Peptides related to the calcium binding domains II and III of calmodulin. Synthesis and calmodulin-like features

Three hexadecapeptides which correspond to the putative Ca2+ binding domains II and III of calmodulin were synthesized employing solid phase methodology. One of the peptides contained an internal cystine bridge which was formed while the corresponding linear peptide was still attached to the polymeric carrier. The interaction of the synthetic peptides with calcium ions was investigated using Tb3+-mediated fluorescence. Binding was of the order Ca12 greater than Ca13 greater than Ca13C (Fig. 1) with binding constants KTb3+ = 0.68 X 10(-5), 0.54 X 10(-5), and 0.21 X 10(-5) M-1 respectively. Biological activity of the compounds was assessed by measuring their stimulatory effect on erythrocyte membrane (Ca2+ + Mg2+)-ATPase activity. For 50% activity as compared with CaM, the concentration of peptides required was for Ca12, Ca13 and Ca13C, 50, 100 and 167 times higher than CaM, respectively. The results suggest that the three synthetic peptides possess certain calmodulin-like features.     

Membranolytic selectivity of cystine-stabilized cyclic protegrins.

To correlate conformational rigidity with membranolytic selectivity of antimicrobial activity and cytotoxicity, we prepared six cyclic analogs of protegrin-1 (PG-1), an 18-residue cationic peptide with a broad-spectrum antimicrobial activity. These cyclic protegrins bear end-to-end peptide bonds together with varying numbers (zero to three) of cross-strand disulfide constraints. The most constrained analog is a cyclic tricystine protegrin (ccPG 3) containing three evenly spaced, parallel disulfide bonds. Antimicrobial assays against 10 organisms in low- and high-salt conditions showed that these cyclic protegrins were broadly active with different antimicrobial profiles against Gram-positive and Gram-negative bacteria, fungi and one tested virus, HIV-1. Compared to PG-1, the cyclic tricystine ccPG 3 displayed approximately a 10-fold decrease in hemolytic activity against human cells and 6- to 30-fold improvement of membranolytic selectivity against six of the 10 tested organisms. In contrast, [DeltaSS]cPG 8, a cyclic protegrin with no disulfide bond, and [DeltaCys6,15]cPG 5, a cyclic mimic of PG-1 with one disulfide bond, exhibited activity spectra, potency, and cytotoxicity similar to PG-1. Circular dichroism showed that cyclic protegrins containing with one to three cystine bonds displayed some degree of beta-strand structures in water/trifluoroethanol or phosphate-buffered solutions. Collectively, our results indicate that cyclic structures are useful in the design of antimicrobial peptides and that an increase in the conformational rigidity of protegrins may confer membranolytic selectivity that dissociates antimicrobial activity from hemolytic activity.     

The structural basis of compstatin activity examined by structure-function-based design of peptide analogs and NMR

We have previously identified compstatin, a 13-residue cyclic peptide, that inhibits complement activation by binding to C3 and preventing C3 cleavage to C3a and C3b. The structure of compstatin consists of a disulfide bridge and a type I beta-turn located at opposite sides to each other. The disulfide bridge is part of a hydrophobic cluster, and the beta-turn is part of a polar surface. We present the design of compstatin analogs in which we have introduced a series of perturbations in key structural elements of their parent peptide, compstatin. We have examined the consistency of the structures of the designed analogs compared with compstatin using NMR, and we have used the resulting structural information to make structure-complement inhibitory activity correlations. We propose the following. 1) Even in the absence of the disulfide bridge, a linear analog has a propensity for structure formation consistent with a turn of a 3(10)-helix or a beta-turn. 2) The type I beta-turn is a necessary but not a sufficient condition for activity. 3) Our substitutions outside the type I beta-turn of compstatin have altered the turn population but not the turn structure. 4) Flexibility of the beta-turn is essential for activity. 5) The type I beta-turn introduces reversibility and sufficiently separates the two sides of the peptide, whereas the disulfide bridge prevents the termini from drifting apart, thus aiding in the formation of the hydrophobic cluster. 6) The hydrophobic cluster at the linked termini is involved in binding to C3 and activity but alone is not sufficient for activity. 7) beta-Turn residues Gln(5) (Asn(5))-Asp(6)-Trp(7)(Phe(7))-Gly(8) are specific for the turn formation, but only Gln(5)(Asn(5))-Asp(6)-Trp(7)-Gly(8) residues are specific for activity. 8) Trp(7) is likely to be involved in direct interaction with C3, possibly through the formation of a hydrogen bond. Finally we propose a binding model for the C3-compstatin complex.     

Neurohypophyseal hormones and behavior: effects of intracerebroventricularly injected hormone analogs in mice.

Neurohypophyseal hormones evoke spontaneous behavioral changes in mice. This study compares the potency of four naturally occuring neurohypophyseal hormones and of ten analogs with amino acid residue replacements selected in such a manner as to cover each residue position of the hormones with the exception of the cystine residue. Peptides were administered intraventricularly and the sum of foraging, scratching and squeaking, recorded at 30 second intervals during a 30 min session, was measured as a function of peptide dose. The most potent group of peptides is represented by the neurohypophyseal hormones as well as the five analogs [Hly⁸] vasopressin, [Δ³-Pro⁷]AVP, [Thi³]LVP, [Abu⁴]AVP and [Abu⁴]LVP. [Leu⁴]LVP showed significant activity but was far less potent than the natural hormones. None of the remaining analogs enhanced activity with an increase in peptide dose. This group included both peptides with C-terminal modifications and those in which the tyrosine (position 2) or the asparagine residue (position 5) of the hormones were substituted by alanine. The neurohypophyseal hormone-induced behavioral results of this study reveal a structure-function relationship, which is in its most important conclusions, identical to the conformation-activity model proposed for endocrine activities of neurohypophyseal peptides.     

Features of a Spatially Constrained Cystine Loop in the p10 FAST Protein Ectodomain Define a New Class of Viral Fusion Peptides

The reovirus fusion-associated small transmembrane (FAST) proteins are the smallest known viral membrane fusion proteins. With ectodomains of only ∼20–40 residues, it is unclear how such diminutive fusion proteins can mediate cell-cell fusion and syncytium formation. Contained within the 40-residue ectodomain of the p10 FAST protein resides an 11-residue sequence of moderately apolar residues, termed the hydrophobic patch (HP). Previous studies indicate the p10 HP shares operational features with the fusion peptide motifs found within the enveloped virus membrane fusion proteins. Using biotinylation assays, we now report that two highly conserved cysteine residues flanking the p10 HP form an essential intramolecular disulfide bond to create a cystine loop. Mutagenic analyses revealed that both formation of the cystine loop and p10 membrane fusion activity are highly sensitive to changes in the size and spatial arrangement of amino acids within the loop. The p10 cystine loop may therefore function as a cystine noose, where fusion peptide activity is dependent on structural constraints within the noose that force solvent exposure of key hydrophobic residues. Moreover, inhibitors of cell surface thioreductase activity indicate that disruption of the disulfide bridge is important for p10-mediated membrane fusion. This is the first example of a viral fusion peptide composed of a small, spatially constrained cystine loop whose function is dependent on altered loop formation, and it suggests the p10 cystine loop represents a new class of viral fusion peptides.     

Mast cell degranulating (MCD) peptide analogs with reduced ring structure

Mast cell degranulating (MCD) peptide, a component of bee venom, is a 22 amino acid peptide with two disulfide bridges. In this first structure-activity study of MCD peptide, three analogs were synthesized and tested: two analogs shortened by omitting sequences 6–10 and 8–13, respectively, and one analog lacking the disulfide bridge between cysteine residues 5 and 19. These analogs were synthesized by solid-phase methods and were compared to MCD peptide in two assays for inflammation: histamine release from mast cells and superoxide anion release from neutrophils. All three analogs produced histamine release, although with only about one fifth of the activity of MCD peptide. Superoxide anion-releasing activity, however, did not parallel histamine release. MCD peptide did not release superoxide anion, while the 6–10 and 8–13 deletion analogs were strong and weak stimulants, respectively, of this anion. CD spectra showed that the secondary structures of the three analogs were very similar to that of MCD peptide, so that a change in secondary structure cannot completely explain the changes in releasing activities. Charge differences between the two deletion analogs and MCD peptide may explain some of the differences in activity. This is the first demonstration that the various activities of MCD peptide can be separated, and provides a lead through which the purported antiinflammatory activity of MCD peptide may possibly be explored in the future.     

Synthesis of cystine peptides 21-25/70-73 and 35-39/56-59 of the beta-subunit of human choriogonadotropin.

Syntheses of two asymmetrical cystine peptides with the amino acid residues 21-25/70-73 and 35-39/56-59, based on the linear amino acid sequence and the disulfide bond assignment in the beta-subunit of human choriogonadotropin (hCG-beta), are described. S-trityl and S-acetamidomethyl peptide fragments of each cystine peptide were prepared in solution phase and were subjected to oxidation with I2/MeOH to form the disulfide bridge. The cystine peptides were characterized by their amino acid analyses and fast atom bombardment mass spectrometry. Immunological characterization by several homologous radioimmunoassay systems showed that peptide 21-25/70-73 had significant hCG, hCG-beta, and hLH activities while peptide 35-39/56-59 failed to reveal any immunoreactivity.     

A facile method for the direct synthesis of lanthionine containing cyclic peptides

A series of disulfide bridged peptides were designed as potential inhibitors of protein-protein interactions. Following solid phase synthesis, completely deprotected linear peptides were first oxidized to their disulfide analogs and then transformed into their lanthionine equivalents via a base-assisted reaction in water. Peptides consisting of cystine bridges of length i, i+3, with and without discrimination of the chiral centers, were studied for this transformation. Lanthionine peptides were also obtained directly from the reduced linear peptides under mild alkaline treatment, and the reaction proceeded via disulfide bond formation. The extent of conversion of a disulfide bridge into its lanthionine counterpart varied according to the primary sequence. Product characterization revealed diastereomeric lanthionine formation. The presence of D-amino acids, peptide conformation, and/or position of the cystine bridge are among the factors determining the facility of this reaction. Elimination of the backbone proton beta to the sulfur atom followed by intramolecular thiol Michael addition is the most likely mechanism for this transformation.     

A facile method for the direct synthesis of lanthionine containing cyclic peptides

Summary A series of disulfide bridged peptides were designed as potential inhibitors of protein-protein interactions. Following solid phase synthesis, completely deprotected linear peptides were first oxidized to their disulfide analogs and then transformed into their lanthionine equivalents via a base-assisted reaction in water. Peptides consisting of cystine bridges of lengthi, i+3, with and without discrimination of the chiral centers, were studied for this transformation. Lanthionine peptides were also obtained directly from the reduced linear peptides under mild alkaline treatment, and the reaction proceeded via disulfide bond formation. The extent of conversion of a disulfide bridge into its lanthionine counterpart varied according to the primary sequence. Product characterization revealed diastereomeric lanthionine formation. The presence of D-amino acids, peptide conformation, and/or position of the cystine bridge are among the factors determining the facility of this reaction. Elimination of the backbone proton beta to the sulfur atom followed by intramolecular thiol Michael addition is the most likely mechanism for this transformation.     

Ribonuclease S-peptide. A model for molecular recognition.

The relationship of structure to function in the recognition of ribonuclease S-peptide by S-protein was studied by several methods. Liquid phase peptide synthesis was employed to generate analogs of S-peptide in which from 1 to 8 residues were deleted from the NH2-terminal end of the S-peptide. Additional derivatives were made by substitutions in the NH2-terminal three amino acids or by modifying the S-peptide analogs by trifluoroacetylation. The analogs were generated in the following way. S-Peptide was cleaved with chymotrypsin. The fragment obtained, RNase(9-20), was purified and lengthened step by step using liquid phase peptide synthesis. A second set of analogs were prepared by cleavage of CF3CO-S-peptide with elastase and the resulting CF3CO-RNase(7-20), similarly lengthened. The various analogs of S-peptide were tested in their capacity to combine with S-protein and regenerate biological activity as measured by Vmax and Kb. This work shows a positive contribution of every one of the first 8 NH2-terminal residues of S-peptide to the molecular recognition of S-protein in the presence of RNA substrate. Substitution of the first 3 residues by alanine or blocking of the free amino groups decreases recognition, indicating that the original primary structure is the most favorable one.     

Non-sequential vasopressin peptides. Stereochemistry and biological activity.

Two cyclic disulfides of structure Cys-Tyr-Arg-Arg-Tyr-Cys-NH2 (1) and Cys-Tyr(Me)-Arg-Arg-Tyr(Me)-Cys-NH2 (2), two nonapeptide derivatives of 1 extended at the C-terminal with Pro-Arg-Gly-NH2 (3) or Pro-D-Arg-Gly-NH2 (4) and derivatives of 3 and 4 having Mpr in position 1, i.e. analogs (5) and (6), respectively, were synthesized, and their stereochemistry and biological activity were studied. All the peptides displayed low dose-dependent uterotonic activity in vitro and antidiuretic activity in vivo. None of the peptides increased the blood pressure of the experimental animals. Compounds 2, 4 and 6 showed a low inhibitory effect on AVP pressor activity; compound 6, in addition, displays a significant and long-lasting vasodepressor effect. NMR measurements indicated the existence of hydrogen bond between the amino acid residues in positions 2,5 and 3,4 of peptides 1 and 2, and side-chain interactions between amino acid residues in positions 2,3 and 4,5 of peptide 1. No such side-chain interactions were detected in peptide 2.     

Synthetic position 5 analogs of adrenocorticotropin fragments and their in vitro lipolytic activity.

Twenty-three analogs of the ACTH-(4-10)-heptapeptide sequence, which forms the "active core" of adrenocorticotropin (ACTH) and related hormones, have been synthesized by the solid-phase method. These analogs all contain structural modifications at or near the 5-glutamic acid residue of ACTH. The peptides were purified to electrophoretic and chromatographic homogeneity. The peptides were assayed for lipolytic activity in an isolated cell system derived from rabbit adipose tissue. In this system, it was determined that residue 5 plays a very important "spacer" role in the peptide, but that this spacer function is not very dependent on the nature of the side chain of the position 5 amino acid. It was found, however, that a number of analogs containing basic residues (arginine or lysine) in position 3 and/or position 5 of ACTH-(3-10) and ACTH-(4-10) fragments have 5 to 10 times the activity of the respective parent peptides. The presence of a latent anionic locus in the rabbit fat-cell receptor for ACTH is suggested by this study.     

Novel cyclic azo-bridged analogs of gonadotropin-releasing hormone.

Five linear analogs of GnRH containing a p-aminophenylalanine (Pap) residue in their sequence and their six corresponding azo-bridged cyclic derivatives were synthesized. The precyclic peptides were prepared on solid-support, while azo-cyclization was performed in solution by diazotization of the p-aminophenylalanine residue followed by intramolecular coupling of the formed diazo salt with either tyrosine or histidine side chains present in the sequence. All peptides were examined for their binding ability to the GnRH receptor expressed on rat pituitary membranes and for their LH-release activity from dispersed rat pituitary cells. Linear analogs 1 i.e [Pap(5)] GnRH and 3, i.e. [Tyr(3), Pap(5)] GnRH, were found to bind to the GnRH receptors only slightly less avidly than native GnRH. Their cyclization, however, led to a marked reduction in the binding capacity, i.e. from IC(50) of 10(-9) M to the 10(-7) M range, and in biopotency, i.e. LH-release. All other linear and cyclic peptides were found to bind selectively to the GnRH receptor only in the low microM range. Only peptide 1 was found comparable to native GnRH in respect to LH-release activity and thus may potentially be a good agonist of the parent peptide. Peptides 1-4, the most potent GnRH receptor binders, were examined for their conformational properties using CD. Cyclic-azo peptides 2 and 4 were further evaluated by NMR spectroscopy in solution combined with molecular modeling. The structural information obtained explains in part the GnRH-like biological activity observed.     

Synthetic peptide derived from alpha-fetoprotein inhibits growth of human breast cancer: investigation of the pharmacophore and synthesis optimization.

Asynthetic peptide that inhibits the growth of estrogen receptor positive (ER+) human breast cancers, growing as xenografts in mice, has been reported. The cyclic 9-mer peptide, cyclo[EMTOVNOGQ], is derived from alpha-fetoprotein (AFP), a safe, naturally occurring human protein produced during pregnancy, which itself has anti-estrogenic and anti-breast cancer activity. To determine the pharmacophore of the peptide, a series of analogs was prepared using solid-phase peptide synthesis. Analogs were screened in a 1-day bioassay, which assessed their ability to inhibit the estrogen-stimulated growth of uterus in immature mice. Deletion of glutamic acid, Glu1, abolished activity of the peptide, but glutamine (Gln) or asparagine (Asn) could be substituted for Glu1 without loss of activity. Methionine (Met2) was replaced with lysine (Lys) or tyrosine (Tyr) with retention of activity. Substitution of Lys for Met2 in the cyclic molecule resulted in a compound with activity comparable with the Met2-containing cyclic molecule, but with a greater than twofold increase in purity and corresponding increase in yield. This Lys analog demonstrated anti-breast cancer activity equivalent to that of the original Met-containing peptide. Therefore, Met2 is not essential for biologic activity and substitution of Lys is synthetically advantageous. Threonine (Thr3) is a nonessential site, and can be substituted with serine (Ser), valine (Val), or alanine (Ala) without significant loss of activity. Hydroxyproline (Hyp), substituted in place of the naturally occurring prolines (Pro4, Pro7), allowed retention of activity and increased stability of the peptide during storage. Replacement of the first Pro (Pro4) with Ser maintains the activity of the peptide, but substitution of Ser for the second Pro (Pro7) abolishes the activity of the peptide. This suggests that the imino acid at residue 7 is important for conformation of the peptide, and the backbone atoms are part of the pharmacophore, but Pro4 is not essential. Valine (Val5) can be substituted only with branched-chain amino acids (isoleucine, leucine or Thr); replacement by d-valine or Ala resulted in loss of biologic activity. Thus, for this site, the bulky branched side chain is essential. Asparagine (Asn6) is essential for activity. Substitution with Gln or aspartic acid (Asp), resulted in reduction of biologic activity. Removal of glycine (Gly8) resulted in a loss of activity but nonconservative substitutions can be made at this site without a loss of activity indicating that it is not part of the pharmacophore. Cyclization of the peptide is facilitated by addition of Gln9, but this residue does not occur in AFP nor is it necessary for activity. Gln9 can be replaced with Asn, resulting in a molecule with similar activity. These data indicate that the pharmacophore of the peptide includes side chains of Val5 and Asn6 and backbone atoms contributed by Thr3, Val5, Asn6, Hyp7 and Gly8. Met2 and Gln9 can be modified or replaced. Glu1 can be replaced with charged amino acids, and is not likely to be part of the binding site of the peptide. The results of this study provide information that will be helpful in the rational modification of cyclo[EMTOVNOGQ] to yield peptide analogs and peptidomimetics with advantages in synthesis, pharmacologic properties, and biologic activity.     

Protein kinase inhibitor-(6-22)-amide peptide analogs with standard and nonstandard amino acid substitutions for phenylalanine 10. Inhibition of cAMP-dependent protein kinase

The minimal structure in the heat-stable inhibitor protein of cAMP-dependent protein kinase required for a low nanomolar potency of inhibition is the peptide Thr6-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-+ ++Ile22-NH2 (PKI-(6-22)-amide). While primary structural determinants for interaction with the protein kinase are distributed throughout the 17 residues of this peptide, we have previously shown that phenylalanine 10 in the NH2-terminal portion is a particularly important determinant for high affinity binding (Glass, D. B., Cheng, H.-C., Mende-Mueller, L., Reed, J., and Walsh, D. A. (1989) J. Biol. Chem. 264, 8802-8810). To investigate this requirement further, peptide analogs of PKI-(6-22)-amide in which various natural and nonstandard amino acids are substituted for phenylalanine 10 have been synthesized and tested for inhibitory potency against the catalytic subunit of the protein kinase. Consistent with the importance of the hydrophobicity of phenylalanine, an alanine 10 substitution analog exhibited a 270-fold decrease in inhibitory potency, whereas the leucine 10 analog lost only 33-fold in activity as compared to the parent peptide PKI-(6-22)-amide. Peptides containing the spatial conformation analogs D-phenylalanine, homophenylalanine, or phenylglycine were 60-120-fold less potent than the parent peptide. Peptides containing various para-substituted phenylalanines at position 10 were only 5-11-fold less potent. One exception to this was (4'-azidophenylalanine 10)PKI-(6-22)-amide, which was nearly equipotent with the parent inhibitor. The most potent analogs were those peptides containing highly aromatic residues at position 10. The 2'-thienylalanine 10, tryptophan (formyl) 10, tryptophan 10, and the 1'-naphthylalanine 10 analogs were 3-fold less potent, equipotent, slightly more potent, and 4-fold more potent than the parent peptide inhibitor, respectively. We conclude that phenylalanine 10 in PKI-(6-22)-amide, and presumably in the native protein inhibitor, interacts through specific hydrophobic and/or aromatic binding to a hydrophobic pocket or cleft near the active site of the protein kinase.     

Study of the binding environment of alpha-factor in its G protein-coupled receptor using fluorescence spectroscopy.

Mating in Saccharomyces cerevisiae is induced by the interaction of alpha-factor (W1H2W3L4Q5L6K7P8G9Q10P11M12Y13) with its cognate G protein-coupled receptor (Ste2p). Fifteen fluorescently labeled analogs of alpha-factor in which the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group was placed at the alphaN-terminus and in side-chains at positions 1, 3, 4, 6, 7, 12 and 13 were synthesized and assayed for biological activity and receptor affinity. Eleven of the analogs retained 6-60% of the biological activity of the alpha-factor, as judged using a growth arrest assay. The binding affinities depended on the position of NBD attachment in the peptide and the distance of the tag from the backbone. Derivatization of the positions 3 and 7 side-chains with the NBD group resulted in analogs with affinities of 17-35% compared with that of alpha-factor. None of the other NBD-containing agonists had sufficient receptor affinity or strong enough emission for fluorescence analysis. The position 3 and 7 analogs were investigated using fluorescence spectroscopy and collisional quenching by KI in the presence of Ste2p in yeast membranes. The results showed that the lambda max of NBD in the position 7 side-chain shifted markedly to the blue (510 nm) when separated by 4 or 6 bonds from the peptide backbone and that this probe was shielded from quenching by KI. In contrast, separation by 3, 5, 10 or more bonds resulted in lambda max ( approximately 540 nm) and collisional quenching constants consistent with increasing degrees of exposure. The NBD group in the position 3 side-chain was also found to be blue shifted (lambda max=520 nm) and shielded from solvent. These results indicate that the position 7 side-chain is likely interacting with a pocket formed by extracellular domains of Ste2p, whereas the side-chain of Trp3 is in a hydrophobic pocket possibly within the transmembrane region of the receptor.     

Solution conformations of bradykinin antagonists modified with Calpha-Calpha cyclized nonaromatic residues

The conformations of four BK antagonists, [D-Arg 0, Hyp3, Thi5, D-Phe7, Acc8]BK (1), Aaa[D-Arg 0, Hyp3, Thi5, D-Phe7, Acc8]BK (2), [D-Arg 0, Hyp3, Thi5, 8, Apc7]BK (3), and Aaa[D-Arg(0), Hyp(3), Thi(5, 8), Apc7]BK (4) were studied by using 2D NMR spectroscopy and MD simulations with time-averaged (TAV) restraints. According to the results of the NMR measurements, the BK antagonists contain 7-30% of minor conformation resulting from cis/trans isomerization of the peptide bonds preceding either Pro or Hyp residues. The major conformation of each peptide possesses all peptide bonds in trans configuration. Peptides modified with the Apc residue at position 7 (peptides 3 and 4) possess a higher percentage of minor isomer.Peptide 1 exhibits the strongest vasodepressor potency among the analogs studied and as a single one forms the betaII-turn in the 2-5 fragment, which is believed to be crucial for antagonistic activity. This peptide is also the most compact. The radius of gyration (Rg) amounts to 6.9 A and is by ca 1.5 A lower than that of the remaining analogs. With peptide 4, the ST-turn of type I within the Ser6-Thi8 fragment was found.     

Structural requirements for the occupancy of pituitary adenylate-cyclase-activating-peptide (PACAP) receptors and adenylate cyclase activation in human neuroblastoma NB-OK-1 cell membranes. Discovery of PACAP(6-38) as a potent antagonist.

In these structure activity studies, the 46 analogs of the 27-amino-acid form of the pituitary-adenylate-cyclase-activating peptide, PACAP(1-27), and the 38-amino-acid form, PACAP(1-38), were either monosubstituted or bisubstituted at positions 1-3, 20 and 21 or N-terminally shortened. All analogs were compared on human neuroblastoma NB-OK-1 cell membranes for their ability to occupy 125I-[AcHis1]PACAP(1-27)-labelled receptors (AcHis, N alpha-acetylhistidine) and to activate adenylate cyclase (in terms of potency and intrinsic activity). The monophasic slope of dose/effect curves on both parameters suggested interaction with one class of PACAP receptor. Residues 28-38 in the C-terminally extended peptide, PACAP(1-38), played a favorable role in recognition, in that receptors coupled to adenylate cyclase were, in general, more sensitive to PACAP(1-38) analogs than to the corresponding PACAP(1-27) analogs. At variance with PACAP(6-27), PACAP(6-38) was well recognized and acted as a potent competitive antagonist (Ki 1.5 nM). Residues 1-3 were all important in enzyme activation: modification of the beta-turn potential gave full agonists (the LAla2 and DAla2 derivatives) or partial agonists (LPhe2 and DPhe2; LArg2 and DArg2; Glu3 and Asn3). Finally, a proper alpha-helix was also important: the combined substitution of Lys21/Lys22 by Gly21/Gly22 decreased the binding affinity sharply.     

Elucidation of the disulfide bridge pattern of the recombinant human growth and differentiation factor 5 dimer and the interchain Cys/Ala mutant monomer

Growth and differentiation factor 5 (GDF5) is involved in many developmental processes such as chondrogenesis and joint and bone formation. A recombinant monomeric human GDF5 mutant rGDF5(C84A) is in vitro as potent as the dimeric native form, and clinical investigations of rGDF5(C84A) are in progress. Native homodimeric GDF5 belongs to the transforming growth factor β (TGF-β) superfamily; each monomer contains a cystine knot formed by three intrachain disulfide bridges, and the monomers are connected via an interchain disulfide bridge. The disulfide bridge pattern of recombinant homodimeric rGDF5 was recently elucidated by X-ray diffraction. A combination of proteolytic degradation with thermolysin, separation of the generated fragments by reverse-phase high-performance liquid chromatography (RP–HPLC), and subsequent analyses of the disulfide-linked peptides by electrospray–mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight (MALDI–TOF) mass spectrometry, amino acid analysis, and Edman degradation led to the unambiguous identification of the disulfide bridge pattern of the monomeric mutant rGDF5(C84A) and of the homodimeric rGDF5 in solution. The cystine knot of homodimeric rGDF5 exhibits the pattern Cys1-Cys5, Cys2-Cys6, and Cys3-Cys7 (three intrachain disulfide bonds), and the monomers are connected by a single interchain disulfide bridge (Cys4-Cys4) in accordance with other members of the TGF-β superfamily. The monomeric mutant rGDF5(C84A) exhibits the same cystine knot pattern as homodimeric rGDF5.