Fragment analogs as better mimics of obestatin

Obestatin is a twenty three amino acid peptide produced in the stomach by post translational modification of the preproghrelin gene. Since its discovery in 2005, many studies have shown that obestatin reduces feed intake and gain in body weight in rodents. Studies from our laboratory have shown the N-terminal thirteen residues mimic obestatin the best and residues 6–18 reduce epididymal fat significantly in adult male mice. In this study we have tried to increase the efficacy of these fragments. As an initial step, we have substituted G(8) with α-aminoisobutyricacid(Aib,U) and F(5) with cyclohexylalanine(Cha) in the N-terminal peptide to obtain two modified peptides and modified the middle fragment (residues 6–18) by substituting both the glycine residues at position 3 and 8 with α-aminoisobutyricacid(U). The rationale being, unusual amino acids could protect the peptides from immediate degradation and Aib would also induce secondary structure in these unstructured peptides. The N-terminal fragment with the G(8)U substitution fared the best. It reduced food intake, gain in body weight, levels of cholesterol and triglycerides in the blood, epididymal and perirenal fat in adult male mice similar to that of obestatin. The middle fragment with G(3,8)U double substitution was the second best.     

Fragments of obestatin as modulators of feed intake, circulating lipids, and stored fat

Obestatin, shown to reduce feed intake and gain in body weight in rodents, is a very attractive candidate to be used against obesity. In this study, we aimed to investigate the relationship between the primary structure and activity of obestatin. Also of interest to us is a peptide of minimal length that closely mimics obestatin. Towards the same, we synthesized rodent obestatin and three overlapping fragments spanning residues 1-13, 6-18, and 11-23 of obestatin. These peptides subsequent to purification and characterization were tested upon adult male mice for their ability to reduce feed intake and gain in body weight. The N-terminal peptide (residues 1-13) mimicked obestatin the closest. The middle fragment (residues 6-18) significantly reduced epididymal fat without much altering feed intake or body weight.     

Synthesis, activity on NK-3 tachykinin receptor and conformational solution studies of scyliorhinin II analogs modified at position 16.

Two analogs of a tachykinin family peptides - scyliorhinin II (ScyII): [Aib(16)]ScyII and [Sar(16)]ScyII were synthesized by the solid-phase method using Fmoc chemistry. Conformational studies in water and DMSO-d(6) on these peptides were performed using a combination of two-dimensional NMR and theoretical conformational analysis. The solution structure of the peptides studied is interpreted as an equilibrium of several conformers with different statistical weights. The structure of [Sar(16)]ScyII in water appeared to be more flexible, especially in the C-terminal fragment. A better defined structure for this analog was obtained in DMSO-d(6), in which the analysis resulted in a family of conformers with similar shapes. Some of these conformers were characterized by the presence of a 3(10)-helix in the N-terminal fragment and middle part of the molecule. The introduction of the Aib residue in position 16 significantly rigidifies the structure. For [Aib(16)]ScyII in both solvent systems very similar populations of conformations were obtained which are characterized by the presence of a 3(10)-helix in the 13-18 fragment. A common structural motif was found in conformationally constrained Cys(7)-Cys(13) fragment, which resembles the Greek letter 'omega'. The differences in the solution structure of the C-terminal fragment of the peptides studied are responsible for their specificity. [Aib(16)]ScyII showed 25% the agonistic activity of selective NK-3 agonist - senktide, but it also showed antagonist effect vs. this peptide, whereas [Sar(16)]ScyII appeared to be a full agonist of NK-3 tachykinin receptor.     

Structure-function relationship studies of bovine parathyroid hormone [bPTH(1-34)] analogues containing alpha-amino-iso-butyric acid (Aib) residues

The N-terminal 1-34 fragments of the parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) elicit the full spectrum of bone-related biological activities of the intact native sequences. It has been suggested that the structural elements essential for bioactivity are two helical segments located at the N-terminal and C-terminal sequences, connected by hinges or flexible points around positions 12 and 19. In order to assess the relevance of the local conformation around Gly(12) upon biological function, we synthesized and characterized the following PTH(1-34) analogues containing Aib residues: (I) A-V-S-E-I-Q-F-nL-H-N-Aib-G-K-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(11), Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (II) A-V-S-E-I-Q-F-nL-H-N-L-Aib-K-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(12),Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (III) A-V-S-E-I-Q-F-nL-H-N-L-G-Aib-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(13), Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (IV) A-V-S-E-I-Q-F-nL-H-N-Aib-Aib-K-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-YNH(2) ([Nle(8,18), Aib(11,12), Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (V) A-V-S-E-I-Q-F-nL-H-N-L-Aib-Aib-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(12,13),Nal(23),Tyr(34)]bPTH(1-34)-NH(2)). (nL= Nle; Nal= L-(2-naphthyl)-alanine; Aib= alpha-amino-isobutyric acid.) The introduction of Aib residues at position 11 in analogue I or at positions 11 and 12 in analogue IV resulted in a 5-20-fold lower efficacy and a substantial loss of binding affinity compared to the parent compound [Nle(8,18), Nal(23),Tyr(34)]bPTH(1-34)-NH(2). Both binding affinity and adenylyl cyclase stimulation activity are largely restored when the Aib residues are introduced at position 12 in analogue II, 13 in analogue III, and 12-13 in analogue V. The conformational properties of the analogues in aqueous solution containing dodecylphosphocholine micelles were studied by CD, two-dimensional (2D) NMR and computer simulations. The results indicated the presence of two helical segments in all analogues, located at the N-terminal and C-terminal sequences. Insertion of Aib residues at positions 12 and 13, or of Aib dyads at positions 11-12 and 12-13, enhances the stability of the N-terminal helix of all analogues. In all analogues the Aib residues are included in the helical segments. These results confirmed the importance of the helical structure in the N-terminal activation domain, as well as of the presence of the Leu(11) hydrophobic side chain in the native sequence, for PTH-like bioactivity.     

Enhanced Protein Thermostability by Ala --> Aib Replacement

The introduction into peptide chains of alpha-aminoisobutyric acid (Aib) has proven to stabilize the helical structure in short peptides by restricting the available range of polypeptide backbone conformations. In order to evaluate the potential stabilizing effect of Aib at the protein level, we have studied the conformational and stability properties of Aib-containing analogs of the carboxy-terminal subdomain 255-316 of thermolysin. Previous NMR studies have shown that this disulfide-free 62-residue fragment forms a dimer in solution and that the global 3D structure of each monomer (3 alpha-helices encompassing residues 260-274, 281-295, and 301-311) is largely coincident with that of the corresponding region in the X-ray structure of intact thermolysin. The Aib analogs of fragment 255-316 were prepared by a semisynthetic approach in which the natural fragment 255-316 was coupled to synthetic analogs of peptide 303-316 using V8-protease in 50% (v/v) aqueous glycerol [De Filippis, V., and Fontana, A. (1990) Int. J. Pept. Protein Res. 35, 219-227]. The Ala residue in position 304, 309, or 312 of fragment 255-316 was replaced by Aib, leading to the singly substituted fragments Ala304Aib, Ala309Aib, and Ala312Aib. Moreover, fragment Ala304Aib/Ala309Aib with a double Ala --> Aib exchange in positions 304 and 309 was produced. Far- and near-UV circular dichroism measurements demonstrated that both secondary and tertiary structures of the natural fragment 255-316 are fully retained upon Ala --> Aib substitution(s). Thermal unfolding measurements, carried out by recording the ellipticity at 222 nm upon heating, showed that the melting temperatures (Tm) of analogs Ala304Aib and Ala309Aib were 2.2 and 5.4 °C higher than that of the Ala-containing natural species (Tm = 63.5 °C), respectively, whereas the Tm of the Ala312Aib analog was lowered by -0.6 °C. The enhanced stability of the Ala304Aib analog can be quantitatively explained on the basis of a reduced backbone entropy of unfolding due to the restriction of the conformational space allowed to Aib in respect to Ala, while the larger stabilization observed for the Ala309Aib analog can be accounted for by both entropic and hydrophobic effects. In fact, whereas Ala304 is a surface residue, Ala309 is shielded from the solvent, and thus the enhanced stability of fragment Ala309Aib is also due to the burial of an additional -CH3 group with respect to the natural fragment. The slightly destabilizing effect of the Ala --> Aib exchange in position 312 appears to derive from unfavorable strain energy effects, since phi and psi values for Ala312 are out of the allowed angles for Aib. Of interest, the simultaneous incorporation of Aib at positions 304 and 309 leads to a significant and additive increase of +8 °C in Tm. The results of this study indicate that the rational incorporation of Aib into a polypeptide chain can be a general procedure to significantly stabilize proteins.     

New tools for the control of peptide conformation and supramolecular chemistry: crown-carrier, C(alpha)-methyl L-DOPA amino acids

The preferred conformation of five, terminally protected, model peptide series to the hexamer level, based on three novel crowned, C(alpha)-methyl L-DOPA amino acids combined with either L-Ala/Aib or Gly/Aib, were assessed in structure supporting solvents using FT-IR absorption, (1)H NMR, and CD techniques. The FT-IR absorption spectra strongly suggest that the contribution of the crowned C(alpha)-tetrasubstituted residue to intramolecular H-bonding is equivalent to that of Aib and is much more significant than that of either L-Ala or Gly. In addition, the (1)H NMR titrations and the CD patterns resemble those typically exhibited by (right-handed) 3(10)-helical structures.     

Structure determination and conformational change induced by tyrosine phosphorylation of the N-terminal domain of the alpha-chain of pig gastric H+/K+-ATPase

It has been well established that phosphorylation is an important reaction for the regulation of protein functions. In the N-terminal domain of the alpha-chain of pig gastric H(+)/K(+)-ATPase, reversible sequential phosphorylation occurs at Tyr 10 and Tyr 7. In this study, we determined the structure of the peptide involving the residues from Gly 2 to Gly 34 of pig gastric H(+)/K(+)-ATPase and investigated the tyrosine phosphorylation-induced conformational change using CD and NMR experiments. The solution structure showed that the N-terminal fragment has a helical conformation, and the peptide adopted two alpha-helices in 50% trifluoroethanol (TFE) solvent, suggesting that the peptide has a high helical propensity under hydrophobic conditions. Furthermore, the CD and NMR data suggested that the structure of the N-terminal fragment becomes more disordered as a result of phosphorylation of Tyr 10. This conformational change induced by the phosphorylation of Tyr 10 might be an advantageous reaction for sequential phosphorylation and may be important for regulating the function of H(+)/K(+)-ATPase.     

Synthesis and structural studies of new analogues of PTH(1–11) containing Cα-tetra-substituted amino acids in position 8

The N-terminal 1–34 fragment of parathyroid hormone (PTH) is fully active in vitro and in vivo and it can reproduce all biological responses characteristic of the native intact PTH. Recently, analogues of PTH(1–11) fragments with helicity-enhancing substitutions have been demonstrated to yield potent analogues of PTH(1–34). The work describes the synthesis, biological activity and structure of analogues of the best modified PTH sequence H-Aib-Val-Aib-Glu-Ile-Gln-Leu-Nle-His-Gln-Har-NH₂ (I). In particular, the effect of the Ala/Aib substitution at positions 1 and 3 as well as of the replacement of Nle in position 8 with d-Nle, l-(αMe)-Nle and d-(αMe)-Nle was studied. The resulting peptides were characterized structurally by CD spectroscopy, solution NMR and MD, and in vitro for activity with respect to the cognate receptor, parathyroid hormone receptor.     

Synthesis and biological activity of nociceptin/orphanin FQ analogues substituted in position 7 or 11 with Calpha,alpha-dialkylated amino acids

Previous structure-activity and NMR studies on nociceptin/orphanin FQ (N/OFQ) demonstrated that Aib substitution of Ala(7) and/or Ala(11) increases the peptide potency through an alpha helix structure induction mechanism. On these bases we synthesised and evaluated pharmacologically in the mouse vas deferens assay a series of N/OFQ-NH(2) analogues substituted in position 7 and 11 with Calpha,alpha-disubstituted cyclic, linear and branched amino acids. None of the 20 novel N/OFQ analogues produced better results than [Aib(7)]N/OFQ-NH(2). Thus, this substitution was combined with other chemical modifications known to modulate peptide potency and/or efficacy generating compound 21 [Nphe(1)Aib(7)Arg(14)Lys(15)]N/OFQ-NH(2) (coded as UFP-111), compound 22 [(pF)Phe(4)Aib(7)Arg(14)Lys(15)]N/OFQ-NH(2) (UFP-112) and compound 23 [Phe(1)Psi(CH(2)-NH)Gly(2)(pF)Phe(4)Aib(7)Arg(14)Lys(15)]N/OFQ-NH(2) (UFP-113). These novel peptides behaved as highly potent NOP receptor ligands showing full (UFP-112) and partial (UFP-113) agonist and pure antagonist (UFP-111) activities in a series of in vitro functional assays performed on pharmacological preparations expressing native as well as recombinant NOP receptors.     

Solution structure of a cathelicidin-derived antimicrobial peptide, CRAMP as determined by NMR spectroscopy.

CRAMP was identified from a cDNA clone derived from mouse femoral marrow cells as a member of cathelicidin-derived antimicrobial peptides. This peptide shows potent antimicrobial activity against gram-positive and gram-negative bacteria but no hemolytic activity against human erythrocytes. CRAMP was known to cause rapid permeabilization of the inner membrane of Escherichia coli. In this study, the structure of CRAMP in TFE/H2O (1 : 1, v/v) solution was determined by CD and NMR spectroscopy. CD spectra showed that CRAMP adopts a mainly alpha-helical conformation in TFE/H2O solution, DPC micelles, SDS micelles and liposomes, whereas it has a random structure in aqueous solution. The tertiary structure of CRAMP in TFE/H2O (1 : 1, v/v), as determined by NMR spectroscopy, consists of two amphipathic alpha-helices from Leu4 to Lys10 and from Gly16 to Leu33. These two helices are connected by a flexible region from Gly11 to Gly16. Previous analysis of series of fragments composed of various portion of CRAMP revealed that an 18-residue fragment with the sequence from Gly16 to Leu33 was found to retain antibacterial activity. Therefore, the amphipathic alpha-helical region from Gly16 to Leu33 of CRAMP plays important roles in spanning the lipid bilayers as well as its antibiotic activity. Based on this structure, novel antibiotic peptides having strong antibiotic activity, with no hemolytic effect will be developed.     

Impromptu Effects of Nt8U with Soyasaponins on Obesity-related Lipid Parameters in High Fat Fed C57BL/6 Mice

International Journal of Peptide Research and Therapeutics - Nt8U is a peptide analog of the N-terminal 13 residue fragment of the satiety peptide obestatin with Gly (8) being replaced by...     

The crystal structure of Afc-containing peptides

A systematic structural analysis of Afc (9-amino-fluorene-9-carboxylic acid) containing peptides is here reported. The crystal structures of four fully protected tripeptides containing the Afc residue in position 2: Z-X(1)-Afc(2)-Y(3)-OMe (peptide a: X = Y = Gly; peptide b: X = Aib, C(alpha, alpha)-dimethylglycine, Y = Gly; peptide c: X = Gly, Y = Aib; peptide d: X = Y = Aib) have been solved by x-ray crystallography. All the results suggest that the Afc residue has a high propensity to assume an extended conformation. In fact, the Afc residue adopts an extended conformation in three peptides examined in this paper (peptides a-c). In contrast, Afc was found in a folded conformation, in the 3(10)-helical region, only in the peptide d, in which it is both preceded and followed by the strong helix promoting Aib.     

Cooperative assembly of a nativelike ubiquitin structure through peptide fragment complexation: energetics of peptide association and folding

Peptide fragments corresponding to the N- and C-terminal portions of bovine ubiquitin, U(1-35) and U(36-76), are shown by NMR to associate in solution to form a complex of modest stability (Kassn approximately 1.4 x 10(5) M(-1) at pH 7.0), with NMR features characteristic of a nativelike structure. The complex undergoes cold denaturation, with temperature-dependent estimates of stability from NMR indicating a DeltaC(p) degrees for fragment complexation in good agreement with that determined for native ubiquitin, suggesting that fragment association results in the burial of a similar hydrophobic surface area. The stability of the complex shows appreciable pH dependence, suggesting that ionic interactions on the surface of the protein contribute significantly. However, denaturation studies of native ubiquitin in the presence of guanidine hydrochloride (Gdn.HCl) show little pH dependence, suggesting that ionic interactions may be "screened" by the denaturant, as recently suggested. Examination of the conformation of the isolated peptide fragments has shown evidence for a low population of nativelike structure in the N-terminal beta-hairpin (residues 1-17) and weak nascent helical propensity in the helical fragment (residues 21-35). In contrast, the C-terminal peptide (36-76) shows evidence in aqueous solution, from some Halpha chemical shifts, for nonnative phi and psi angles; nonnative alpha-helical structure is readily induced in the presence of organic cosolvents, indicating that tertiary interactions in both native ubiquitin and the folded fragment complex strongly dictate its structural preference. The data suggest that the N-terminal fragment (1-35), where interaction between the helix and hairpin requires the minimum loss of conformational entropy, may provide the nucleation site for fragment complexation.     

The domain organization of streptokinase: nuclear magnetic resonance, circular dichroism, and functional characterization of proteolytic fragments.

Streptococcus equisimilis streptokinase (SK) is a bacterial protein of unknown tertiary structure and domain organization that is used extensively to treat acute myocardial infarction following coronary thrombosis. Six fragments of SK were generated by limited proteolysis with chymotrypsin and purified. NMR and CD experiments have shown that the secondary and tertiary structure present in the native molecule is preserved within all fragments, except the N-terminal fragment SK7. NMR spectra demonstrate the presence in SK of three structurally autonomous domains and a less structured C-terminal "tail." Cleavage within the N-terminal domain generates an N-terminal fragment, SK7, which remains noncovalently associated with the remainder of the molecule; in isolation, SK7 adopts an unfolded conformation. The abilities of these fragments to induce active site formation within human plasminogen upon formation of their heterodimeric complex were assayed. The lowest mass SK fragment exhibiting Plg-dependent activator activity was shown to be SK27 (mass 27,000, residues 147-380), which contains both central and C-terminal domains, although this activity was reduced approximately 6,000-fold relative to that of full-length SK. The activity of a 36,000 mass fragment, SK36 (residues 64-380), which differs from SK27 in possessing a portion of the N-terminal domain, was reduced to 0.1-1.0% of that of SK. Other fragments (masses 7,000, 11,000, 16,000, 17,000, 25,000, and 26,000), representing either single domains or single domains extended by portions of other domains, were inactive. However, SK7 (residues 1-63), at a 100-fold molar excess concentration, greatly potentiated the activities of SK27 and SK36, by up to 50- and > 130-fold, respectively. These findings demonstrate that all of SK's three domains are essential for native-like SK activity. The central and C-terminal domains mediate plasminogen-binding and active site-generating functions, whereas the N-terminal domain mediates an activity-potentiating function.     

Conformational studies of two bradykinin antagonists by using two-dimensional NMR techniques and molecular dynamics simulations

The solution conformations of two potent antagonists of bradykinin (Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9), [Aca(-1),DArg0,Hyp3,Thi5,DPhe7,(N-Bzl)Gly8]BK (1) and [Aaa(-1),DArg0,Hyp3,Thi5,(2-DNal)7,Thi8]BK (2), were studied by using 2D NMR spectroscopy in DMSO-d6 and molecular dynamics simulations. The NMR spectra of peptide 1 reveals the existence of at least two isomers arising from isomerization across the DPhe7-(N-Bzl)Gly8 peptide bond. The more populated isomer possesses the cis peptide bond at this position. The ratio of cis/trans isomers amounted to 7:3. With both antagonists, the NMR data indicate a beta-turn structure for the Hyp3-Gly4 residues. In addition, for peptide 2, position 2,3 is likely to be occupied by turn-like structures. The cis peptide bond between DPhe7 and (N-Bzl)Gly8 in analogue 1 suggests type VI beta-turn at position 7,8. The molecular dynamics runs were performed on both peptides in DMSO solution. The results indicate that the structure of peptide 1 is characterized by type VIb beta-turn comprising residues Ser6-Arg9 and the betaI or betaII-turn involving the Pro2-Thi5 fragment, whereas peptide 2 shows the tendency towards the formation of type I beta-turn at position 2,3. The structures of both antagonists are stabilized by a salt bridge between the guanidine moiety of Arg1 and the carboxyl group of Arg9. Moreover, the side chain of DArg0 is apart of the rest of molecule and is not involved in structural elements except for a few calculated structures.     

Solution structure of a K+-channel blocker from the scorpion Tityus cambridgei

A new K(+)-channel blocking peptide identified from the scorpion venom of Tityus cambridgei (Tc1) is composed of 23 amino acid residues linked with three disulfide bridges. Tc1 is the shortest known toxin from scorpion venom that recognizes the Shaker B K(+) channels and the voltage-dependent K(+) channels in the brain. Synthetic Tc1 was produced using solid-phase synthesis, and its activity was found to be the same as that of native Tc1. The pairings of three disulfide bridges in the synthetic Tc1 were identified by NMR experiments. The NMR solution structures of Tc1 were determined by simulated annealing and energy-minimization calculations using the X-PLOR program. The results showed that Tc1 contains an alpha-helix and a 3(10)-helix at N-terminal Gly(4)-Lys(10) and a double-stranded beta-sheet at Gly(13)-Ile(16) and Arg(19)-Tyr(23), with a type I' beta-turn at Asn(17)-Gly(18). Superposition of each structure with the best structure yielded an average root mean square deviation of 0.26 +/- 0.05 A for the backbone atoms and of 1.40 +/- 0.23 A for heavy atoms in residues 2 to 23. The three-dimensional structure of Tc1 was compared with two structurally and functionally related scorpion toxins, charybdotoxin (ChTx) and noxiustoxin (NTx). We concluded that the C-terminal structure is the most important region for the blocking activity of voltage-gated (Kv-type) channels for scorpion K(+)-channel blockers. We also found that some of the residues in the larger scorpion K(+)-channel blockers (31 to 40 amino acids) are not involved in K(+)-channel blocking activity.     

13 C solid-state NMR study of the 13 C-labeled peptide, (E)8 GGLGGQGAG(A)6 GGAGQGGYGG as a model for the local structure of Nephila clavipes dragline silk (MaSp1) before and after spinning

We prepared the water soluble model peptide, (E)(8) GGLGGQGAG(A)(6) GGAGQGGYGG, to throw light on the local structure of spidroin 1 (MaSpl) protein in spider dragline silk of Nephila clavipes before and after spinning. Solution (13) C NMR showed that the conformation of the peptide in aqueous solution was essentially random coil. Solid-state NMR was used to follow conformation-dependent (13) C chemical shifts in (13) C selectively labeled versions of the peptide. The peptide lyophilized from an aqueous solution at neutral pH (hereafter referred to as "without acid treatment)"was used to mimic the state of the spidroin stored in the spider's silk gland while the peptide precipitated from the acidic solution ("with acid treatment") was used to simulate the role of acid treatment in inducing conformation change in the natural spinning process. In without acid treatment, the fraction of random coil conformation was lowest in the N-terminal region (residues 15-18) when compared with the C-terminus. The conformational change produced by the acid treatment occurred in the sequence, G(15) AG(A)(6) GGAG(27), interposed between pairs of Gly residues pairs, Gly(12,13), and Gly(29,30). The acid treated peptide showed a remarkable decrease in the fraction of random coil conformation from A(20) to A(23) in the poly-Ala region when compared with the peptide without acid treatment. These observations taken together suggest that the peptide can be used as a model for studying the localization of the conformation change in spider silk fibroin in the natural spinning and the role of acid treatment in this process.     

Conformation-activity relationship of tachykinin neurokinin A (4-10) and of some [Xaa8] analogues.

NKA (4-10), the C-terminal heptapeptide fragment (Asp-Ser-Phe-Val-Gly-Leu-Met-NH2) of tachykinin NKA, is more active than the parent native compound in the interaction with the NK-2 receptor. Substitution of Gly8 with the more flexible residue beta-Ala8 increases its selectivity with respect to other two known receptors (NK-1 and NK-3), whereas substitution with either D-Ala8 or GABA8 deprives the peptide of its biological activity. These findings can be interpreted by a conformational analysis based on NMR studies in DMSO-d6 and in a DMSO-d6/H2O cryoprotective mixture combined with internal energy calculations. NKA(4-10) is characterized by a structure containing a type I beta-turn extending from Ser5 to Gly8, followed by a gamma-turn centered on Gly8, whereas for [beta-Ala8]NKA(4-10) is possible to suggest a type I beta-turn extending from Ser5 to beta-Ala8, followed by a C8 turn comprising beta-Ala8 and Leu9 and by another beta-turn extending from beta-Ala8 to the terminal NH2. The preferred conformation of [beta-Ala8]NKA(4-10) is not compatible with models for NK-1 and NK-3 agonists proposed on the basis of rigid peptide agonists [Levian-Teitelbaum et al. (1989) Biopolymers 28, 51-64; Sumner & Ferretti (1989) FEBS Lett. 253, 117-120]. The preferred solution conformation of [beta-Ala8]NKA(4-10) may thus be considered as a likely bioactive conformation for NK-2 selective peptides.     

Reconstitution of a native-like SH2 domain from disordered peptide fragments examined by multidimensional heteronuclear NMR

The N-terminal SH2 domain from the p85alpha subunit of phosphatidylinositol 3' kinase is cleaved specifically into 9- and 5-kD fragments by limited proteolytic digestion with trypsin. The noncovalent SH2 domain complex and its constituent tryptic peptides have been investigated using high-resolution heteronuclear magnetic resonance (NMR). These studies have established the viability of the SH2 domain as a fragment complementation system. The individual peptide fragments are predominantly unstructured in solution. In contrast, the noncovalent 9-kD + 5-kD complex shows a native-like (1)H-(15)N HSQC spectrum, demonstrating that the two fragments fold into a native-like structure on binding. Chemical shift analysis of the noncovalent complex compared to the native SH2 domain reveals that the highest degree of perturbation in the structure occurs at the cleavage site within a flexible loop and along the hydrophobic interface between the two peptide fragments. Mapping of these chemical shift changes on the structure of the domain reveals changes consistent with the reduction in affinity for the target peptide ligand observed in the noncovalent complex relative to the intact protein. The 5-kD fragment of the homologous Src protein is incapable of structurally complementing the p85 9-kD fragment, either in complex formation or in the context of the full-length protein. These high-resolution structural studies of the SH2 domain fragment complementation features establish the suitability of the system for further protein-folding and design studies.     

Synthesis and study of cyclic and linear analogs of neurotensin

New cyclic analogues of neurotensin (NT): [cyclo (13----8), Gly8]NT-(8-13), [cyclo (13----7), Gly7]NT-(7-13), [cyclo (13----5 epsilon), Lys5]NT-(5-13), [cyclo (13----4 epsilon), Lys4]NT-(4-13), and their linear precursors have been synthesized. The latter (protected linear compounds) were prepared by solid-phase peptide synthesis, and cyclization was attained by using diphenylphosphoryl azide. Cyclization of C-terminal hexa- and octapeptide fragments of NT was found to lead to cycloanalogues possessing high depressor activity. As judged by CD spectral data in aqueous solution, the cyclohexapeptide analogue has a relatively rigid conformation different from its linear counter-part and the NT-(9-13) fragment, whereas NT, its cyclohepta- and cyclononapeptides have random structure.