Biologically active calcitonin analogs which have minimal interactions with phospholipids

A number of peptide hormones have been shown to contain amphipathic helical segments capable of binding to phospholipids. This conformational feature has been associated with increased biological activity of these hormones. We demonstrate, however, that two calcitonin analogs, [Gly8,Ala16]-des-Leu19 salmon calcitonin and des-1-amino-[Ala1,7,Gly8]-des-Leu19 salmon calcitonin have minimal interactions with phospholipids. Neither of these peptides acquire any increased helical content in the presence of dimyristoylphosphatidylglycerol and these peptides have only weak effects in altering the phase transition properties of this lipid. Therefore, although the presence of a phospholipid-induced amphipathic helical sequence may enhance the biological activity, it is not required for activity.     

鲑鱼降钙素及其类似物的合成

鲑鱼降钙素及其类似物的合成潘和平王良友陈正英王芳王会信（军事医学科学院基础医学研究所,北京１００８５０）ＳｙｎｔｈｅｓｉｓｏｆＳａｌｍｏｎＣａｌｃｉｔｏｎｉｎａｎｄＩｔｓＡｎａｌｏｇｓＰａｎＨｅ－ＰｉｎｇＷａｎｇＬｉａｎｇ－ＹｏｕＣｈｅｎＺｈｅｎｇ－...     

Presence of an amphipathic helical segment and its relationship to biological potency of calcitonin analogs

The conformational properties of a number of calcitonin analogs were studied by circular dichroism. The ability of dimyristoylphosphatidylglycerol, lysophosphatidylcholine or sodium dodecyl sulfate to induce the formation of more highly ordered structures in these peptides was also assessed by circular dichroism. In all cases sodium dodecyl sulfate induced the largest change in the circular dichroism spectra of the peptides. Salmon calcitonin and its analogs were slightly more helical in the presence of the anionic phospholipid than in the presence of the zwitterionic detergent lysophosphatidylcholine while the reverse is true for human calcitonin and its analogs. Some of the calcitonin analogs convert turbid suspensions of phosphatidylglycerol to a clear solution from which the phospholipid is no longer readily sedimentable by centrifugation. Several of the physical properties of these peptides could be correlated with their biological activity. Generally peptides which showed no hypocalcemic activity had the least negative mean residue ellipticities at 222 nm. Only biologically active analogs were able quantitatively to solubilize dimyristoyl-phosphatidylglycerol and in this solubilized form the peptides have a higher helical content. More active derivatives exhibit larger increases in helix content in the presence of this phospholipid. Inactive analogs had the least negative mean residue ellipticities at 222 nm in the presence of lysophosphatidylcholine or sodium dodecyl sulfate. Thus, the ability of a calcitonin analog to form structures of higher helical content in the presence of amphiphiles is a requirement for the analog to exhibit high potency in assays of biological activity.     

Functional relationship between receptor binding and biological activity for analogs of mammalian and salmon gonadotropin-releasing hormones in the pituitary of goldfish (Carassius auratus)

The relationship between gonadotropin-releasing hormone (GnRH) receptor binding and biological activity in the goldfish pituitary for mammalian and salmon GnRH (sGnRH) analogs with structural modification at the C terminus involving replacement of glycine amide with an alkyl amine and replacement of the Gly6 residue with D amino acids was examined. The GnRH receptor binding data were analyzed with a computerized curve-fitting program (LIGAND) for a single as well as two classes of binding sites; analysis based on one site fit estimated binding affinity and capacity for one class of binding site, and analysis based on two-site fit estimated binding affinity and capacity for two classes of binding sites (high-affinity/low-capacity and low-affinity/high-capacity binding sites). The estimated receptor affinity values were then used to determine the correlation between binding affinity and gonadotropin (GTH)-release potency in vitro. The highest correlation between biological activity and receptor binding affinity was obtained for the high-affinity/low-capacity binding sites and GnRH analogs containing Trp7 and Leu8 residues (i.e., the salmon GnRH structural format) (R = 0.940 +/- 0.150). For the same group of GnRH analogs, there was no significant correlation between the relative GTH-release potency and binding affinity of the low-affinity/high-capacity sites (R = 0.159 +/- 0.434), or that obtained from a one-site fit (R = 0.198 +/- 0.431). Similarly, for mammalian GnRH analogs, significant correlation between binding affinity and biological activity (R = 0.406 +/- 0.049) was only obtained for the high-affinity sites, although the degree of correlation was significantly lower than that obtained for salmon GnRH analogs. The present findings provide strong support for the hypothesis that high-affinity GnRH receptors are involved in the control of GTH release in the goldfish pituitary. In addition, the results demonstrate clearly that the presence of Trp7, Leu8 residues in salmon GnRH molecule, a native peptide in goldfish, is important for recognition of the ligand by the GnRH receptors in the goldfish pituitary, and that structural modifications at positions 6 and 10 in this peptide can increase receptor binding affinity and biological activity at the pituitary level. The most active sGnRH analog identified to date is [D-Arg6, Pro9-NEt]-sGnRH.     

The hydrophobic moment of the amphipathic helix of salmon calcitonin and biological potency

The formation of an amphipathic helix in the central portion of calcitonin contributes to the potency of this hormone. We have synthesized a number of analogs of salmon calcitonin, containing deletions in the region of the peptide which is thought to form an amphipathic helix. There is no direct relationship between the hydrophobic moment of the helix and the biological activity of the peptide. For example, salmon des-Leu19-calcitonin and des-Ser13-calcitonin both have lower helical hydrophobic moments but have greater or equal biological potency compared with the native hormone. We suggest that other conformational features, such as flexibility and helix-forming potential, are also important in determining biological potency.     

Inhibition of starfish oocyte maturation by leupeptin analogs, potent trypsin inhibitors

Inhibition of subsite-substituted leupeptin analogs, potent trypsin inhibitors, on 1-methyladenine-induced germinal vesicle breakdown was investigated in a starfish, Asterina pectinifera. Of benzyloxycarbonyl(Z)-Leu-P2-argininals, the analog with Ser at P2 residue was the strongest inhibitor, and those with Pro, Leu greater than Thr greater than Gly were followed in this order. In Z-P3-Ser-argininals, ranking of the inhibitory ability was as follows: Phe greater than Leu much greater than Pro greater than Ala at P3 residue. Among 11 analogs synthesized, Z-Phe-Ser-argininal showed the strongest inhibition. The inhibitory potency of the analog was 100-fold stronger than that of leupeptin (acetyl-Leu-Leu-argininal). Thus, trypsin-like enzyme possessing a narrow subsite specificity participates in oocyte maturation in the starfish.     

Study of a series of analogs of salmon calcitonin in which alanine replaces leucine

Leucine residues at positions 12, 16 and 19 of salmon calcitonin were systematically replaced by alanine either one, two or three at a time. Substitution of Ala at positions 12 or 16 had the greatest effect on the structure of the peptide and on the ability of the peptide to attain structures of higher helical content in the presence of lipid. Despite the similar effects on the conformational properties, the effects on activity are markedly different for analogs with Ala substitutions at positions 12 and 16. The Ala12 derivatives are much less active while the Ala16 derivatives are slightly more active than the parent hormone. In contrast to the effects of substitutions at these positions, substitution of Ala at position 19 has relatively little effect on activity or on conformation. These results demonstrate that different regions of the calcitonin molecule have different conformational requirements for maximal activity.     

Structure–activity study of macropin,a novel antimicrobial peptide from the venom of solitary bee Macropis fulvipes (Hymenoptera: Melittidae)

A novel antimicrobial peptide, designated macropin (MAC‐1) with sequence Gly‐Phe‐Gly‐Met‐Ala‐Leu‐Lys‐Leu‐Leu‐Lys‐Lys‐Val‐Leu‐NH₂, was isolated from the venom of the solitary bee Macropis fulvipes. MAC‐1 exhibited antimicrobial activity against both Gram‐positive and Gram‐negative bacteria, antifungal activity, and moderate hemolytic activity against human red blood cells. A series of macropin analogs were prepared to further evaluate the effect of structural alterations on antimicrobial and hemolytic activities and stability in human serum. The antimicrobial activities of several analogs against pathogenic Pseudomonas aeruginosa were significantly increased while their toxicity against human red blood cells was decreased. The activity enhancement is related to the introduction of either l ‐ or d ‐lysine in selected positions. Furthermore, all‐d analog and analogs with d ‐amino acid residues introduced at the N‐terminal part of the peptide chain exhibited better serum stability than did natural macropin. Data obtained by CD spectroscopy suggest a propensity of the peptide to adopt an amphipathic α‐helical secondary structure in the presence of trifluoroethanol or membrane‐mimicking sodium dodecyl sulfate. In addition, the study elucidates the structure–activity relationship for the effect of d ‐amino acid substitutions in MAC‐1 using NMR spectroscopy. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Solution structure and biological activity of recombinant salmon calcitonin S-sulfonated analog

Salmon calcitonin S-sulfonated analog (abbreviated as [S-SO(3)(-)]rsCT) was prepared by introducing two sulfonic groups into the side chains of Cys1 and Cys7 of recombinant salmon calcitonin. The hypocalcemic potency of this open-chain analog is 5500IU/mg, which is about 30% higher than that (4500IU/mg) of the wild type. The solution conformation of [S-SO(3)(-)]rsCT was studied in aqueous trifluoroethanol solution by CD, 2D-NMR spectroscopy, and distance geometry calculations. In the mixture of 60% TFE and 40% water, the peptide assumes an amphipathic alpha-helix in the region of residues 4-22, which is one turn longer than that of the native sCT. The structural feature analysis of the peptide revealed the presence of hydrophobic surface composed of five hydrophobic side chains of residues Leu4, Leu9, Leu12, Leu16, and Leu19, and a network of salt-bridges that consisted of a tetrad of oppositely charged side chains (Cys7-SO(3)(-)-Lys11(+)-Glu15(-)-Lys18(+)). The multiple salt bridges resulted in the stabilization of the longer amphipathic alpha-helix. Meanwhile, the higher hypocalcemic potency of the peptide could be attributed to the array of hydrophobic side chains of five leucine residues of the amphipathic alpha-helix.     

Spatial proximity between a photolabile residue in position 19 of salmon calcitonin and the amino terminus of the human calcitonin receptor

Calcitonins are 32-amino acid peptide hormones with both peripheral and central actions mediated via specific cell surface receptors, which belong to the class II subfamily of G protein-coupled receptors. Understanding receptor function, particularly in terms of ligand recognition by calcitonin receptors, may aid in the rational design of calcitonin analogs with increased potency and improved selectivity. To directly identify sites of proximity between calcitonin and its receptor, we carried out photoaffinity labeling studies followed by protein digestion and mapping of the radiolabeled photoconjugated receptor. A fully active salmon calcitonin analog [Arg(11,18),Bpa19]sCT, incorporating a photolabile p-benzoyl-L-phenylalanine into position 19 of the ligand, has been used to demonstrate spatial proximity between residue 19 of the peptide and the amino-terminal extracellular domain of the receptor. Cyanogen bromide cleavage together with endoproteinase Asp-N digestion indicated that binding was predominantly to the region delimited by receptor residues Cys134 and Met187. Binding to this fragment was supported further by cyanogen bromide-digestion of receptors that were mutated to remove the predicted cleavage site at Met133 (M133A, M133L). Binding within the 54-amino acid fragment was refined further by digestion with endoproteinase Lys-C to the 8-amino acid region corresponding to Cys134-Lys141. These results provide the first direct demonstration of a contact domain between salmon calcitonin and its receptor and will contribute toward modeling of the calcitonin-receptor interface.     

Effects of lipid structure on peptide-lipid interactions. Complexes of salmon calcitonin with phosphatidylglycerol and with phosphatidic acid

The interactions of salmon calcitonin with a number of phospholipids are studied by electron microscopy, circular dichroism and the leakage of carboxyfluorescein. At room temperature, calcitonin reacts strongly with dimyristoylphosphatidylglycerol and egg phosphatidic acid, while only moderate or no interaction is observed with several other phospholipids. The interaction is judged by the dissolution of the phospholipid dispersion and by electron microscopic observation and is in general concomitant with an increase in the helical content of the peptide. The electrostatic charge and the transition temperature of each of the phospholipids are important factors in determining the extent of reaction with salmon calcitonin. An exception is the sulphatide from bovine brain. The resulting morphology of the complex formed between salmon calcitonin and phosphatidic acid is quite different from that formed with phosphatidylglycerol. In the case of phosphatidylglycerol and most other negatively charged phospholipids, disc-shaped complexes are observed under the electron microscope by negative staining. The calcitonin- DMPG complexes are about 7 nm thick and their diameter increases with an increasing lipid-to-peptide ratio. In contrast, phosphatidic acids form spherical complexes with salmon calcitonin causing large multilamellar structures to spontaneously break-up into smaller particles of about 10 to 20 nm in diameter independent of the lipid-to-peptide ratio. The contrasting effects of salmon calcitonin on the morphology of these two phospholipids is explicable by consideration of the size of the lipid headgroup. Phosphatidic acid can accommodate the peptide without rupture of the bilayer, while the larger headgroup of phosphatidylglycerol requires the bilayer to rupture. This model is supported by studies of calcitonin-induced leakage of carboxyfluorescein from sonicated vesicles of 75% egg phosphatidylcholine and 25% either egg phosphatidic acid, egg phosphatidylglycerol or dimyristoylphosphatidylglycerol . There was a much greater increase in carboxyfluorescein leakage from phosphatidylglycerol-containing vesicles induced by salmon calcitonin demonstrating the greater ability of the peptide to rupture bilayers containing this phospholipid.     

Role of residue 478 as a determinant of the substrate specificity of cytochrome P450 2B1.

Two allelic variants and eight site-directed mutants of cytochrome P450 2B1 differing at residue 478 have been expressed in COS cells and assayed for androstenedione hydroxylase activities. The 478Gly and 478Ala variants and five mutants (Ser, Thr, Val, Ile, and Leu) exhibited 16 beta-OH:16 alpha-OH ratios ranging from 0.7 to 9.3, whereas the Pro, Glu, and Arg mutants were expressed but inactive. The seven samples active toward androstenedione also exhibited testosterone 16 beta-OH:16 alpha-OH ratios ranging from 0.4 to 2.3. With both steroids, the Gly variant had the highest 16 beta-hydroxylase activity, and the 16 beta-OH:16 alpha-OH ratio increased with the size of aliphatic size chains (Ala, Val, and Ile/Leu). The highest ratio of androgen 15 alpha:16-hydroxylation was observed with the Ser mutant. On the basis of previous work indicating decreased susceptibility of the 478Ala variant in liver microsomal and reconstituted systems to inactivation by chloramphenicol analogs, methodology was refined for monitoring enzyme inactivation in COS cell microsomes. The Gly and Ala variants were inactivated by chloramphenicol with similar rate constants, whereas the Ser and Val mutants were inactivated more slowly, and the Leu mutant was refractory. Only the Gly variant was inactivated by the chloramphenicol analog N-(2-p-nitrophenethyl)chlorofluoroacetamide. Thus, the side chain of residue 478 appears to be a major determinant of enzyme inactivation as well as of androgen hydroxylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and biological activity of linear and cyclic enkephalins modified at the Gly3-Phe4 amide bond

As part of our continuing effort to define structure-activity relationships for enkephalin and design enzymatically resistant analogs, we report the synthesis and biological activities of linear and cyclic enkephalin analogs modified at the Gly3-Phe4 amide bond. The partial retro-inverso enkephalin analog Tyr-D-Ala-gGly-(R,S)-mPhe-Leu-NH2 and its cyclic counterpart, Tyr-cyclo[D-A2 bu-gGly-(R,S)-mPhe-Leu-], were synthesized as diastereomeric mixtures using solution methodology. The racemic benzylmalonate allowed the linear analog to be synthesized by fragment coupling at the reversed bond. Cyclization of the second analog was carried out at high concentration, eliminating formation of polymer by the use of an insoluble base. All gem-diaminoalkyl residues were prepared by conversion of peptidyl amides with benzene iodonium bis(trifluoroacetate). Diastereomers of both compounds were separable by reverse phase HPLC but those of the linear compound racemized rapidly under conditions of testing and were therefore tested together. All analogs tested had activities ranging from 6 to 14% of the activity of Leu enkephalin, indicating that the Gly3-Phe4 amide bond is important, though not crucial, for receptor binding.     

Effects of lipid structure on peptide-lipid interactions complexes of salmon calcitonin with phosphatidylglycerol and with phosphatidic acid

The interactions of salmon cacitonin with a number of phospholipids are studied by electron microscopy, circular dichroism and the leakage of carboxyfluorescein. At room temperature, calcitonin reacts strongly with dimyristoylphosphatidylglycerol and egg phosphatidic acid, while only moderate or no interaction is observed with several other phospholipids. The interaction is judged by the dissolution of the phospholipid dispersion and by electron microscopic observation and is in general concomitant with an increase in the helical content of the peptide. The electrostatic charge and the transition temperature of each of the phospholipids are important factors in determining the extent of reaction with salmon calcitonin. An exception is the sulphatide from bovine brain. The resulting morphology of the complex formed between salmon calcitonin and phosphatidic acid is quite different from that formed with phosphatidylglycerol. In the case of phosphatidylglycerol and most other negatively charged phospholipids, disc-shaped complexes are observed under the electron microscope by negative staining. The calcitonin-DMPG complexes are about 7 nm thick and their diameter increases with an increasing lipid-to-peptide ratio. In contrast, phosphatidic acids form spherical complexes with salmon calcitonin causing large multilamellar structures to spontaneously break-up into smaller particles of about 10 to 20 nm in diameter independent of the lipid-to-peptide ratio. The contrasting effects of salmon calcitonin on the morphology of these two phospholipids is explicable by consideration of the size of the lipid headgroup. Phosphatidic acid can accommodate the peptide without rupture of the bilayer, while the larger headgroup of phosphatidylglycerol requires the bilayer to rupture. This model is supported by studies of calcitonin-induced leakage of carboxyfluorescein from sonicated vesicles of 75% egg phosphatidylcholine and 25% either egg phosphatidic acid, egg phosphatidylglycerol or dimyristoylphosphatidylglycerol. There was a much greater increase in carboxyfluorescein leakage from phosphatidylglycerol-containing vesicles induced by salmon calcitonin demonstrating the greater ability of the peptide to rupture bilayers containing this phospholipid.     

Antimicrobial activity of leucine‐substituted decoralin analogs with lower hemolytic activity

Linear cationic α‐helical antimicrobial peptides are promising chemotherapeutics. Most of them act by different mechanisms, making it difficult to microorganisms acquiring resistance. Decoralin is an example of antimicrobial peptide; it was described by Konno et al. and presented activity against microorganisms, but with pronounced hemolytic activity. We synthesized leucine‐substituted decoralin analogs designed based on important physicochemical properties, which depend on the maintenance of the amphiphilic α‐helical tendency of the native molecule. Peptides were synthesized, purified, and characterized, and the conformational studies were performed. The results indicated that the analogs presented both higher therapeutic indexes, but with antagonistic behavior. While [Leu]¹⁰‐Dec‐NH₂ analog showed similar activity against different microorganisms (c.a. 0.4–0.8 μmol L^?1), helical structuration, and some hemolytic activity, [Leu]⁸‐Dec‐NH₂ analog did not tend to helical structure and presented antimicrobial activities two orders higher than the other two peptides analyzed. On the other hand, this analog showed to be the less hemolytic (MHC value = 50.0 μmol L^?1). This approach provided insight for understanding the effects of the leucine substitution in the amphiphilic balance. They led to changes on the conformational tendency, which showed to be important for the mechanism of action and affecting antimicrobial and hemolytic activities. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Structure-activity studies on cysteine-substituted neurokinin A analogs.

A complete series of analogs of tyrosine modified neurokinin A ([Tyr1]-NKA or [Tyr0]-NKA) has been synthesized by substituting each natural residue with 1-Cys. These analogs were tested for their ability to bind recombinant neurokinin-2 (NK-2) receptor. Substitution of Phe6 with Cys completely abolished binding of the analog to the receptor. Substitution of residues in the carboxyl-terminal region of the peptide (Met10, Leu9, Gly8, Val7) and Asp4 with Cys gave reductions in binding affinity of between 23- and 250-fold. Molecular dynamics simulations of these analogs suggest that changes in peptide structure and flexibility are not large contributors to the losses in receptor binding affinity. Reductions in binding affinity are therefore more confidently ascribed to losses of peptide-receptor interactions.     

NMR investigations of structural and dynamics features of natively unstructured drug peptide – salmon calcitonin: implication to rational design of potent sCT analogs

Backbone dynamics and conformational properties of drug peptide salmon calcitonin have been studied in aqueous solution using nuclear magnetic resonance (NMR). Although salmon calcitonin (sCT) is largely unfolded in solution (as has been reported in several circular dichroism studies), the secondary H^α chemical shifts and three bond H^N–H^α coupling constants indicated that most of the residues of the peptide are populating the α‐helical region of the Ramachandran (?, ψ) map. Further, the peptide in solution has been found to exhibit multiple conformational states exchanging slowly on the NMR timescale (10²–10³ s^?1), inferred by the multiple chemical shift assignments in the region Leu4–Leu12 and around Pro23 (for residues Gln20–Tyr22 and Arg24). Possibly, these slowly exchanging multiple conformational states might inhibit symmetric self‐association of the peptide and, in part, may account for its reduced aggregation propensity compared with human calcitonin (which lacks this property). The ¹⁵N NMR‐relaxation data revealed (i) the presence of slow (microsecond‐to‐millisecond) timescale dynamics in the N‐terminal region (Cys1–Ser5) and core residues His17 and Asn26 and (ii) the presence of high frequency (nanosecond‐to‐picosecond) motions in the C‐terminal arm. Put together, the various results suggested that (i) the flexible C‐terminal of sCT (from Thr25–Thr31) is involved in identification of specific target receptors, (ii) whereas the N‐terminal of sCT (from Cys1–Gln20) in solution – exhibiting significant amount of conformational plasticity and strong bias towards biologically active α‐helical structure – facilitates favorable conformational adaptations while interacting with the intermembrane domains of these target receptors. Thus, we believe that the structural and dynamics features of sCT presented here will be useful guiding attributes for the rational design of biologically active sCT analogs. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

The C region of human insulin-like growth factor (IGF) I is required for high affinity binding to the type 1 IGF receptor

We have produced and characterized the binding properties of three structural analogs of human insulin-like growth factor I (hIGF-I). These analogs are [1-62]hIGF-I, an analog lacking the carboxyl-terminal 8-amino acid D region of hIGF-I; [1-27, Gly4, 38-70]hIGF-I, an analog in which residues 28-37 of the C region of hIGF-I are replaced by a 4-reside glycine bridge; and [1-27,Gly4,38-62]hIGF-I, an analog with the C region glycine replacement and a D region deletion. The removal of the D region of hIGF-I has little effect on binding to the type 1 and type 2 insulin-like growth factor (IGF) receptors. [1-62]hIGF-I has 2-fold higher affinity for the insulin receptor and 4-fold higher affinity for IGF serum-binding proteins. The replacement of the C region of hIGF-I with a four-glycine span results in a 30-fold loss of affinity for the type 1 IGF receptor. However this analog has near normal affinity for the type 2 IGF receptor, the insulin receptor, and IGF serum-binding proteins. Incorporating the C region glycine replacement and the D region deletion into one analog does not affect binding to either the type 2 receptor or to IGF serum-binding proteins. As predicted from the single deletion analogs [1-27,Gly4,38-62]hIGF-I has reduced affinity for the type 1 IGF receptor (approximately 40-fold) and increased affinity for the insulin receptor (5-fold). These data indicate that determinants in the C region of hIGF-I are involved in maintaining high affinity binding to the type 1 IGF receptor and that neither the C region nor the D region are required for high affinity binding to the type 2 IGF receptor or to IGF serum-binding proteins.     

Modulating calcitonin fibrillogenesis: an antiparallel alpha-helical dimer inhibits fibrillation of salmon calcitonin

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 alpha-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.     

Conformational and biological properties of partial sequences of salmon calcitonin

According to the Chou-Fasman rules for predicting the secondary structures of proteins, the 12-20 portion of salmon calcitonin should adopt an alpha helical conformation. These residues would form an amphipathic helix and contribute to the solubilization of certain phospholipids by the peptide. Circular dichroism was used to assess the extent that peptide segments of salmon calcitonin fold into structures of higher helical content in the presence of dimyristoylphosphatidylglycerol, lysolecithin or sodium dodecyl sulfate. All of the segments studied are carboxyl terminal amides as is the native, intact, salmon calcitonin. Salmon calcitonin segments 11-23 or 12-23 form no more helical structure in the presence of lipids or detergents than does a segment comprising the hydrophilic carboxyl terminal residues 22-32 which is not predicted to adopt a helical conformation. Even a larger segment containing residues 12-32 does not exhibit a great increase in helical content in the presence of lipids or detergents, and it causes only a small broadening of the phase transition of dimyristoylphosphatidylglycerol. In contrast, a preparation with an equivalent molar ratio of dimyristoylphosphatidylglycerol to the salmon calcitonin segment 1-23 exhibits a very marked broadening of the phase transition, similar to what is found with the 32 amino acid native hormone. This amino terminal segment also adopts a conformation of higher helical content than even the intact hormone. This 1-23 segment is the only one studied that showed significant interaction with lipids, and it is also the only one which exhibited any hypocalcemic activity.(ABSTRACT TRUNCATED AT 250 WORDS)