Structural basis for binding of accessory proteins by the appendage domain of GGAs

The Golgi-associated, gamma-adaptin-related, ADP-ribosylation-factor binding proteins (GGAs) and adaptor protein (AP)-1 are adaptors involved in clathrin-mediated transport between the trans-Golgi network and endosomal system. The appendage domains of GGAs and the AP-1 gamma-adaptin subunit are structurally homologous and have been proposed to bind to accessory proteins via interaction with short sequences containing phenylalanines and acidic residues. Here we present the structure of the human GGA1 appendage in complex with its cognate binding peptide from the p56 accessory protein (DDDDFGGFEAAETFD) as determined by X-ray crystallography. The interaction is governed predominantly by packing of the first two phenylalanine residues of the peptide with conserved basic and hydrophobic residues from GGA1. Additionally, several main chain hydrogen bonds cause the peptide to form an additional beta-strand on the edge of the preexisting beta-sheet of the protein. Isothermal titration calorimetry was used to assess the affinities of different peptides for the GGA and gamma-appendage domains.     

Characterization of a putative fusogenic sequence in the E2 hepatitis G virus protein

With the aim of better understanding the fusion process mediated by the envelope proteins of the hepatitis G virus (HGV/GBV-C), we have investigated the interaction with model membranes of two overlapping peptides [(267-284) and (279-298)] belonging to the E2 structural protein. The peptides were compared for their ability to perturb lipid bilayers by means of different techniques such as differential scanning calorimetry and fluorescence spectroscopy. Furthermore, the conformational behaviour of the peptides in different membrane environments was studied by Fourier-transform infrared spectroscopy and circular dichroism. The results showed that only the E2(279-298) peptide sequence was able to bind with high affinity to negatively charged membranes, to permeabilize efficiently negative lipid bilayers, to induce haemolysis, and to promote inter-vesicle fusion. This fusogenic activity could be related to the induced peptide conformation upon interaction with the target membrane.     

Energetics of target peptide binding by calmodulin reveals different modes of binding

Thermodynamic parameters of interactions of calcium-saturated calmodulin (Ca(2+)-CaM) with melittin, C-terminal fragment of melittin, or peptides derived from the CaM binding regions of constitutive (cerebellar) nitric-oxide synthase, cyclic nucleotide phosphodiesterase, calmodulin-dependent protein kinase I, and caldesmon (CaD-A, CaD-A*) have been measured using isothermal titration calorimetry. The peptides could be separated into two groups according to the change in heat capacity upon complex formation, DeltaC(p). The calmodulin-dependent protein kinase I, constitutive (cerebellar) nitric-oxide synthase, and melittin peptides have DeltaC(p) values clustered around -3.2 kJ.mol(-1).K(-1), consistent with the formation of a globular CaM-peptide complex in the canonical fashion. In contrast, phosphodiesterase, the C-terminal fragment of melittin, CaD-A, and CaD-A* have DeltaC(p) values clustered around -1.6 kJ.mol(-1).K(-1), indicative of interactions between the peptide and mostly one lobe of CaM, probably the C-terminal lobe. It is also shown that the interactions for different peptides with Ca(2+)-CaM can be either enthalpically or entropically driven. The difference in the energetics of peptide/Ca(2+)-CaM complex formation appears to be due to the coupling of peptide/Ca(2+)-CaM complex formation to the coil-helix transition of the peptide. The binding of a helical peptide to Ca(2+)-CaM is dominated by favorable entropic effects, which are probably mostly due to hydrophobic interactions between nonpolar groups of the peptide and Ca(2+)-CaM. Applications of these findings to the design of potential CaM inhibitors are discussed.     

Opioid peptide interactions with lipid bilayer membranes.

The interaction of the delta-opioid receptor selective peptides, cyclic [D-Pen2, D-Pen5]-enkephalin [DPDPE] and its acyclic analog, DPDPE(SH)2, with neutral phospholipid bilayer membranes was examined by permeability and calorimetry measurements. The permeabilities were accomplished by entrapping either peptide inside of unilamellar liposomes (composed of a mixture of a molar ratio 65:25:10 phosphatidylcholine/phosphatidylethanolamine/cholesterol) then monitoring the peptide efflux through the bilayer. The initial permeability of DPDPE (first 12 h) averaged over four experiments was (0.91 +/- 0.47).10(-12) cm s-1. In contrast the average permeability of the acylic DPDPE(SH)2 was (4.26 +/- 0.23).10(-12) cm s-1. The effect of these peptides on the phase transition, Tm, of 1,2-dipalmitoylphosphatidylcholine (DPPC) bilayers was examined by high sensitivity differential scanning calorimetry. The Tm, the calorimetric enthalpy, and the van 't Hoff enthalpy of DPPC were not significantly altered by the presence of DPDPE, whereas the calorimetric data for DPPC with DPDPE(SH)2 showed a small, yet significant, increase (0.2 degrees C) in the Tm with a 30% decrease in the cooperative unit. Both the permeability and calorimetry data reveal a stronger peptide-membrane interaction in the case of the more flexible acyclic peptide.     

A quantitative study of the in vitro binding of the C-terminal domain of p21 to PCNA: affinity, stoichiometry, and thermodynamics

Proliferating cell nuclear antigen (PCNA) plays an essential role in DNA replication, repair, and control of cell proliferation, and its activity can be modulated by interaction with p21(Waf1/Cip1) [Cox, L. S., (1997) Trends Cell Biol. 7, 493-497]. This protein-protein interaction provides a particularly good model target for designing therapeutic agents to treat proliferative disorders such as cancer. In this study, the formation of complexes between PCNA and peptides derived from the C-terminus of p21 has been investigated at the molecular level and quantified using a competitive PCNA binding assay and isothermal titration calorimetry (ITC). The affinity constant for the interaction between p21 (141-160) peptide and PCNA has been determined to be 1.14 x 10(7) M(-)(1), corresponding to a K(d) of 87.7 nM. Measurement of the interaction of truncation and substitution analogues based on the p21 (141-160) sequence with PCNA revealed that the N-terminal part (residues 141-152) of the above peptide is the minimum recognition motif, required for PCNA binding. Truncation of the C-terminal region p21 (153-160), though, inhibited significantly the ability of the peptides to compete with the full-length p21 (141-160) for binding to PCNA. Alanine mutation of Met 147 or Asp 149 completely abolished or significantly decreased, respectively, the level of the PCNA binding and the inhibition of SV40 DNA replication. Comparison of the data obtained by the competitive PCNA binding assay and the ITC measurements demonstrated the usefulness of this assay for screening for compounds that could modulate the PCNA-p21 interaction. Using this assay, we have screened rationally designed peptides for binding to PCNA and interruption of the PCNA-p21 (141-160) complex. As a result of this screening, we have identified a 16-residue peptide (consensus motif 1 peptide) with the following sequence: SAVLQKKITDYFHPKK. Consensus motif 1 peptide and p21 (141-160) have similar affinities for binding PCNA and abilities to inhibit in vitro replication of DNA originated from SV40. Such peptides could prove useful in assessing p21-mimetic strategies for cancer treatment.     

A transmembrane peptide from the human EGF receptor: behaviour of the cytoplasmic juxtamembrane domain in lipid bilayers

Solid state (2)H NMR spectroscopy was employed to study peptides related to the transmembrane domain of the human epidermal growth factor receptor, for insight into the interaction of its cytoplasmic juxtamembrane domain with the membrane surface. Since such receptors have clusters of (+)charged amino acids in this region, the effect of (-)charged phosphatidylserine at the concentration found naturally in the cytoplasmic leaflet (15 mol%) was considered. Each peptide contained 34 amino acids, which included the hydrophobic 23 amino acid stretch thought to span the membrane and a ten amino acid segment beyond the 'cytoplasmic' surface. Non-perturbing deuterium probe nuclei were located within alanine side chains in intramembranous and extramembranous portions. (2)H NMR spectra were recorded at 35 degrees C and 65 degrees C in fluid lipid bilayers consisting of (zwitterionic) 1-palmitoyl-2-oleoylphosphatidylcholine, with and without 15 mol% (anionic) phosphatidylserine. The cationic extramembranous portion of the receptor backbone was found to be highly rotationally mobile on a time scale of 10(-4)-10(-5) s in both types of membrane - as was the alpha-helical intramembranous portion. Deuterium nuclei in alanine side chains (-CD(3)) detected modest changes in peptide backbone orientation and/or dynamics related to the presence of 1-stearoyl-2-oleoylphosphatidylserine: in the case of the extramembranous portion of the peptide these seemed related to lipid charge. Temperature effects on the peptide backbone external to the membrane were qualitatively different from effects on the helical transmembrane domain - likely reflecting the different physical constraints on these peptide regions and the greater flexibility of the extramembranous domain. Effects related to lipid charge could be detected in the spectrum of CD(3) groups on the internally mobile side chain of Val(650), six residues beyond the membrane surface.     

The effects of Leu or Val residues on cell selectivity of α-helical peptides

In this study, the peptides were designed to compare the effect of multiple Leu or Val residues as the hydrophobic side of an α-helical model on their structure, function, and interaction with model membranes. The Leu-rich peptides displayed 4- to 16-fold stronger antimicrobial activity than Val-rich peptides, while Val-containing peptides showed no haemolysis and weak cytotoxicity. The peptides LR and VR showed an α-helical-rich structure under a membranemimicking environment. Different cell selectivity for Leu- or Val-containing peptides correlated with the targeted cell membranes. The Leu-rich peptide LR(W) and Val-rich peptide VR(W) interacted preferentially with negatively charged phospholipids over zwitterionic phospholipids. VR(W) displayed no interaction with zwitterionic phospholipids, which was consistent with its lack of haemolytic activity. The ability of LR to depolarize bacterial cells was much greater than that of VR. Val- and Leu-rich peptides appeared to kill bacteria in a membrane-targeted fashion, with different modes of action. Leu-rich peptides appeared to be active via a membrane-disrupting mode, while Val-rich peptides were active via the formation of small channels.     

Conformational properties of the beta(400-436) and beta(400-445) C-terminal peptides of porcine brain tubulin.

Two peptides from the C-terminal region of the major beta-tubulin isotype (400-436 and 400-445) that include the critical areas for interaction with MAP2 and tau were examined to determine their conformations in aqueous solution. Despite a high theoretical potential for alpha-helix formation, CD spectroscopy showed that these peptides consisted primarily of random coil with some reverse turn. This was unaffected by the presence of counterions to the negatively charged side chains (Ca2+, Mg2+), but did change when the side-chain charges were neutralized by lowering the pH; under these conditions, the alpha-helix content of the longer peptide rose to 25% and the C-terminal truncated peptide to 15%. The peptides also adopt alpha-helical structure in the presence of trifluoroethanol, the truncated peptide again attaining a lower maximum percentage. The beta(400-445) peptide was also studied by 1-D and 2-D NMR techniques. The results indicate that at pH 5.6 or 7 in an aqueous solution the peptide is extremely flexible and lacks regular secondary structure, consistent with the CD results. Both peptides inhibited microtubule-associated protein-stimulated tubulin assembly, with the longer peptide being about 4 times as inhibitory as the smaller peptide. Neither was inhibitory in the absence of microtubule-associated proteins, indicating that interaction with this species was necessary for inhibition. The greater activity of the longer peptide could be due to the extra negative charges in this peptide and/or the greater tendency of this peptide to form an alpha-helical structure under the appropriate conditions.     

A comparison of the interaction of glucagon, human parathyroid hormone-(1-34)-peptide and calcitonin with dimyristoylphosphatidylglycerol and with dimyristoylphosphatidylcholine

The interaction of glucagon, human parathyroid hormone-(1-34)-peptide and salmon calcitonin with dimyristoylphosphatidylglycerol (DMPG) and with dimyristoylphosphatidylcholine (DMPC) was studied as a function of pH and temperature. The effect of lipid on the secondary structure of the peptide was assessed by circular dichroism and the effect of the peptide on the phase transition properties of the lipid was studied using differential scanning calorimetry. Some peptides interact more strongly with anionic than with zwitterionic phospholipids. This does not require an overall positive charge on the peptide. Increased thermal stability is observed in complexes formed between cationic peptides and anionic lipids. Particularly marked effects of glucagon and human parathyroid hormone-(1-34)-peptide on the phase transition properties of DMPG at pH 5 have been observed. The transition temperature is raised over 10 degrees C at a lipid/peptide molar ratio of less than 30:1 and the transition enthalpy is increased over 2-fold. These effects do not occur with any basic peptide and were not observed with metorphinamide, molluscan cardioexcitatory neuropeptide or myelin basic protein. The results demonstrate that certain peptides can affect the phase transition properties of lipids in a manner similar to divalent cations. The overall hydrophobicities of these peptides can be evaluated by their partitioning between aqueous and organic solvents. None of the above three peptide hormones partition into the organic phase. However, a closely related peptide, human calcitonin, does exhibit substantial partitioning into the organic phase. Nevertheless, human calcitonin has a weaker interaction with both DMPC and DMPG than does salmon calcitonin. The effects of human calcitonin on the phase transition of DMPC are qualitatively different from those of salmon calcitonin in that the human form more readily eliminates the pretransition but causes less change in the main transition. Like overall charge, overall hydrophobicity is not an overwhelming factor in determining the ability of peptides to interact with phospholipids but rather more specific interactions are required for strong complexes to form.     

Characterization of the structure and membrane interaction of the antimicrobial peptides aurein 2.2 and 2.3 from Australian southern bell frogs

The structure and membrane interaction of the antimicrobial peptide aurein 2.2 (GLFDIVKKVVGALGSL-CONH(2)), aurein 2.3 (GLFDIVKKVVGAIGSL-CONH(2)), both from Litoria aurea, and a carboxy C-terminal analog of aurein 2.3 (GLFDIVKKVVGAIGSL-COOH) were studied to determine which features of this class of peptides are key to activity. Circular dichroism and solution-state NMR data indicate that all three peptides adopt an alpha-helical structure in the presence of trifluoroethanol or lipids such as 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and a 1:1 mixture of DMPC and 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DMPG). Oriented circular dichroism was used to determine the orientation of the peptides in lipid bilayers over a range of concentrations (peptide/lipid molar ratios (P/L) = 1:15-1:120) in DMPC and 1:1 DMPC/DMPG, in the liquid crystalline state. The results demonstrate that in DMPC all three peptides are surface adsorbed over a range of low peptide concentrations but insert into the bilayers at high peptide concentrations. This finding is corroborated by (31)P-solid-state NMR data of the three peptides in DMPC, which shows that at high peptide concentrations the peptides perturb the membrane. Oriented circular dichroism data of the aurein peptides in 1:1 DMPC/DMPG, on the other hand, show that the peptides with amidated C-termini readily insert into the membrane bilayers over the concentration range studied (P/L = 1:15-1:120), whereas the aurein 2.3 peptide with a carboxy C-terminus inserts at a threshold concentration of P/L* between 1:80 and 1:120. Overall, the data presented here suggest that all three peptides studied interact with phosphatidylcholine membranes in a manner which is similar to aurein 1.2 and citropin 1.1, as reported in the literature, with no correlation to the reported activity. On the other hand, both aurein 2.2 and aurein 2.3 behave similarly in phosphatidylcholine/phosphatidylglycerol (PC/PG) membranes, whereas aurein 2.3-COOH inserts less readily. As this does not correlate with reported activities, minimal inhibitory concentrations of the three peptides against Staphylococcus aureus (strain C622, ATCC 25923) and Staphylococcus epidermidis (strain C621--clinical isolate) were determined. The correlation between structure, membrane interaction, and activity are discussed in light of these results.     

Thermodynamic and structural analysis of interactions between peptide ligands and SEB

Staphylococcal enterotoxin B (SEB) is an exotoxin produced by Staphylococcus aureus and commonly associated with food poisoning. In this study, SEB‐binding peptides were identified by screening a phage displayed peptide library. The binding of peptides to SEB was tested with isothermal titration calorimetry (ITC) and of the five selected peptides, three showed affinity to SEB, with one measured to have the highest affinity constant (10⁵ M^?1). ITC revealed that the interaction of peptide ligands with SEB was driven entropically and the binding was dominated by hydrophobic interactions. Circular dichroism (CD) measurements and molecular dynamics (MD) simulations, together, give a structural insight into the interaction of peptides with SEB. While SEB binding peptides showed random coil structure before binding, after complex formation they had more ordered structures. The peptide with highest affinity to SEB showed stable conformation during MD simulation. Taken together, our approach about thermodynamic and structural characterization of peptide ligands can be used to develop aptamers, with high affinity and selectivity, for biosensor applications. Copyright © 2009 John Wiley & Sons, Ltd.     

Energetics and partition of two cecropin-melittin hybrid peptides to model membranes of different composition

The energetics and partition of two hybrid peptides of cecropin A and melittin (CA(1-8)M(1-18) and CA(1-7)M(2-9)) with liposomes of different composition were studied by time-resolved fluorescence spectroscopy, isothermal titration calorimetry, and surface plasmon resonance. The study was carried out with large unilamellar vesicles of three different lipid compositions: 1,2-dimyristoil-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DMPG), and a 3:1 binary mixture of DMPC/DMPG in a wide range of peptide/lipid ratios. The results are compatible with a model involving a strong electrostatic surface interaction between the peptides and the negatively charged liposomes, giving rise to aggregation and precipitation. A correlation is observed in the calorimetric experiments between the observed events and charge neutralization for negatively charged and mixed membranes. In the case of zwitterionic membranes, a very interesting case study was obtained with the smaller peptide, CA(1-7)M(2-9). The calorimetric results obtained for this peptide in a large range of peptide/lipid ratios can be interpreted on the basis of an initial and progressive surface coverage until a threshold concentration, where the orientation changes from parallel to perpendicular to the membrane, followed by pore formation and eventually membrane disruption. The importance of negatively charged lipids on the discrimination between bacterial and eukaryotic membranes is emphasized.     

Thermodynamics of binding interactions between bovine beta-lactoglobulin A and the antihypertensive peptide beta-Lg f142-148

The binding capacity of bovine beta-lactoglobulin variant A (beta-Lg A) for six peptides derived from beta-Lg was evaluated using an ultrafiltration method under the following conditions: pH 6.8, 40 degrees C, and a beta-Lg A/peptide molar ratio of 1:5. Only peptides beta-Lg f102-105, f142-148, and f69-83 bound in significant amounts to beta-Lg A corresponding to 1.5, 1.1, and 0.7 mol of peptide per mole of beta-Lg A, respectively. The interaction between beta-Lg A and the antihypertensive peptide beta-Lg f142-148 was investigated further by isothermal titration calorimetry. The binding isotherms at pH 6.8 and 25 degrees C confirmed that beta-Lg f142-148 bound to beta-Lg A and that the interaction followed a sequential three-site binding model with constants of association of 2 x 10(3), 1 x 10(3), and 0.4 x 10(3) M(-1) for the first, second, and third binding sites, respectively. The enthalpy of binding was exothermic for the first and second binding sites and endothermic for the third binding site. Binding of the peptide to all three sites was spontaneous as shown by the negative free energy values. These results show for the first time that beta-Lg A can bind bioactive peptides. This potential could be exploited to transport bioactive peptides and protect them in the gastrointestinal tract following their oral administration as nutraceuticals.     

High-resolution NMR studies of fibrinogen-like peptides in solution: interaction of thrombin with residues 1-23 of the A alpha chain of human fibrinogen

The interaction of the following human fibrinogen-like peptides with bovine thrombin was studied by use of one- and two-dimensional NMR techniques in aqueous solution: Ala(1)-Asp-Ser-Gly-Glu-Gly-Asp-Phe(8)-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg(16 )- Gly(17)-Pro-Arg(19)-Val(20)-Val-Glu-Arg (F10), residues 1-16 of F10 (fibrinopeptide A), residues 17-23 of F10 (F12), residues 1-20 of F10 (F13), residues 6-20 of F10 with Arg(16) replaced by a Gly residue (F14), and residues 6-19 of F10 with Arg(16) replaced by a Leu residue (F15). At pH 5.3 and 25 degrees C, the Arg(16)-Gly(17) peptide bonds of both peptides F10 and F13 were cleaved instantaneously in the presence of 0.6 mM thrombin, whereas the cleavage of the Arg(19)-Val(20) peptide bonds in peptides F12, F13, and F14 took over 1 h for completion. On the basis of observations of line broadening, fibrinopeptide A was found to bind to thrombin. While resonances from residues Ala(1)-Glu(5) were little affected, binding of fibrinopeptide A to thrombin caused significant line broadening of NH and side-chain proton resonances within residues Asp(7)-Arg(16). There is a chain reversal within residues Asp(7)-Arg(16) such that Phe(8) is brought close to the Arg(16)-Gly(17) peptide bond in the thrombin-peptide complex, as indicated by transferred NOEs between the aromatic ring protons of Phe(8) and the C alpha H protons of Gly(14) and the C gamma H protons of Val(15). A similar chain reversal was obtained in the isolated peptide F10 at a subzero temperature of -8 degrees C. The titration behavior of Asp(7) in peptide F13 does not deviate from that of the reference peptide, N-acetyl-Asp-NHMe at both 25 and -8 degrees C, indicating that no strong interaction exists between Asp(7) and Arg(16) or Arg(19). Peptides with Arg(16) replaced by Gly and Leu, respectively, i.e., F14 and F15, were also found to bind to thrombin but with a different conformation, as indicated by the absence of the long-range NOEs observed with fibrinopeptide A. Residues Asp(7)-Arg(16) constitute an essential structural element in the interaction of thrombin with fibrinogen.     

Synthetic partial extension peptides of P-450(SCC) and adrenodoxin precursors: effects on the import of mitochondrial enzyme precursors

Various portions of the extension peptides of P-450(SCC) precursor were chemically synthesized. The effects of these peptides on the import of enzyme precursors into mitochondria were examined. Peptides SEP1-15 and SEP1-20, corresponding to the amino terminal portion of the extension peptides, strongly inhibited the import of P-450(SCC) precursor into mitochondria. These peptides were effective at concentrations above 30 microM, and complete inhibition was observed at 100 microM. SEP1-11, which is shorter than SEP1-15 and SEP1-20, showed very weak inhibition. SEP25-39, which corresponds to the carboxy terminal portion of the extension peptide, did not affect the import of the precursor. The import of P-450(11 beta) and adrenodoxin precursors were also inhibited by SEP1-15. Another peptide, AEP1-14, which corresponds to the amino terminal portion of the extension peptide of adrenodoxin precursor, was also synthesized. The peptide inhibited the import of both adrenodoxin and P-450(SCC) precursors into mitochondria. The import of malate dehydrogenase was also inhibited by SEP1-15 and AEP1-14. The rate of the internalization of the precursor into mitochondria was decreased by the peptides. The amount of the precursor bound to the surface of mitochondria and the processing of adrenodoxin precursor were not affected. The respiratory activities of isolated mitochondria were not influenced by SEP1-15 even at 100 microM. We conclude that the inhibitory activities of the synthetic partial extension peptides on the import of enzyme precursors into mitochondria require the presence of about fifteen amino acid residues in the amino terminal portion of the extension peptides, and the inhibition of the import by the peptides was dependent on the blockage of the internalization of the precursors into mitochondria.     

Interactions between charged polypeptides and nonionic surfactants

The influence of molecular characteristics on the mutual interaction between peptides and nonionic surfactants has been investigated by studying the effects of surfactants on amphiphilic, random copolymers of alpha-L-amino acids containing lysine residues as the hydrophilic parts. The hydrophobic residues were either phenylalanine or tyrosine. The peptide-surfactant interactions were studied by means of circular dichroism spectroscopy and binding isotherms, as well as by 1D and 2D NMR. The binding of surfactant to the peptides was found to be a cooperative process, appearing at surfactant concentrations just below the critical micellar concentration. However, a certain degree of peptide hydrophobicity is necessary to obtain an interaction with nonionic surfactant. When this prerequisite is fulfilled, the peptide mainly interacts with self-assembled, micelle-like surfactant aggregates formed onto the peptide chain. Therefore, the peptide-surfactant complex is best described in terms of a necklace model, with the peptide interacting primarily with the palisade region of the micelles via its hydrophobic side chains. The interaction yields an increased amount of alpha-helix conformation in the peptide. Surfactants that combine small headgroups with a propensity to form small, nearly spherical micelles were shown to give the largest increase in alpha-helix content.     

Identification of peptide antagonists to glycoprotein Ibalpha that selectively inhibit von Willebrand factor dependent platelet aggregation

GPIbalpha is an integral membrane protein of the GPIb-IX-V complex found on the platelet surface that interacts with the A1 domain of von Willebrand factor (vWF-A1). The interaction of GPIbalpha with vWF-A1 under conditions of high shear stress is the first step in platelet-driven thrombus formation. Phage display was used to identify peptide antagonists of the GPIbalpha-vWF-A1 interaction. Two nine amino acid cysteine-constrained phage display libraries were screened against GPIbalpha revealing peptides that formed a consensus sequence. A peptide with sequence most representative of the consensus, designated PS-4, was used as the basis for an optimized library. The optimized selection identified additional GPIbalpha binding peptides with sequences nearly identical to the parent peptide. Surface plasmon resonance of the PS-4 parent and two optimized synthetic peptides, OS-1 and OS-2, determined their equilibrium dissociation GPIbalpha binding constants ( K Ds) of 64, 0.74, and 31 nM, respectively. Isothermal calorimetry corroborated the K D of peptide PS-4 with a resulting affinity value of 68 nM. An ELISA demonstrated that peptides PS-4, OS-1, and OS-2 competitively inhibited the interaction between the vWF-A1 domain and GPIbalpha-Fc in a concentration-dependent manner. All three peptides inhibited GPIbalpha-vWF-mediated platelet aggregation induced under high shear conditions using the platelet function analyzer (PFA-100) with full blockade observed at 150 nM for OS-1. In addition, OS-1 blocked ristocetin-induced platelet agglutination of human platelets in plasma with no influence on platelet aggregation induced by several agonists of alternative platelet aggregation pathways, demonstrating that this peptide specifically disrupted the GPIbalpha-vWF-A1 interaction.     

Peptidyl dipeptidases (Ance and Acer) of Drosophila melanogaster: major differences in the substrate specificity of two homologs of human angiotensin I-converting enzyme

Drosophila melanogaster angiotensin converting enzyme (Ance) and angiotensin converting enzyme related (Acer) are single domain homologs of mammalian peptidyl dipeptidase A (angiotensin I-converting enzyme) whose physiological substrates have not as yet been identified. We have investigated the in vitro substrate specificities of the two peptidases towards a variety of insect and mammalian peptides. Ance was generally much better than Acer at hydrolyzing peptides of 5-13 amino acids in length. Only two of the peptides, [Leu(5)]enkephalinamide and leucokinin-I were cleaved faster by Acer. Increasing NaCl concentration had opposite affects on the cleavage of [Leu(5)]enkephalin and [Leu(5)]enkephalinamide by Acer, decreasing the activity towards [Leu(5)]enkephalin but increasing the activity towards [Leu(5)]enkephalinamide. Of the insect peptides tested, the tachykinin-related peptide, Lom TK-1, proved to be the best substrate for Ance with a k(cat)/K(m) ratio of 0.122s(-1) microM(-1). However, in comparison, the D. melanogaster tachykinins, DTK-1, DTK-2, DTK-3 and DTK-4 were poor Ance substrates. DTK-5 was the best substrate of this family, but the apparent high K(m) for hydrolysis by Ance suggested that this peptide would not be a natural Ance substrate. This low affinity for DTK-5 is the likely reason why the peptide was not rapidly degraded in D. melanogaster hemolymph, where Ance was shown to be a major peptide-degrading activity.     

Synthetic, immunological and structural studies on repeat unit peptides of Plasmodium falciparum antigens

Using solid phase methodology, we have synthesized five peptides (16-18 residues long) corresponding to repeat sequences of four antigens of a human malarial parasite, Plasmodium falciparum. Three of these antigens (RESA, FIRA, and ABRA) are found in the asexual blood-stages of the parasite, while the remaining one (CSP) is found in the sporozoites. The synthetic peptides, conjugated to bovine serum albumin, elicited high levels of antibodies in rabbits, and these antibodies were found to cross-react with the heterologous peptides. The degree of cross-reactivity, as estimated in an ELISA, was quite remarkable among all the peptides. The peptide corresponding to the RESA tetrapeptide repeat was found to be the most immunogenic and highly cross-reactive. For this reason this tetrapeptide repeat unit, peptide 1, may be a suitable candidate for inclusion in a multiple epitope polypeptide vaccine design. Conformational studies using circular dichroism spectroscopy show that these peptides have similar conformational characteristics with a common feature of approximately 30% and approximately 50% helical content water and TFE respectively. Theoretical predictions regarding conformation using the Chou-Fasman method have also been presented.     

QSAR analysis of antimicrobial and haemolytic effects of cyclic cationic antimicrobial peptides derived from protegrin-1

In this paper we quantitatively analyse antimicrobial and haemolytic activities of porcine protegrin-1 (PG-1) mimetics-cyclic cationic peptides with beta-hairpin fold synthesised by Robinson et al. [Bioorg. Med. Chem.2005, 13, 2055]. The presented QSAR models, which use molecular properties related to possible mechanisms of cell membrane disruption that can be easily calculated from available data on amino acids, rationalize the relationships between sequences and antimicrobial and haemolytic potencies of the cyclic peptides. The best models obtained by application of genetic function approximation algorithm correlate antimicrobial potencies to the peptide's charge and amphipathicity index, while the haemolytic effect correlates well with the lipophilicity of residues forming the nonpolar face of the beta-hairpin. The models permit selection of site-directed residue substitutions leading to simultaneous optimization of antimicrobial and haemolytic potencies. Examples of such residue substitutions in the nonpolar face of a symmetric cyclic beta-hairpin PG-1 analogue with an ideal amphipathic structure are given.