Amphipathic control of the 310‐/α‐helix equilibrium in synthetic peptides

Abstract: A series of short, amphipathic peptides incorporating 80% C^α,C^α‐disubstituted glycines has been prepared to investigate amphipathicity as a helix‐stabilizing effect. The peptides were designed to adopt 3₁₀‐ or α‐helices based on amphipathic design of the primary sequence. Characterization by circular dichroism spectroscopy in various media (1 : 1 acetonitrile/water; 9 : 1 acetonitrile/water; 9 : 1 acetonitrile/TFE; 25 mm SDS micelles in water) indicates that the peptides selectively adopt their designed conformation in micellar environments. We speculate that steric effects from ith and ith + 3 residues interactions may destabilize the 3₁₀‐helix in peptides containing amino acids with large side‐chains, as with 1‐aminocyclohexane‐1‐carboxylic acid (Ac₆c). This problem may be overcome by alternating large and small amino acids in the ith and ith + 3 residues, which are staggered in the 3₁₀‐helix.     

Structures of neuropeptide γ from goldfish and mammalian neuropeptide γ, as determined by 1H NMR spectroscopy

Abstract: Neuropeptide γ belongs to tachykinin families which have a common C‐terminal amino acid sequence (Phe‐X‐Leu‐Met‐NH₂) and which induce various biological responses including salivation, hypotension, and contraction of gastrointestinal, respiratory, and urinary smooth muscle. In the present study, we present the solution structures of neuropeptide γ (NPγ) from gold fish (G‐NPγ) and mammalian NPγ (M‐NPγ), as determined by nuclear magnetic resonance (NMR) spectroscopy in 50% trifluoroethanol (TFE)/water (1 : 1, v/v) solution and 200 mm sodium dodecyl sulfate (SDS) micelles. In aqueous TFE solution, G‐NPγ has a α‐helical conformation in the region of His¹²–Met²¹ and a short helix in the N‐terminal region, and has a β‐turn from Arg⁹ to Arg¹¹ in between. In aqueous TFE solution, M‐NPγ also has α‐helical conformations both in the C‐terminal region and the N‐terminal region and a β‐turn from His⁹ to Arg¹¹ in between. In SDS micelle, the structure of G‐NPγ contains a stable α‐helix from His¹² to Met²¹ and a β‐turn from Arg⁹ to Arg¹¹, while M‐NPγ has a short helix from Ser¹⁶ to Met²¹. The region from His¹² to Met²¹ corresponds to the amino acid sequence of neurokinin A. Neuropeptide γ may act as a precursor of neurokinin A and the post‐translational processing of this peptide involves the enzymatic attack of the basic β‐turn region from residue 9 to residue 11 in the middle. From our relaxation study, it could be suggested that in fish system G‐NPγ induces the biological actions corresponding to those of substance P in mammalian system. The structures of G‐NPγ and M‐NPγ contain α‐helical structures at the C‐terminus and this helix seems to promote the affinity for NK₁ and/or NK₂ receptor.     

Antimicrobial Properties of Brevinin‐2‐Related Peptide and its Analogs: Efficacy Against Multidrug‐Resistant Acinetobacter baumannii

Brevinin‐2 related peptide (B2RP; GIWDTIKSMG¹⁰KVFAGKILQN²⁰L.NH₂), first isolated from skin secretions of the mink frog Lithobates septentrionalis, shows broad‐spectrum antimicrobial activity but its therapeutic potential is limited by moderate hemolytic activity. The peptide adopts an α‐helical conformation in a membrane‐mimetic solvent but amphipathicity is low. Increasing amphipathicity together with hydrophobicity by the substitutions Lys¹⁶→Leu and Lys¹⁶→Ala increased hemolytic activity approximately fivefold without increasing antimicrobial potency. The substitution Leu¹⁸→Lys increased both cationicity and amphipathicity but produced decreases in both antimicrobial potency and hemolytic activity. In contrast, increasing cationicity of B2RP without changing amphipathicity by the substitution Asp⁴→Lys resulted in a fourfold increase in potency against Escherichia coli [minimal inhibitory concentration (MIC) = 6 μm ) and twofold increases in potency against Staphylococcus aureus (MIC = 12.5 μm ) and Candida albicans (MIC = 6 μm ) without changing significantly hemolytic activity against human erythrocytes (LC₅₀ = 95 μm ). The emergence of antibiotic‐resistant strains of the Gram‐negative bacterium Acinetobacter baumannii constitutes a serious risk to public health. B2RP (MIC = 3–6 μm ) and [Lys⁴]B2RP (MIC = 1.5–3 μm ) potently inhibited the growth of nosocomial isolates of multidrug‐resistant Acinetobacter baumannii. Although the analogs [Lys⁴, Lys¹⁸]B2RP and [Lys⁴, Ala¹⁶, Lys¹⁸]B2RP showed reduced potency against Staphylococcus aureus, they retained activity against Acinetobacter baumannii (MIC = 3–6 μm ) and had very low hemolytic activity (LC₅₀ > 200 μm ).     

Relationship between the tertiary structures of mastoparan B and its analogs and their lytic activities studied by NMR spectroscopy

Abstract: Mastoparan B (MP‐B), an antimicrobial cationic tetradecapeptide amide isolated from the venom of the hornet Vespa basalis, is an amphiphilic α‐helical peptide. MP‐B possesses a variety of biological activities, such as mast cells degradation histamine release, erythrocyte lysis and inhibition of the growth of Gram‐positive and Gram‐negative bacteria. In order to study the relationship between the structure and the biological activity of MP‐B, we used four analogs by replacing amino acids with alanine. Tertiary structures of MP‐B and its analogs in 2,2,2‐trifluoroethanol (TFE)‐containing aqueous solution have been determined by NMR spectroscopy and molecular modeling. The results indicate that [Ala⁴]MP‐B and [Ala¹²]MP‐B with higher hydrophobicity adopt a higher content of amphiphilic helical structures, and have better antimicrobial and hemolytic activities than MP‐B. However, [Ala³]MP‐B and [Ala⁹]MP‐B with lower hydrophobicity have disordered structures. [Ala³]MP‐B and [Ala⁹]MP‐B have low antimicrobial activity and much less hemolytic activity relative to MP‐B. It is likely that tryptophan residue in MP‐B and appropriate hydrophobicity of MP‐B to induce α‐helical structure is essential for the antibacterial and hemolytic activity of MP‐B. This study can aid understanding of the structure–activity relationship of MP‐B and to design peptides to possess lytic activity.     

Effects of Na2SO4 on hydrophobic and electrostatic interactions between amphipathic α‐helices

Abstract: The effects of 2 molal Na₂SO₄ at neutral pH on hydrophobic and electrostatic interactions between amphipathic α‐helices were investigated by circular dichroism spectroscopy. The amphipathic peptides that were studied included LEK (acetyl‐ L EE L KKK L EE L KKK L EE L ‐NH₂) and LEE (acetyl‐ L EE L EEE L EE L EEE L EE L ‐NH₂). In phosphate buffer at neutral pH, only LEK adopted a predominantly α‐helical conformation, attributable to glu^––lys⁺ interactions where a major contribution is evidently a hydrogen bond (Biochemistry 32 : 9668–9676). Despite the presence of lys⁺ in the e and g′ positions of the abcdefg heptad repeat, LEK exhibited mean‐residue ellipticities at 222 nm ([θ]₂₂₂) which were dependent on peptide concentration, indicating the presence of a coiled coil. In the presence of 2 molal Na₂SO₄ at 25–75°C, the helical content of LEK increased, with the greatest increase observed at 75°C. The value of the ellipticity ratio R ([θ]₂₂₂/[θ]₂₀₈) of LEK in 2 molal Na₂SO₄ also increased, indicating a stronger interhelical association. At 50°C and 75°C, LEK remained predominantly α‐helical. In phosphate buffer at neutral pH, LEE was mainly random coil. In the presence of 2 molal Na₂SO₄, however, the peptide formed α‐helices that associated to form a coiled coil. At 50°C and 75°C, LEE became predominantly random coil but the remaining α‐helices were still associating. These results are consistent with the strengthening of interhelical hydrophobic interactions and the absence of screening of helix‐stabilizing and helix‐destabilizing electrostatic interactions in amphipathic α‐helices by Na₂SO₄.     

Antibacterial,antitumor and hemolytic activities of α‐helical antibiotic peptide,P18 and its analogs

Abstract: The α‐helical antibiotic peptide (P18: KWKLFKKIPKFLHLAKKF‐NH₂) designed from the cecropin A(1–8)–magainin 2 (1?12) hybrid displayed strong bactericidal and tumoricidal activity without inducing hemolysis. The effect of the Pro⁹ residue at central position of P18 on cell selectivity was investigated by Pro⁹ → Leu or Pro⁹ → Ser substitution. Either substitution markedly reduced the antibacterial activity of P18 and increased hemolysis, although it did not significantly affect cytotoxicity against human transformed tumor and normal fibroblast cells. These results suggest that a proline kink in α‐helical antibiotic peptide P18 serves as a hinge region to facilitate ion channel formation on bacterial cell membranes and thus plays an important role in providing high selectivity against bacterial cells. Furthermore, to investigate the structure?antibiotic activity relationships of P18, a series of N‐ or C‐terminal deletion and substitution analogs of P18 were synthesized. The C‐terminal region of P18 was related to its antibiotic activity and α‐helical conformation on lipid membranes rather than N‐terminal one. Higher α‐helicity of the peptides was involved in the hemolytic and antitumor activity rather than antibacterial activity. Except for [L⁹]‐P18 and [S⁹]‐P18, all the designed peptides containing a Pro residue showed potent antibacterial activity, although they did not induce a cytolytic effect against human erythrocyte and normal fibroblast cells at the concentration required to kill bacteria. In particular, P18 and some analogs (N‐1, N‐2, N‐3, N‐3L and N‐4L) with potent bactericidal and tumoricidal activity and little or no normal cell toxicity may serve as an attractive candidate for the development of novel anti‐infective or antitumor agents.     

Three‐dimensional solution structure of a disulfide bond isomer of the human insulin‐like growth factor‐I

Abstract: The solution structure of a disulfide bond isomer of human insulin‐like growth factor‐I (IGF‐I) was determined using homonuclear NMR methods. A total of 292 interatomic distance constraints, including 12 related to the disulfide bridges, was used in the distance geometry calculations. The determined structures contain two helical rods corresponding to the sequence regions, Ala⁸–Cys¹⁸ and Leu⁵⁴–Cys⁶¹. Comparison with the previously determined structure of native human IGF‐I revealed partial correspondence of the secondary structure (helices I: Ala⁸–Cys¹⁸ and helices III: Leu⁵⁴–Cys⁶¹) and internal packing. Helix II in native human IGF‐I (residues Gly⁴²–Cys⁴⁸) is disrupted in the isomer. A similar relationship has been described between the structure of native insulin and a homologous disulfide isomer, suggesting that these alternative folds represent general features of insulin‐like sequences. In each case the precision of the distance geometry ensemble is low due in part to resonance broadening and a paucity of NOEs relative to other globular proteins of this size. These observations suggest that tertiary structure of the isomer is not highly ordered. Comparison of the biological activities of native and the disulfide bond isomer of human IGF‐I highlight the importance of Tyr²⁴, Phe²⁵, Phe⁴⁹–Cys⁵² and Phe¹⁶ in binding to the IGF‐I receptor or specific IGFBPs. The relationship of this proposed receptor‐binding surface of human IGF‐I to those of insulin is discussed.     

Synthesis of 17O isotope labeled Leu-enkephalin and 17O n.m.r.*

Oxygen-17 isotope was introduced into the α-carboxyl group of glycine, 1-phenylalanine, 1-leucine and 1-tyrosine by acid catalyzed exchange of ¹⁷O from H₂¹⁷O or by acid hydrolysis of respective amino acid methyl esters in H₂¹⁷O. Quantitative enrichment of glycine was achieved by acid hydrolysis of amino acetonitrile in H₂¹⁷O. For α-amino protection in amino acids t-butoxycarbonyl (Boc) group was employed for ¹⁷O labeled enkephalin synthesis. Five analogues of Leu-enkephalins (I–V) labeled with ¹⁷O at different amino acid residues were synthesized by solid phase method. ¹⁷O n.m.r. spectra were measured at 24.4 and 67.8 MHz for Leu-enkephalins ¹⁷O labeled at Gly² and Phe⁴ positions. A downfield shift was observed for ¹⁷O labeled Gly² Leu-enkephalin upon heating. This shift is indicative of the rupture of intramolecular hydrogen bonds. The preliminary results confirm the hypothesis that an intramolecular hydrogen bond exists between the carbonyl group of Gly² and NH group of Leu⁵.     

Conformational analysis by NMR and distance geometry techniques of a peptide mimetic of the third helix of the Antennapedia homeodomain*

Abstract: The Antennapedia homeodomain structure consists of four helices. The helices II and III are connected by a tripeptide that forms a turn, and constitute the well‐known helix‐turn‐helix motif. The recognition helix penetrates the DNA major groove, gives specific protein–DNA contacts and forms direct, or water‐mediated, intermolecular hydrogen bonds. It was suggested that helix III (and perhaps also helix IV) might represent the recognition helix of Antennapedia homeodomain, which makes contact with the surface of the major groove of the DNA. In an attempt to clarify the helix III capabilities of assuming an helical conformation when separated from the rest of the protein, we carried out the structural determination of the recognition helix III in different solvent media. The conformational study of fragments 42–53, where residues W48 and F49, not involved in the protein–DNA interaction, were substituted by two alanines, was conducted in sodium dodecyl sulfate (SDS), trifluoroethanol (TFE) and TFE/water, using circular dichroism, nuclear magnetic resonance (NMR) and distance geometry (DG) techniques. The fragment assumes a well‐defined secondary structure in TFE and in TFE/water (90/10, v/v) with an α‐helix encompassing residues 4–9, while in TFE/water (70/30, v/v) a less regular structure was found. The DG results in the micellar system evidence the presence of a distorted α‐helical conformation involving residues 4–8. Our results reveal that the isolated Antennapedia recognition helix III tend to preserve in solution the α‐helical conformation even if separated from the rest of the molecule.     

NMR structure of a minimum activity domain of human parathyroid peptide hormone: structural origin of receptor activation

Abstract: Parathyroid hormone (PTH) which increases osteoblast numbers and bone formation by activating bone‐lining cells to osteoblasts plays an important role in calcium and phosphate homeostasis and bone remodeling by activating PTH receptors. To determine the structural origin of a minimum activity domain of hPTH, we initiated a detailed structural determination of the hPTH^H14 in aqueous solution using NMR spectroscopy. Circular dichroism and NMR data demonstrated that hPTH^H14 maintains a typical helical conformation in both membrane‐mimicking environments and 30% TFE solution. The solution structure clearly showed that the residues from Ser³ to Leu¹¹ of hPTH^H14 formed a stable helical structure, especially having charged side‐chains oriented in opposite directions relative to one another for optimum interaction with the receptor molecule.     

NMR and CD conformational studies of the C‐terminal 16‐peptides of Pseudomonas aeruginosa c551 and Hydrogenobacter thermophilus c552 cytochromes

Abstract: The 16‐amino acid sequences of the C‐terminal helices of the homologous bacterial cytochromes c₅₅₁ from Pseudomonas aeruginosa and c₅₅₂ from Hydrogenobacter thermophilus were synthesized and their solution structure studied. Circular dichroism and NMR experiments in aqueous solution have shown the presence of α‐helices and 3₁₀‐helices. The populations of helical structures in phosphate buffer, pH 3.5, 293 K, were 21% for c₅₅₁ and 20% for c₅₅₂, but increased to 56.7 and 48%, respectively, in 50% aqueous 2,2,2‐trifluoroethanol. An isodichroic point was observed at 203 nm in CD spectra for the helix/coil transition in mixtures of water/2,2,2‐trifluoroethanol. NMR spectra in phosphate buffer show the presence of both α‐ and 3₁₀‐helical structures. In water/2,2,2‐trifluoroethanol (50 : 50) α‐helices are predominant. CD temperature‐dependency studies indicate that both peptides exhibit the same cooperativity for the transition in water/2,2,2‐trifluoroethanol (50 : 50). The experimental data show that the amino acid substitutions do not favor heat resistance of the secondary structure of the c₅₅₂ C‐terminal helix at the local level. Instead, they optimize nonlocal contacts of the polypeptide chain, which stabilize the tertiary structure in the native protein.     

Synthesis and immunological studies of α‐conotoxin chimera containing an immunodominant epitope from the 268–284 region of HSV gD protein

Abstract: We have synthesized and characterized new chimeric peptides by inserting an epitope of the glycoprotein D (gD) of herpes simplex virus (HSV) serotype 1 as ‘guest’ sequence in the ‘host’ structure of α‐conotoxin GI, a 13‐residue peptide (ECCNPACGRHYSC) isolated from the venom of Conus geographus. The 276–284 region of HSV gD‐1 selected for these studies is highly hydrophilic and adopts a β‐turn. The α‐conotoxin GI also contains a β‐turn in the 8–12 region, stabilized by two disulfide bridges at positions 2–7 and 3–13. Thus, the tetramer sequence of α‐conotoxin, ⁸Arg‐His‐Tyr‐Ser¹² has been replaced by Asp‐Pro‐Val‐Gly (DPVG), identified previously as the epitope core. The syntheses were performed by Fmoc strategy on Rink resin and DTNB or air oxidation were applied for the formation of the first 3–13 disulfide bond in the presence of guanidinium hydrochloride. For the formation of the second disulfide Cys²‐Cys⁷ three different oxidation procedures [iodine in 95% acetic acid, air oxidation in dimethyl sulfoxide/1 m HCl or Tl(tfa)₃ in trifluoroacetic acid (TFE)] were compared. The high‐performance liquid chromatography purified peptides were characterized by electrospray mass spectrometry and amino acid analysis. The bicyclic HSV‐α‐[Tyr¹]‐conotoxin chimeric peptide and native α‐conotoxin GI showed similar circular dichroism spectra in phosphate‐buffered saline (PBS) and in a PBS‐TFE 1 : 1 (v/v) mixture, which might suggest that these compounds also share similar secondary structures. In immunologic studies the characteristics of the primary and of the memory immunoglobulin (Ig) M‐ and IgG‐type antibody responses showed that the bicyclic HSV‐α‐[Tyr¹]‐conotoxin chimera is capable to induce strong antibody responses in C57/Bl/6 mice but was poorly immunogenic in CBA and BALB/c mice. Data obtained with the C57/Bl/6 serum indicate that the polyclonal antibodies recognize the DPVG motif presented in the bicyclic HSV‐α‐[Tyr¹]‐conotoxin and some reactivity was also found with the monocyclic but not with the linear form of the chimera. Results with two IgM type monoclonal antibodies from a bicyclic HSV‐α‐[Tyr¹]‐conotoxin immunized C57/Bl/6 mouse also point to the specific interaction with the DPVG sequence. Taken together these studies suggest, that the relative intensity of DPVG‐specific responses was found to be dependent on the mouse strain and on the conformation of the chimeric molecules. We found that the IgM monoclonal antibodies are able to recognize the linear DPVG sequence, while the majority of IgG antibodies is directed to the same motif in a conformation stabilized by double cyclization.     

Importance of the leucine side-chain to the spasmogenic activity and binding of Substance P analogues

Previous studies with Substance P (SP) antagonists (GR 71251, [d Pro⁹, Pro¹⁰, Trp¹¹]SP and d Pro⁹, MeLeu¹⁰, Trp¹¹]SP) have suggested the existence in the guinea-pig ileum (GPI) of two distinct tachykinin receptors associated with the contractile responses of [Pro⁹]SP and septide. In addition [Apa^9-10]SP, a glycine-substituted analogue of SP with a carba bond between residues 9 and 10, [Gly⁹-ψ(CH₂-CH₂)-Gly¹⁰SP = [Apa^9-10]SP, was shown to belong to the ‘septide family’ (low affinity for NK-1 specific binding sites and high potency in the GPI). In order to establish the importance of the isopropyl side-chain in position 10, the binding potencies and activities of [Gly⁹-ψ(CH₂-CH₂)-Gly¹⁰]SP, [Ala¹⁰]SP, [Gly⁹-ψ(CH₂-CH₂)-Leu¹⁰]SP and [Gly⁹-ψ(CH₂-CH₂)-d Leu¹⁰]SP were compared. Conformational behaviour of active peptides with a carba bond was analyzed by NMR and modelisation studies. This study with agonists demonstrated that undecapeptides substituted in position 10 in the SP sequence also enabled discrimination of NK-1 receptors from receptors responsible for the spasmogenic activities of peptides belonging to the ‘septide family’. [Gly⁹-ψ(CH₂-CH₂)- Leu¹⁰]SP is ahighly potent NK-1 agonist, [Gly⁹-ψ(CH₂-CH₂)-Gly¹⁰]SP acts on the septide-sensitive receptor, and [Ala¹⁰]SP is a mixed agonist.     

Specificity of 17β-oestradiol and benzo[α]pyrene oxidation by polymorphic human cytochrome P4501B1 variants substituted at residues 48, 119 and 432

1. Eight human cytochrome P4501B1 (CYP1B1) allelic variants, namely Arg⁴⁸Ala¹¹⁹Leu⁴³², Arg⁴⁸Ala¹¹⁹Val⁴³², Gly⁴⁸Ala¹¹⁹Leu⁴³², Gly⁴⁸Ala¹¹⁹Val⁴³², Arg⁴⁸Ser¹¹⁹Leu⁴³², Arg⁴⁸Ser¹¹⁹Val⁴³², Gly⁴⁸Ser¹¹⁹Leu⁴³² and Gly⁴⁸Ser¹¹⁹Val⁴³² (all with Asn⁴⁵³), were expressed in Escherichia coli together with human NADPH-P450 reductase and their catalytic specificities towards oxidation of 17β-oestradiol and benzo[α]pyrene were determined. 2. All of the CYP1B1 variants expressed in bacterial membranes showed Fe²⁺·CO versus Fe²⁺ difference spectra with wavelength maxima at 446nm and they reacted with antibodies raised against recombinant human CYP1B1 in immunoblots. The ratio of expression of the reductase to CYP1B1 in these eight preparations ranged from 0.2 to 0.5. 3. CYP1B1 Arg⁴⁸ variants tended to have higher activities for 17β-oestradiol 4-hydroxylation than Gly⁴⁸ variants, although there were no significant variations in 17β-oestradiol 2-hydroxylation activity in these eight CYP1B1 variants. Interestingly, ratios of formation of 17β-oestradiol 4-hydroxylation to 2-hydroxylation by these CYP1B1 variants were higher in all of the Val⁴³² forms than the corresponding Leu⁴³² forms. 4. In contrast, Leu⁴³² forms of CYP1B1 showed higher rates of oxidation of benzo[α]pyrene (to the 7,8-dihydoxy-7,8-dihydrodiol in the presence of epoxide hydrolase) than did the Val⁴³² forms. 5. These results suggest that polymorphic human CYP1B1 variants may cause some altered catalytic specificity with 17β-oestradiol and benzo[α]pyrene and may influence susceptibilities of individuals towards endogenous and exogenous carcinogens.     

Importance of the C‐terminal phenylalanine of gastrin for binding to the human CCK2 receptor

Abstract: The importance of the C‐terminal Phe of gastrin and structural requirements at position 17 for binding to the human CCK₂ receptor were assessed using analogs of [Leu¹⁵]G(11?17). The following peptides were synthesized, Ac[Leu¹⁵]G(11?17), Ac[Leu¹⁵]G(11?16)NH₂, [Leu¹⁵]G(11?17), [Leu¹⁵,Ala¹⁷]G(11?17), [Leu¹⁵,Abu¹⁷]G(11?17), [Leu¹⁵,Val¹⁷]G(11?17), [Leu¹⁵,Leu¹⁷]G(11?17), [Leu¹⁵,Cha¹⁷]G(11?17), [Leu¹⁵,Trp¹⁷]G(11?17), [Leu¹⁵,Tic¹⁷]G(11?17), [Leu¹⁵, d ‐Phe¹⁷]G(11?17) and [Leu¹⁵,p‐X‐Phe¹⁷]G(11?17), where X = F, Cl, Br, I, OH, CH₃, NH₂ and NO_2. Competition binding experiments with [³H]CCK‐8 were performed using human CCK₂ receptors stably expressed in CHO cells. Phe¹⁷ was shown to be important for binding. A hydrophobic side‐chain larger than Leu is required at position 17 but aromaticity does not appear to be essential. Constraint of the aromatic side‐chain either in the g(+) or g(–) conformation, as in the case of Tic, results in a significant decrease in affinity. In addition, the peptide conformation induced by incorporation of d ‐Phe decreases binding. The size and electron withdrawing/donating properties of the para substituent are not important for interaction with the receptor. The current study shows that the use of des‐Phe analogs of gastrin is not a viable strategy for development of antagonists for the human CCK₂ receptor.     

Conformational studies of a potent Leu11, D-Trp12-containing lactam-bridged parathyroid hormone-related protein-derived antagonist

Human parathyroid hormone-related protein (PTHrP) is expressed in various tissues where it acts as an endocrine/paracrine factor involved in cellular growth, differentiation and development of fetal skeleton. As for parathyroid hormone (PTH), which is the hormone responsible for regulation of extracellular calcium homeostasis, the N-terminal 1-34 fragment can reproduce the full spectrum of calciotropic activities inherent in full-length PTH. Truncation of six amino acid residues from the N-terminus of both hormone sequences generates 7-34 fragments which act as weak antagonists. Although PTH(7-34) is a pure antagonist, PTHrP(7-34) acts as partial agonist against the receptor shared by both hormones, the PTH/PTHrP receptor. In the current study, we analyzed the conformation of [Leu¹¹,d -Trp¹²,Lys²⁶,Asp³⁰]PTHrP(7-34)NH₂ (hybrid-lactam) in a 1:1 mixture of H₂O/TFE-d₃ at pH 4 by circular dichroism, nuclear magnetic resonance and distance geometry calculations. This weak antagonist (K_b= 650 nm ) combines two modifications: Leu¹¹,d -Trp¹² (K_b= 5.1 nm ), reported to eliminate partial agonism and enhance potency, and Lys²⁶-Asp³⁰ lactamization (K_b= 31 nm ), aimed to stabilize the helical structure of the principal binding domain attributed to residues 25-34. The helical content in 30% trifluoroethanol is 88%, i.e., higher than the corresponding linear analog, and comprises the d -Trp¹²-Thr³³ segment. This hybrid lactam contains a rigid helical segment spanning the 14-18 sequence followed by a hinge motif around Arg^19-20, but the sequence 14-18 forms a stable helix. In all potent lactam-containing, PTHrP-derived agonists and antagonists studied so far, the dominant structural motif consists of two helical domains at the two ends of the sequence and of two hinge regions centered around Gly¹²-Lys¹³ and Arg¹⁹. The weakly active agonists and antagonists do not exhibit the “hinge” around position 19. These findings suggest that the presence and location of discrete hinge regions that connect the N- and C-terminal helices are essential for generating the bioactive conformation of ligands for the PTH/ PTHrP receptor.     

Determination of stereochemistry stability coefficients of amino acid side‐chains in an amphipathic α‐helix

Abstract: We describe here a systematic study to determine the effect on secondary structure of d ‐amino acid substitutions in the nonpolar face of an amphipathic α‐helical peptide. The helix‐destabilizing ability of 19 d ‐amino acid residues in an amphipathic α‐helical model peptide was evaluated by reversed‐phase HPLC and CD spectroscopy. l ‐Amino acid and d ‐amino acid residues show a wide range of helix‐destabilizing effects relative to Gly, as evidenced in melting temperatures (ΔT_m) ranging from ?8.5°C to 30.5°C for the l ‐amino acids and ?9.5°C to 9.0°C for the d ‐amino acids. Helix stereochemistry stability coefficients defined as the difference in T_m values for the l ‐ and d ‐amino acid substitutions [(ΔT_m′ = T_mL and T_mD)] ranging from 1°C to 34.5°C. HPLC retention times [Δt_R(X_L?X_D)] also had values ranging from ?0.52 to 7.31 min at pH 7.0. The helix‐destabilizing ability of a specific d ‐amino acid is highly dependent on its side‐chain, with no clear relationship to the helical propensity of its corresponding l ‐enantiomers. In both CD and reversed‐phase HPLC studies, d ‐amino acids with β‐branched side‐chains destabilize α‐helical structure to the greatest extent. A series of helix stability coefficients was subsequently determined, which should prove valuable both for protein structure‐activity studies and de novo design of novel biologically active peptides.     

The solution structure of uperin 3.6, an antibiotic peptide from the granular dorsal glands of the Australian toadlet,Uperoleia mjobergii

Abstract: Uperin 3.6 (GVIDA⁵AKKVV¹⁰NVLKN¹⁵LF-NH₂) is a wide-spectrum antibiotic peptide isolated from the Australian toadlet, Uperoleia mjobergii. With only 17 amino acid residues, it is smaller than most other wide-spectrum antibiotic peptides isolated from amphibians. In 50% (by vol.) trifluoroethanol, an NMR study and structure calculations indicate that uperin 3.6 adopts a well-defined amphipathic α-helix with distinct hydrophilic and hydrophobic faces. Examination of the activities of synthetic modifications of uperin 3.6 reveal that the three lysine residues are essential for antibiotic activity.