Conformational aspects of gastrin-related peptides: A circular dichroism study

The conformational properties of a series of gastrin-related peptides in aqueous solution and in 2,2,2-trifluoroethanol (TFE) have been investigated by CD measurements. In aqueous solution the peptides Leu³²-HG-34 (human big gastrin), Nle¹⁵-HG-17 (human little gastrin), and Nle¹¹-HG-13 assume a random-coil structure in the pH range 3–7. In TFE the three hormones fold into partially ordered structures, consisting of mixtures of α-helix, β-form and random coil. Comparison with the CD properties of the shorter gastrin peptides HG-4 (tetragastrin), N^α-Boc-HG-5 (pentagastrin), and HG-7 (heptagastrin) indicates that the biologically important C-terminal sequence Trp-Met-Asp-Phe-NH₂ in TFE does not maintain the same geometry upon elongation of the chain at the N-terminus from 4 to 34 residues. Thus, the various conformations in solution of the gastrin peptides examined do not provide a structural explanation for their very similar biological activity. Therefore, we hypothesize that the C-terminal tetrapeptide amide folds into an “active” structure only upon interaction with the receptor.     

Interaction of metal ions with gastrointestinal hormones: Binding studies of Mg++ to biologically active analogs of little gastrin and minigastrin

The interaction of magnesium ions with Des-Trp¹-Nle¹²-minigastrin I (Nle¹¹-HG-13) and Nle¹¹⁵-little gastrin I (Nle¹⁵-HG-17) has been studied by CD and spectrophotometric techniques in trifluoroethanol. Spectrophotometric titrations using murexide as a metallochromic indicator showed that there are three binding sites for magnesium ions in Nle¹¹-HG-13, with binding constants of the order of (6 ± 2) × 10⁶, (1.7 ± 0.5) × 10⁶, and (5.0 ± 0.5) × 10⁵M^?1. These figures have been independently confirmed by CD measurements in the far-uv in the presence of increasing amounts of magnesium ions. Elongation of the peptide chain from Nle¹¹-HG-13 to Nle¹⁵-HG-17 does not provide any additional binding site for the metal ions. In both hormones, we have observed different responses in the near- and fur-uv CD properties with regard to added magnesium. The intensity of the CD bands in the aromatic region changes cooperatively with the ion/hormone molar ratio. These findings lead us to conclude that at the C-terminal, the biologically important sequence, -Trp-Nle-Asp-Phe-Nh₂, is directly involved in the interaction with magnesium.     

Interaction of calcium ions with peptide hormones of the gastrin family

The interactions of Des-Trp¹-Nle¹²-minigastrin I (Nle¹¹-HG-13) and Nle¹⁵-little gastrin I (Nle¹⁵-HG-17) with calcium ions have been investigated in water and in trifluoroethanol solution using uv and CD absorption techniques. Both hormones strongly interact with Ca²⁺ in the organic medium. In the case of Nle¹¹-HG-13, the near-uv chiroptical properties (dominated by the transitions of the Trp residue in the C-terminal tetrapeptide sequence) indicate that three metal ions per mole of hormone are involved in the binding process. From the different response of near-uv and far-uv CD properties to the addition of calcium and from the change of the CD spectra in the aromatic absorption region, it is concluded that the biologically important C-terminal sequence is directly involved in the interaction with calcium. Elongation of the peptide chain from Nle¹¹-HG-13 to Nle¹⁵-HG-17 (Nle¹⁵-little gastrin I) does not provide any additional binding site for calcium ions. The change of the CD properties in the near- and far-uv indicates that three metal ions per mole of hormone are involved in the binding process. The dichroic absorption in the aromatic region indicates that only one of the two Trp residues of the little gastrin analog is sensitive to the presence of calcium. On the assumption that the variation of the CD properties is proportional to the extent of calcium binding, the binding constants K₁, K₂, and K₃ have been estimated roughly. Two similar sets of binding constants have been found, with K₁ ≥ 10⁶M^?1 and K₃ of the order of 10⁵M^?1, indicating similar binding sites in the two hormones with high affinity for calcium ions.     

Human somatotropin: Noncovalent interactions of the natural NH2-terminal 1–134 segment with synthetic analogs of the COOH-terminal fragment

Three analogs of the COOH-terminal fragments of human somatotropin (HGH), namely [Nle¹⁷⁰,Ala^165,182]-HGH-(150–187), [Nle¹⁷⁰,Ala^165,182]-HGH-(152–187), and [Nle¹⁷⁰,Ala^165,182]-HGH-(154–187), have been synthesized by the solid-phase method. The synthetic analogs were complemented with the natural NH₂-terminal fragment [Cys(Cam)⁵³]-HGH-(1–134) to form recombinants with HGH activities, as revealed by the rabbit liver membrane receptor binding and the Nb2 lymphoma cell assays.     

Human somatotropin: semisynthesis of the hormone by noncovalent interaction of the natural NH2-terminal fragment with a synthetic analog of the COOH-terminal fragment

Complementation of the natural [Cys (Cam)⁵³]-HGH-(1–134) fragment with synthetic analogs of COOH-terminal fragments of human somatotropin (HGH), namely [Nle¹⁷⁰, Ala^165,182]-HGH-(140–182) and [Nle¹⁷⁰,Ala^165,182]-HGH-(140–187) has been investigated. It was found that the synthetic fragment, [Nle¹⁷⁰,Ala^165,182]-HGH-(140–187), gave a recombinant with about 40% HGH radioreceptor-binding activity. When compared with the natural recombinant, the semisynthetic hormone exhibited similar receptor-binding activities. The natural and semisynthetic recombinants were indistinguishable in radioimmunoassay. The α-helical content of the semisynthetic recombinant was completely restored in comparison with that of the native hormone as revealed by circular dichroism spectra. On the other hand, attempts to obtain a recombinant with the synthetic [Nle¹⁷⁰,Ala^165,182]-HGH-(140–182) were unsuccessful. The synthesis of [Nle¹⁷⁰,Ala^165,182]-HGH-(140–182) and [Nle¹⁷⁰,Ala^165,182]-HGH-(140–187) is herein described.     

The Conformational Analysis of Substance P Analogs Using High-Field NMR Techniques

Abstract

High-field nuclear magnetic resonance measurements were carried out on substance P fragments SP_4–11 [pGlu⁵]-SP_5–11 and [pGlu⁶]SP_6–11 both at 400 and at 500 MHz. A spectral simulation was carried out on two of these peptides and the coupling constants were interpreted in terms of the conformations. The J_NH-CHa coupling constants are all ～8 Hz, with the exception of glycine, indicating no preferred conformation for the backbone. For the amino acids other than p-Glu, a comparison of the coupling constant data suggests the same relative rotamer populations for the side chains. Proton longitudinal relaxation time data were measured for all three peptides and support the above conclusions.     

Adrenocorticotropin/α-Melanocyte-Stimulating Hormone (ACTH/MSH)-Like Peptides Modulate Adenylate Cyclase Activity in Rat Brain Slices: Evidence for an ACTH/MSH Receptor-Coupled Mechanism

Abstract: The regulation of adenylate cyclase activity by adrenocorticotropin/α-melanocyte–stimulating hormone (ACTH/MSH)-like peptides was investigated in rat brain slices using a superfusion method. Adenylate cyclase activity was concentration-dependently increased by ACTH-(1–24), α-MSH (EC₅₀ values 16 and 6 nM, respectively), and [Nle⁴,D-Phe⁷]α-MSH (EC₅₀ value 1.6 nM), in the presence of forskolin (1 μM, optimal concentration). 1-9-Dideoxy-forskolin did not augment the response of adenylate cyclase to ACTH-(1–24). Various peptide fragments were tested for their ability to enhance [³H]cyclic AMP production. [Nle⁴,D-Phe⁷]α-MSH increased [³H]cyclic AMP formation with a maximal effect of 30% and was more potent than ACTH-(1–24), ACTH-(1–16)-NH₂, α-MSH, ACTH-(1–13)-NH₂, [MetO⁴]α-MSH, [MetO₂⁴,D-Lys⁸,Phe⁹]ACTH-(4–9), ACTH-(7–16)-NH₂, ACTH-(1–10), and ACTH-(11–24), in order of potency. This structure–activity relationship resembles that found for the previously described peptide-induced display of excessive grooming. ACTH-(1–24) stimulated adenylate cyclase activity in both striatal (maximal effect, ?20%) and septal slices (maximal effect, ?40%), but not in hippocampal or cortical slices. Lesioning of the dopaminergic projections to the striatum did not result in a diminished effect of [Nle⁴,D-Phe⁷]α-MSH on [³H]cyclic AMP accumulation, which indicates that the ACTH/MSH receptor–stimulated adenylate cyclase is not located on striatal dopaminergic terminals. ACTH-(1–24) did not affect the dopamine D₁ or D₂ receptor–mediated modulation of adenylate cyclase activity. Based on the present data, we suggest that the binding of endogenous ACTH or α-MSH to a putative ACTH/MSH receptor in certain brain regions leads to the activation of a signal transduction pathway using cyclic AMP as a second messenger.     

Identification by photoaffinity labeling of the extracellular N-terminal domain of PAC1 receptor as the major binding site for PACAP

Pituitary adenylate cyclase-activating polypeptide (PACAP) exerts many crucial biological functions through the interaction with its specific PAC1 receptor (PAC1-R), a class B G protein-coupled receptor (GPCR). To identify the binding sites of PACAP in the PAC1-R, three peptide derivatives containing a photoreactive p-benzoyl-phenylalanine (Bpa) residue were developed. These photosensitive PACAP analogs were fully biologically active and competent to displace radiolabeled Ac-PACAP27 from the PAC1-R. Subsequently, the ¹²⁵I-labeled photoprobes were used to anchor the PAC1-R expressed in Chinese hamster ovary cells. Photolabeling led to the formation of two protein complexes of 76 and 67 kDa, representing different glycosylated forms of the receptor. Proteinase and chemical cleavages of the peptide-receptor complexes revealed that ¹²⁵I[Bpa⁰, Nle¹⁷]PACAP27, ¹²⁵I[Bpa⁶, Nle¹⁷]PACAP27 and ¹²⁵I[Nle¹⁷, Bpa²²]PACAP27 covalently labeled the Ser⁹⁸ - Met¹¹¹ segment, the Ser¹²⁴ - Glu¹²⁵ dipeptide and the Ser¹⁴¹ - Met¹⁷² fragment, respectively. Taking into account the topology of the PAC1-R, these segments are mainly located within the extracellular N-terminal domain, indicating that this PAC1-R domain is the major binding site of PACAP27. The present study constitutes the first characterization of the binding domains of PACAP to its specific receptor and suggests heterogeneity within the binding mode of peptide ligands to class B GPCRs.     

Total synthesis,purification, and characterization of human [Phe(p-CH2SO3Na)52, Nle32,53,56, Nal55]-CCK20–58, [Tyr52, Nle32,53,56,Nal55]-CCK-58, and [Phe(p-CH2SO3Na)52, Nle32,53,56, Nal55]-CCK-58

The synthesis of [Phe(p-CH₂SO₃Na)⁵², Nle^32,53,56 Nal⁵⁵]-CCK_20–58, [Tyr⁵², Nle^32,53,56, Nal⁵⁵]-CCK-58 and of [Phe(p-CH₂SO₃Na)⁵², Nle^32,53,56, Nal⁵⁵]-CCK-58 using the (9-fluorenylmethyloxy)-carbonyl (Fmoc) strategy on a 2,4-DMBHA resin is described. The crude peptide preparations were extremely complex when analyzed by RP-HPLC, capillary zone electrophoresis (CZE), and ion-exchange chromatography (IE-FPLC). We found that the most effective strategy for purification included cation-exchange chromatography followed by a RP-HPLC desalting step. The highly purified peptides (purity greater than 90%) were characterized by RP-HPLC, size exclusion HPLC (SEC), IE-FPLC, CZE, mass spectrometry, amino acid analysis, and Edman sequence analysis {for [Tyr⁵², Nle^32,53,56, Nal⁵⁵]-CCK-58}. The results demonstrate the applicability of the 2,4-DMBHA resin for Fmoc solid-phase synthesis of long peptides amides (58 residues in length in this case) as well as the efficacy of an FPLC/RP-HPLC approach for the purification of very long, heterogeneous crude peptides, allowing a true assessment of the biological properties of these analogs to be carried out. [Phe(p-CH₂SO₃Na)⁵², Nle^32,53,56, Nal⁵⁵]-CCK_20–58 was less than 1% as potent as CCK-8 while [Tyr⁵², Nle^32,53,56, Nal⁵⁵]-CCK-58 and [Phe(p-CH₂SO₃Na)⁵², Nle^32,53,56, Nal⁵⁵]-CCK-58 were inactive at the doses tested (<0.01%).     

Conformational and molecular modeling studies of beta-cyclodextrin-heptagastrin and the third extracellular loop of the cholecystokinin 2 receptor

The conformational features of a conjugate of the C-terminus of human gastrin (HG[11-17]), the shortest gastrin sequence retaining biological function, with beta-cyclodextrin ([Nle(15)]-HG[11-17]-betaCD) were determined by NMR spectroscopy in an aqueous solution of dodecylphosphocholine (DPC) micelles. The peptide-betaCD conjugate displays a binding affinity and activation profile comparable to those of HG[11-17] at the cholecysokinin 2 (CCK(2)) receptor, the G protein-coupled receptor responsible for the gastrointestinal function of gastrin. The structure of the peptide consisted of a well-defined beta-turn between Gly(13) and Asp(16) of gastrin. The structural preferences of [Nle(15)]-HG[11-17]-betaCD in DPC micelles and the 5-doxylstearate-induced relaxation of the (1)H NMR resonances support a membrane-associated receptor recognition mechanism. Addition of [Nle(15)]-HG[11-17]-betaCD to the third extracellular loop domain of the CCK(2) receptor, CCK(2)-R(352-379), generated a number of intermolecular nuclear Overhauser enhancements (NOEs) and chemical shift perturbations. NOE-restrained MD simulations of the [Nle(15)]-HG[11-17]-betaCD-CCK(2)-R complex produced a topological orientation in which the C-terminus was located in a shallow hydrophobic pocket near the confluence of TM2 and -3. Despite the steric bulk and physicochemical properties of betaCD, the [Nle(15)]-HG[11-17]-betaCD-CCK(2)-R complex is similar to the CCK-8-CCK(2)-R complex determined previously, providing insight into the mode of ligand binding and the role of electrostatic interactions.     

Parathyroid hormone stimulates adenylate cyclase in rat cerebral microvessels

We have studied the effect of parathyroid hormone (PTH) on adenylate cyclase of microvessels isolated from rat cerebral cortex. Native bovine (b) PTH-(1–84), the synthetic amino-terminal fragment bPTH-(1–34) and the synthetic analog [Nle⁸, Nle¹⁸, Tyr³⁴]-bPTH- (1–34) amide stimulated adenylate cyclase in a dose-dependent manner with apparent ED₅₀ values of 16 nM, 6.3 nM and 15 nM respectively. The stimulation by bPTH was greatly enhanced by guanosine triphosphate. The PTH antagonist, [Nle⁸, Nle¹⁸, Tyr³⁴]-bPTH-(3–34) amide inhibited the action of bPTH-(1–84) and bPTH-(1–34). In summary, PTH stimulated adenylate cyclase in rat cerebral microvessels in a very similar manner to its stimulation in the renal cortex.     

Specificity of receptors to vasoactive intestinal peptide in guinea pig brain.

The specific binding of VIP to guinea pig brain membranes was tested by 1/ the ability of eight VIP and secretin analogs and fragments to inhibit the binding of ¹²⁵I-VIP and 2/ the capacity of the same peptides to influence basal and VIP-stimulated adenylate cyclase activities. Among all peptides tested, only VIP, secretin, [Val⁵] secretin, and [Gln⁹, Asn¹⁵] secretin (5–27) were able to inhibit ¹²⁵I-VIP binding. The adenylate cyclase activity was stimulated by VIP, secretin and [Val⁵] secretin. [Gln⁹, Asn¹⁵] secretin (5–27) although inactive per se was able to inhibit the VIP-stimulated adenylate cyclase activity competitively.     

Metal ion binding affinities of gastrin and CCK in membrane mimetic environments

The fully active gastrin and CCK analogues [Nle¹⁵]-gastrin- 17 and [Nle, Thr]-CCK-9 were analysed for their Ca²⁺ and Tb³⁺ affinities in various membrane mimetic conditions. In TFE both gastrin and CCK exhibited high affinities for calcium and terbium. At saturation level identical metal ion/peptide ratios were determined with Ca²⁺ and Tb³⁺, i.e. R = 3 for gastrin and R = 1 for CCK, confirming the very similar coordination properties of the two metal ions. The conformational effects of both metal ions were found to be very similar with a disordering effect in the case of gastrin and a conformational transition to β-turn type structure in the case of CCK. In order to mimic more properly physiological conditions, similar experiments were performed in the prsence of phospholipid bilayers. No interaction of the peptides with the bilayers was observed even in the presence of phospholipid bilayers. No interaction of the peptides with the bilayers was observed even in the presence of mmolar Ca²⁺ concentrations. Induced lipid interaction via N-terminal lipodervatization of gastrin and CCK allowed to translocate quantitatively the two hormones into phospholipid bilayers and to examine the effect of extravesicular Ca²⁺ on the conformation of the peptide headgroups and on their display at the water/lipid interphase. The CCK moiety of the lipo-CCK inserted into phospholipid bilayers interacts with the lipid phase and addition of Ca²⁺ enhances the clustering of the peptide headgroups in a more β-sheet type conformation. Conversely, insertion of lipo-gastrin into the bilayers leads to full exposure of the gastrin headgroup to the bulk water in predominantly random coil structure. Again Ca²⁺ provokes aggregation. As the lipo-peptide/phospholipid system still represents only an artificial model, it remains hazardous to derive a biological relevance from these data. The significantly higher affinity of lanthanide ions than Ca²⁺ for the peptides could well play a role in the inhibibitory activity of lanthanum on the signal transduction of the CCK family of hormones.     

Methoxinine – an alternative stable amino acid substitute for oxidation‐sensitive methionine in radiolabelled peptide conjugates

Radiolabelled peptides with high specificity and affinity towards receptors that are overexpressed by tumour cells are used in nuclear medicine for the diagnosis (imaging) and therapy of cancer. In some cases, the sequences of peptides under investigations contain methionine (Met), an amino acid prone to oxidation during radiolabelling procedures. The formation of oxidative side products can affect the purity of the final radiopharmaceutical product and/or impair its specificity and affinity towards the corresponding receptor. The replacement of Met with oxidation resistant amino acid analogues, for example, norleucine (Nle), can provide a solution. While this approach has been applied successfully to different radiolabelled peptides, a Met → Nle switch only preserves the length of the amino acid side chain important for hydrophobic interactions but not its hydrogen‐bonding properties. We report here the use of methoxinine (Mox), a non‐canonical amino acid that resembles more closely the electronic properties of Met in comparison to Nle. Specifically, we replaced Met¹⁵ by Mox¹⁵ and Nle¹⁵ in the binding sequence of a radiometal‐labelled human gastrin derivative [d ‐Glu¹⁰]HG(10‐17), named MG11 (d ‐Glu‐Ala‐Tyr‐Gly‐Trp‐Met‐Asp‐Phe‐NH₂). A comparison of the physicochemical properties of ¹⁷⁷Lu‐DOTA[ X ¹⁵]MG11 ( X = Met, Nle, Mox) in vitro (cell internalization/externalization properties, receptor affinity (IC₅₀), blood plasma stability and logD) showed that Mox indeed represents a suitable, oxidation‐stable amino acid substitute of Met in radiolabelled peptide conjugates. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

1H-NMR studies on substance P hexapeptide analogs

¹H-nmr studies of [pGlu⁶]SP_6–11, [gpGlu⁶,mPhe⁷]SP_6–11, and [pGlu⁶,N-CH₃Phe⁷]SP_6–11 in DMSO-d₆ reveal characteristic chemical shifts, ³J_NH-αCH, temperature dependence, as well as deuterium exchange half-times. Marked similarities are revealed for the two first analogs, whereas the N-methylated analog is clearly different. Possible conformations are considered.     

Respirable powder formulation of a shortened vasoactive intestinal peptide analog for treatment of airway inflammatory diseases

The aim of present study was to develop a respirable powder (RP) of a shortened vasoactive intestinal peptide (VIP) analog for inhalation. VIP and C‐terminally truncated VIP analogs were synthesized with a solid‐phase method. A structure‐activity relationship (SAR) study was carried out in terms with binding and relaxant activities of the peptides. Prepared RP formulation of a shortened VIP analog was physicochemically characterized by morphological, in vitro aerodynamic, and pharmacological assessments. The SAR study demonstrated that the N‐terminal 23 amino acid residues were required for biological activity of VIP. Upon chemical modification of VIP(1–23), [R^{15, 20, 21}, L¹⁷]‐VIP(1–23) was newly developed, which had higher binding activity in rat lung and smooth muscle relaxant effect in mouse stomach than VIP(1–23). The [R^{15, 20, 21}, L¹⁷]‐VIP(1–23)‐based RP, [R^{15, 20, 21}, L¹⁷]‐VIP(1–23)/RP, exhibited fine in vitro inhalation performance. Airway inflammation evoked by sensitization of antigen in rats was attenuated by pre‐treatment with the [R^{15, 20, 21}, L¹⁷]‐VIP(1–23)/RP at a dose of 50 μg‐[R^{15, 20, 21}, L¹⁷]‐VIP(1–23)/rat as evidenced by a 70% reduction of recruited inflammatory cells in bronchoalveolar lavage fluid. On the basis of these results, [R^{15, 20, 21}, L¹⁷]‐VIP(1–23)/RP might be a promising agent for treatment of airway inflammatory diseases.     

Isolation and partial amino acid sequence of A 78 kDa porcine gastrin-binding protein

• 1.1. A 78 kDa protein (p78) has been partially purified from washed membranes isolated from the corpus of porcine gastric mucosa. The purification was monitored by covalent cross-linking of iodinated [Nle¹⁵]-gastrin; 17.
• 2.2. A single N-terminal sequence extending for 33 amino acids was obtained from the p78 preparation. Partial sequences totalling 192 amino acids were also obtained from 14 tryptic and 3 Staphylococcal V8 peptides.
• 3.3. 10 peptides plus the N-terminal sequence were derived from a previously unsequenced protein which was distantly related to the product of the E. coli fadB gene (Baldwin G. S. (1993) Comp. Biochem. Physiol. 104B, 55–61). The remaining 7 peptides were derived from the gb-subunit of the gastric H⁺/K⁺-ATPase.
• 4.4. The gastrin-binding activity remained in association with p78, and could be separated from the P-subunit of the gastric H⁺K⁺-ATPase, during chromatography on tomato lectin-Sepharose.
• 5.5. We conclude that p78 binds gastrin, and is a novel member of the enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase family of enzymes.

     

D-isomeric replacements within the 6-9 core sequence of Ac-[Nle4]-alpha-MSH4-11-NH2: a topological model for the solution conformation of alpha-melanotropin

Analogs of Ac-[Nle⁴]-α-MSH_4–11-NH₂ and Ac-[Nle⁴, D -Phe⁷]-α-MSH_4–11-NH₂ were prepared with D -isomeric replacements at the His⁶, Arg⁸, and Trp⁹ residues. The requirement for an indole moiety at position 9 also was evaluated by replacement with L -leucine in both parent fragment analogs. D -isomeric replacements at positions 6 and 8 in either series were detrimental to biological potency in frog (Rana pipiens) and lizard skin (Anolis carolinensis) in vitro melanotropic assays. However, Ac-[Nle⁴, D -Trp⁹]-α-MSH_4–11-NH₂ and Ac-[Nle⁴, D -Phe⁷, D -Trp⁹]-α-MSH_4–11-NH₂ were equipotent and 10 × more potent than Ac-[Nle⁴]-α-MSH_4–11-NH₂, respectively, in the lizard skin bioassay, and 30 and 1900 times more potent in the frog skin bioassay. Ac-[Nle⁴, D -Phe⁷, D -Trp⁹]-α-MSH_4–11-NH₂ was 3 × more potent than α-MSH in the frog skin bioassay. Proton nmr studies in aqueous solution revealed a marked preservation of the backbone conformation of these linear analogs. Chemical-shift variations due to the through-space anisotropic influence of the core aromatic amino acid residues permitted evaluation of side-chain topology. The observed topology was consistent with nonhydrogen-bonded β-like structure (? = ?139°, ψ = +135° for L -amino acids; ? = +139°, ψ = ?135° for D -amino acids) as the predominant solution conformation. The biological and conformational data suggest that high melanotropic potency requires a close spatial arrangement of the His⁶, Phe⁷, and Arg⁸ side chains.     

Membrane-active properties of α-MSH analogs: aggregation and fusion of liposomes triggered by surface-conjugated peptides

Reaction of the melanotropin hormone analogs [Nle⁴,D-Phe⁷]-α-MSH and [Nle⁴,D-Phe⁷]-α-MSH(4-10), which were extended at their N-terminus by a thiol-functionalized spacer arm, with preformed liposomes containing thiol-reactive (phospho)lipid derivatives resulted in the aggregation of the vesicles and in a partial leakage of their inner contents. This aggregation/leakage effect, which was only observed when the peptides were covalently conjugated to the surface of the liposomes, was correlated with the fusion of the vesicles as demonstrated by the observed decrease in resonance energy transfer between probes in a membrane lipid mixing assay. A limited fusion was confirmed by monitoring the mixing of the liposome inner contents (formation of 1-aminonaphthalene-3,6,8-trisulfonic acid/p-xylene bis(pyridinium bromide) complex). The membrane-active properties of the peptides could be correlated with changes in the fluorescence emission spectra of their tryptophan residue, which suggested that after their covalent binding to the outer surface of the liposomes they can partition within the core of the bilayers. A blue shift of 10 nm was observed for [Nle⁴,D-Phe⁷]-α-MSH which was correlated with an increase in fluorescence anisotropy and with changes in the accessibility of the coupled peptide as assessed by the quenching of fluorescence of its tryptophan residue by iodide (Stern-Volmer plots). These results should be related to the previously described capacity of α-MSH, and analogs, to interact with membranes and with the favored conformation of these peptides which, via a β-turn, segregate their central hydrophobic residues into a domain that could insert into membranes and, as shown here, trigger their destabilization.