.4 (not shown) indicated the presence of strong αCHi–NHi+1 connectivities along the whole chain of hCT and sCT, and weak NHi–NHi+1 connectivities concentrated in the 12–20 region and in the Cys1–Cys7 loop. Furthermore, we noticed the presence of scattered αCHi–NHi+2 and βCHi–NHi+1 connectivities. This finding argues for the presence of a structure fluctuating between an extended chain and a sequence of turns located in the central region of CT (30Wüthrich K. NMR of Proteins and Nucleic Acids. John Wiley & Sons, Inc., New York1986: 162-170Google Scholar). We synthesized the LAsCT mutant to lengthen and stabilize the helix. A detailed structural study by NMR spectroscopy and calculations of LAsCT in the presence of SDS2 indicated that LAsCT does have a longer (Leu4–Gly28) and more stable helix than does sCT (Thr6–Tyr22). What structure does it assume in water? Fig. 3 shows the CD spectra of LAsCT at pH 7.4 and 295 K in water (broken line) and in SDS (continuous line). Estimation of the secondary structure by the k2d neural network algorithm (18Andrade M.A. Chacón P. Morán F. Protein Eng. 1993; 6: 383-390Crossref PubMed Scopus (952) Google Scholar, 19Merelo J.J. Andrade M.A. Prieto A. Morán F. Neurocomputing. 1994; 6: 443-454Crossref Scopus (124) Google Scholar) suggests the presence of ∼20% α-helix in water. In SDS, a dominant α-helix is clearly discernable, as two minima with high ellipticity values are observed at 220 and 208 nm. We estimated the presence of 56% α-helix, significantly lower than the 75% obtained by NMR studies. Therefore, although secondary structure estimation from CD spectra is less reliable than from NMR, we think that the detected helical percentage (∼20%) indicates a sufficient stability to promote the dimer. Possible fibril formation by LAsCT was examined (with time course) at pH 3.3 and 7.4 for freshly dissolved peptide at different concentrations (0.031, 0.060, and 0.18 mm). The ellipticity at 205 nm was monitored for 24 h without any appreciable variation of the signal. We also compared the CD spectra of the samples above with corresponding concentrations derived from a sample of 0.59 mm kept at room temperature for 8 months without showing macroscopic evidence of aggregation. The spectra from the two sets are identical, so we concluded that the fibrillation time for LAsCT is a slow process, similar to that reported for sCT (8Arvinte T. Cudd A. Drake A.F. J. Biol. Chem. 1993; 268: 6415-6422Abstract Full Text PDF PubMed Google Scholar). Gel filtration experiments indicated that at pH 3.3 and 7.4, the elution patterns of LAsCT are symmetric and correspond to an apparent dimeric molecular weight. Finally, the denaturation curve at pH 7.4 is very similar to that observed for sCT, confirming the hydrophobic nature of the interactions stabilizing the dimer. Therefore, we conclude that, except for the longer helical segment, the solution behavior of LAsCT is identical to that of sCT. LAsCT Forms an Antiparallel α-Helix Dimer in Water—The NMR spectral assignment of LAsCT was carried out at pH 3.3 and pH 7.4 by the homonuclear 1H NMR approach (30Wüthrich K. NMR of Proteins and Nucleic Acids. John Wiley & Sons, Inc., New York1986: 162-170Google Scholar). Identification of the complete spin systems of all 32 residues was based on TOCSY and NOESY experiments. The peptide secondary structure was delineated from qualitative analysis of the sequential (αCHi–NHi+1 and NHi–NHi+1) and medium range (αCHi–NHi+n, 1 < n < 4, and αCHi–βCHi+3) NOEs. Fig. 4A summarizes the observed NOEs at pH 7.4 and at a concentration of 0.59 mm. The central region of the peptide shows intense NHi–NHi+1 NOEs, whereas the αCHi–NHi+1 connectivities are weaker, implying a generally helical structure (30Wüthrich K. NMR of Proteins and Nucleic Acids. John Wiley & Sons, Inc., New York1986: 162-170Google Scholar). This was clearly identified by αCHi-NHi+3, αCHi–NHi+4 and αCHi–βCHi+3 appearing in the region Leu12–Tyr22 (Fig. 4A); the lack of such connectivities at the N- and C-terminal regions suggests a less organized structure. The presence of a helix in the Leu12–Tyr22 region is also supported by 3JHNα < 6 Hz (30Wüthrich K. NMR of Proteins and Nucleic Acids. John Wiley & Sons, Inc., New York1986: 162-170Google Scholar). Although without doubt the helical nature in the 12–22 region is established via sequential and medium range NOEs, the differentiation between an α-helix and a 310-helix is, however, more difficult. Actually, the fact that αCHi–NHi+2 connectivities, not observable in an α-helix, are generally detected along the chain and in particular, in the Leu12–Tyr22 region, suggests that we have a 310-helix. On the other hand, the presence of αCHi–NHi+4 connectivities (Leu12–Leu16, Ser13–His17, His17–Thr21, and Lys18–Tyr22), not observable in a 310-helix, also demonstrates that this part of the peptide could take up an α-helix. The simultaneous presence of NOEs peculiar to α- and 310-helices can be interpreted as evidence of flexibility (31Otting G. Qian Y. Müller M. Affolter M. Gehring W. Wüthrich K. EMBO J. 1988; 7: 4305-4309Crossref PubMed Scopus (109) Google Scholar). Calculations (see below), however, indicate a preference for the α-helix. The helical wheel diagram (Fig. 4B) indicates that Leu12, Leu16, and Leu19, and Ser13, His17, and Thr21 are positioned on opposite sides of the helix, indicating an amphipatic helix whose hydrophobic face is composed of leucine residues. Three-dimensional structures of LAsCT were generated as described under “Experimental Procedures.” From the initial 60 structures, 30 were selected for further refinement. They had no violations of the upper and lower bounds of the NMR distance restraints greater than 0.10 and 0.12 nm, respectively, and did not predict any unobserved NOEs. They were subjected to restrained energy minimization using the AMBER package (see “Experimental Procedures”). The energies of these refined structures were all in the narrow range from -3,225 to -3,775 kJ·mol-1. Refinement produced a decrease of the overall energy, but the NOE restraint energies underwent a small increase upon minimization. The overall agreement among individual conformers can be seen by global root mean square (r.m.s.) deviation. The average r.m.s. deviation between the 12 best structure pairs is 0.074 ± 0.016 nm for the backbone atoms in the 9–22 region, and 0.128 ± 0.036 nm including all heavy atoms of the helical region. The average sum of all violations for these structures is 0.76 nm, whereas the average distance restraint violation is 0.0067 nm. Fig. 5A shows a superposition of the polypeptide backbone for the first 12 best structures of LAsCT: a unique backbone fold is obtained for residues 9–22, whereas lack of convergence is instead observed in the C terminus of the hormone and, to a lesser degree, in the N-terminal region. This reflects the absence of a sufficient number of NOEs, mainly due to the inherent flexibility of the molecule (14Amodeo P. Motta A. Strazzullo G. Castiglione Morelli M.A. J. Biomol. NMR. 1999; 13: 161-174Crossref PubMed Scopus (33) Google Scholar). In fact, the distribution of the observed NOEs along the LAsCT sequence indicates that the average number of constra" @default.
W2000147308 created "2016-06-24" @default.
W2000147308 creator A5053875999 @default.
W2000147308 creator A5061315099 @default.
W2000147308 date "2004-02-01" @default.
W2000147308 modified "2023-09-28" @default.
W2000147308 title "Modulating Calcitonin Fibrillogenesis" @default.
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