Human calcitonin (hCT) has been investigated by NMR at 400 MHz in DMSOd6 and in an 85% DMSOd6-15% 1H2O (v/v) cryoprotective mixture. All backbone and side-chain resonances have been assigned, and the secondary structure has been determined in both solvents. In DMSOd6, the simultaneous presence of d alpha N, dNN, and some specific weak medium-range nuclear Overhauser effects, together with the amide temperature coefficients and the analysis of the NH-alpha CH spin-spin coupling constants, indicates that hCT is highly flexible but with three domains (comprising segments Asn3-Gly10, Gln14-Thr21, and Thr25-Ala31) in extended conformations which dynamically transform into isolated beta turns in the N- and C-terminal regions and into adjacent tight turns, resembling a 3(10) helix structure, in the central part. The DMSO-water mixture rigidifies the polypeptide chain, favoring an ordered, extended conformation. NOESY data indicate the presence of a short double-stranded antiparallel beta sheet in the central region made by residues 16-21 and connected by a two-residue hairpin loop formed by residues 18 and 19. Two tight turns, formed by residues 3-6 and 28-31, were also identified. The central beta sheet does not favor an amphipathic distribution of the residues as found for salmon calcitonin [Motta, A., Castiglione Morelli, M. A., Goud, N., & Temussi, P. A. (1989) Biochemistry 28, 7998-8002]. This is in agreement with the smaller tendency of hCT to form the amphipathic alpha helix, postulated to be responsible for the interaction of hCT with lipids. The possible role of the cis-trans isomerism of Pro is discussed.

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The pentapeptide Boc-Val-delta Phe-Gly-delta Phe-Val-OME, containing two dehydro-phenylalanine (delta Phe) residues, has been synthesized and its structure investigated. In the crystalline state, the molecule adopts a right-handed 3(10)-helical conformation stabilized by two intramolecular hydrogen bonds between CO of Val1 and NH of delta Phe4, and between CO of delta Phe2 and NH of Val5, respectively. NMR measurements are consistent with the presence of 3(10)-helical structures also in acetonitrile and dimethylsulphoxide solution: the distances between backbone protons estimated from NOE connectivities are in overall agreement with those observed in the solid state; the chemical shifts of the amide protons show the smaller temperature coefficients for the NHs that in solid state are involved in intramolecular hydrogen bonds. The CD spectra in acetonitrile, chloroform, methanol and dimethylsulphoxide display exciton couplets of bands corresponding to the delta Phe electronic transition at 280 nm; the sign of the bands is consistent with the presence of helical structures having a prevalent left-handed screw sense. Addition of 1,1,1,3,3,3-hexafluoro-propan-2-ol gives rise to the gradual appearance of a couplet of opposite sign, suggesting the helix reversal from left-handed sense to right-handed sense. The conformational behaviour is discussed on the basis of the specific sequence of the peptide.

Tuftsin, a linear tetrapeptide (Thr-Lys-Pro-Arg), corresponding to the sequence 289-292 of the heavy chain of leukokinin, has been the object of intensive SAR studies during the past 30 years, owing to its numerous biological activities and to the possibility of generating a novel anticancer drug. A cyclic tuftsin analogue, c-[T-K-P-R-G], has biological activity 50 times higher than that of the parent linear peptide. Here we present a conformational study of c-[T-K-P-R-G] based on NMR data in a cryoprotective DMSO/water mixture. The preferred conformation is a type VIa turn centered on the K-P residues. The orientation of the side chains of the two basic residues (K and R) may represent the essential feature of the bioactive conformation of tuftsin. A possible role of tuftsin as a DNA binding motif is suggested by the similarity of the bioactive conformation of c-[T-K-P-R-G] and of the beta-turn conformation proposed by Suzuki for the [T,S]-P-K-R motif.

trifluoroacetic acid to remove the protecting group. The same treatments as above gave the monomer trifluoroacetate.The monomer salt was dissolved in DMF or Me2SO at the concentration listed in Table II. To the solution was added with shaking 1.2 equiv of triethylamine. The system of high concentration of the monomer salt became immediately complete gel. In High-Resolution Nmr of Random dl Copolypeptides 831 this case the polymerization was allowed without shaking. The system of lower concentration of the monomer salt kept a liquid state throughout polycondensation. After the polymerization the solid or the viscous liquid was triturated with ACN and the polymer obtained was filtered, extracted repeatedly by methanol, washed with diethyl ether, and dried.