Understanding the antimicrobial properties/activity of an 11-residue Lys homopeptide by alanine and proline scan.

Previous work demonstrated that lysine homopeptides adopt a polyproline II (PPII) structure. Lysine homopeptides with odd number of residues, especially with 11 residues (K11), were capable of inhibiting the growth of a broader spectrum of bacteria than those with an even number. Confocal studies also determined that K11 was able to localize exclusively in the bacterial membrane, leading to cell death. In this work, the mechanism of action of this peptide was further analyzed focused on examining the structural changes in bacterial membrane induced by K11, and in K11 itself when interacting with bacterial membrane lipids. Moreover, alanine and proline scans were performed for K11 to identify relevant positions in structure conformation and antibacterial activity. To do so, circular dichroism spectroscopy (CD) was conducted in saline phosphate buffer (PBS) and in lipidic vesicles, using large unilamellar vesicles (LUV), composed of 2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) or bacterial membrane lipid. Antimicrobial activity of K11 and their analogs was evaluated in Gram-positive and Gram-negative bacterial strains. The scanning electron microscopy (SEM) micrographs of Staphylococcus aureus ATCC 25923 exposed to the Lys homopeptide at MIC concentration showed blisters and bubbles formed on the bacterial surface, suggesting that K11 exerts its action by destabilizing the bacterial membrane. CD analysis revealed a remarkably enhanced PPII structure of K11 when replacing some of its central residues by proline in PBS. However, when such peptide analogs were confronted with either DMPG-LUV or membrane lipid extract-LUV, the tendency to form PPII structure was severely weakened. On the contrary, K11 peptide showed a remarkably enhanced PPII structure in the presence of DMPG-LUV. Antibacterial tests revealed that K11 was able to inhibit all tested bacteria with an MIC value of 5 µM, while proline and alanine analogs have a reduced activity on Listeria monocytogenes. Besides, the activity against Vibrio parahaemolyticus was affected in most of the alanine-substituted analogs. However, lysine substitutions by alanine or proline at position 7 did not alter the activity against all tested bacterial strains, suggesting that this position can be screened to find a substitute amino acid yielding a peptide with increased antibacterial activity. These results also indicate that the PPII secondary structure of K11 is stabilized by the interaction of the peptide with negatively charged phospholipids in the bacterial membrane, though not being the sole determinant for its antimicrobial activity.

Structural glance into a novel anti-staphylococcal peptide.

Novel antimicrobial peptides are valuable molecules for developing anti-infective drugs to counteract the contemporary spread of microbial drug-resistance. Here we focus on a novel peptide (RKWVWWRNR-NH2) derived from the fragment 107-115 of the human lysozyme that displays a 20-fold increase in anti-staphylococcal activity. The conformational analysis of this peptide and its interaction with model lipidic phases-as assayed by circular dichroism and fluorescence spectroscopy-show a noteworthy spectral change, which might be related to its anti-staphylococcal activity. The secondary structure of peptide [K(108)W(111)] 107-115 hLz was dramatically affected through a single substitution at position 111 (Ala by Trp). Therefore, this conformational change might improve the interaction of the novel peptide with the bacterial plasma membrane. These results highlight the role of peptide secondary structure and the distribution of polar/nonpolar residues for the effective interaction of this peptide with the bacterial plasma membrane, a key step for triggering its lethal effect. This knowledge may contribute to the rational design of a new generation of antimicrobial peptides with increased efficacy developed from natural sources by simple screening tools.

Design of Affinity Chromatography Peptide Ligands Through Combinatorial Peptide Library Screening.

In this chapter, a protocol to design affinity chromatography matrices with short peptide ligands immobilized for protein purification is described. The first step consists of the synthesis of a combinatorial peptide library on the hydroxymethylbenzoyl (HMBA)-ChemMatrix resin by the divide-couple-recombine (DCR) method using the Fmoc chemistry. Next, the library is screened with the protein of interest labeled with a fluorescent dye or biotin. Subsequently, peptides contained on positive beads are identified by tandem matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS/MS), and those sequences showing greater consensus are synthesized in larger quantities and immobilized on chromatographic supports. Finally, target protein adsorption on peptide affinity matrices is evaluated through equilibrium adsorption isotherms and breakthrough curves.

Domino reactions with fluorinated five-membered heterocycles. alpha-Trifluoromethyl alpha-amino acids with unsaturated side-chains.

Alpha-trifluoromethyl alpha-amino acids with unsaturated side-chains have been prepared from 5-fluoro-4-trifluoromethyloxazole and allyl, propargyl as well as terpene alcohols in a one-pot procedure.

Synthesis in vitro of a seven amino acid peptide encoded in the leader RNA of Rous sarcoma virus.

Sequences of avian retroviral RNAs suggest that short open reading frames in the putatively untranslated leader sequences might direct the synthesis of small peptides. Previous analyses indicate that translation of Rous sarcoma virus (RSV) RNA in vitro faithfully reflects translation of the viral RNA in the chick cell. Accordingly, we sought to determine if the heptapeptide LP1, encoded in the open reading frame closest to the 5' end of RSV RNA, could be synthesized in vitro since this would strongly suggest that it might also be synthesized in vivo. Here we confirm that RSV RNA directs the synthesis of LP1 in rabbit reticulocyte lysates. LP1 is rapidly degraded in the lysate by an aminopeptidase activity. On the basis of the following observations, we propose that the open reading frame encoding LP1 plays a role in the life cycle of avian retroviruses. The LP1 open reading frame is ubiquitous with respect to position and length in 12 strains of avian retrovirus. In the amino acid sequences of the 12 strains, only three of the seven residues are invariant. On the basis of the conservation of the -3 and +4 nucleotides flanking the AUG codon, the strengths of initiation for translation of LP1 are approximately the same in the different viruses. The LP1 open reading frame is positioned in front of sites on retrovirus RNA that are required for initiation of cDNA synthesis and for packaging of the RNA into mature virus.

Lysine-50 is a likely site for anchoring the plasminogen N-terminal peptide to lysine-binding kringles.

Interactions between the kringle 4 (K4) domain of human plasminogen (Pgn) and segments of the N-terminal Glu1-Lys77 peptide (NTP) have been investigated via 1H-NMR at 500 MHz. NTP peptide stretches devoid of Lys residues but carrying an internal Arg residue show negligible affinity toward K4 (equilibrium association constant Ka < 0.05 mM(-1)). In contrast, while most fragments containing an internal Lys residue exhibit affinities comparable to that shown by the blocked Lys derivative Nalpha-acetyl-L-lysine-methyl ester (Ka approximately 0.2 mM(-1), peptides encompassing Lys50O consistently show higher Ka values. Among the investigated linear peptides, Nalpha-acetyl-Ala-Phe-Tyr-His-Ser-Ser-Lys5O-Glu-Gln-NH2 (AcAFYHSK5OEQ-NH2) exhibits the strongest interaction with K4 (Ka approximately 1.4 mM(-1)), followed by AcYHSK50EQ-NH2 (Ka approximately 0.9 mM(-1)). Relative to the wild-type sequence, mutated hexapeptides exhibit lesser affinity for K4. When a Lys50 --> Ser mutation was introduced (==> AcYHSS50EQ-NH2), binding was abolished. The Ile27-lle56 construct (L-NTP) contains the Lys50 site within a loop constrained by two cystine bridges. The propensity of recombinant Pgn K1 (rK1) and K2 (rK2) modules, and of Pgn fragments encompassing the intact K4 and K5 domains, for binding L-NTP, was investigated. We find that L-NTP interacts with rK1, rK2, K4, and K5-all lysine-binding kringles-in a fashion that closely mimics what has been observed for the Glul-HSer57 N-terminal fragment of Pgn (CB-NTP). Thus, both the constellation of kringle lysine binding site (LBS) aromatic residues that are perturbed upon complexation of L-NTP and magnitudes of kringle-L-NTP binding affinities (rK1, Ka approximately 4.3 mM(-1); rK2, Ka approximately 3.7 mM(-1; K4, Ka approximately 6.4 mM(1); and K5, Ka approximately 2.1 mM(-1)) are essentially the same as for the corresponding kringle-CB-NTP pairs. Molecular modeling studies suggest that the Glu39-Lys50 stretch in NTP generates an area that complements, both topologically and electrostatically, the solvent-exposed kringle LBS surface.

Structural/functional properties of the Glu1-HSer57 N-terminal fragment of human plasminogen: conformational characterization and interaction with kringle domains.

The Glu1-Val79 N-terminal peptide (NTP) domain of human plasminogen (Pgn) is followed by a tandem array of five kringle (K) structures of approximately 9 kDa each. K1, K2, K4, and K5 contain each a lysine-binding site (LBS). Pgn was cleaved with CNBr and the Glul-HSer57 N-terminal fragment (CB-NTP) isolated. In addition, the Ile27-Ile56 peptide (L-NTP) that spans the doubly S-S bridged loop segment of NTP was synthesized. Pgn kringles were generated either by proteolytic fragmentation of Pgn (K4, K5) or via recombinant gene expression (rK1, rK2, and rK3). Interactions of CB-NTP with each of the Pgn kringles were monitored by 1H-NMR at 500 MHz and values for the equilibrium association constants (Ka) determined: rK1, Ka approximately 4.6 mM(-1); rK2, Ka approximately 3.3 mM(-1); K4, Ka approximately 6.2 mM-'; K5, K, 2.3 mM(-1). Thus, the lysine-binding kringles interact with CB-NTP more strongly than with Nalpha-acetyl-L-lysine methyl ester (Ka < 0.6 mM(-l), which reveals specificity for the NTP. In contrast, CB-NTP does not measurably interact with rK3. which is devoid of a LBS. CB-NTP and L-NTP 1H-NMR spectra were assigned and interproton distances estimated from 1H-1H Overhauser (NOESY) experiments. Structures of L-NTP and the Glul-Ile27 segment of CB-NTP were computed via restrained dynamic simulated annealing/energy minimization (SA/EM) protocols. Conformational models of CB-NTP were generated by joining the two (sub)structures followed by a round of constrained SA/EM. Helical turns are indicated for segments 6-9, 12-16, 28-30, and 45-48. Within the Cys34-Cys42 loop of L-NTP, the structure of the Glu-Glu-Asp-Glu-Glu39 segment appears to be relatively less defined, as is the case for the stretch containing Lys5O within the Cys42-Cys54 segment, consistent with the latter possibly interacting with kringle domains in intact Glul-Pgn. Overall, the CB-NTP and L-NTP fragments are of low regular secondary structure content-as indicated by UV-CD spectra- and exhibit fast amide 1H-2H exchange in 2H2O, suggestive of high flexibility.

Inhibition of HIV-2(ROD) replication in a lymphoblastoid cell line by the alpha1-antitrypsin Portland variant (alpha1-PDX) and the decRVKRcmk peptide: comparison with HIV-1(LAI).

We investigated the effects of alpha1-antitrypsine Portland variant (alpha1-PDX) and decanoylRVKRchloromethylketone (decRVKRcmk) on HIV-2(ROD) replication in the Jurkat lymphoblastoid cell line. To this end, cells were stably transfected with the alpha1-PDX (J-PDX) and used as targets for HIV-2(ROD) infection. Controls were prepared with the empty vector (J-pcDNA3). HIV-2(ROD) and HIV-1(LAI) replications were significantly inhibited and delayed in the presence of the alpha1-PDX protein. When decRVKRcmk was used at 35 microM, inhibition rates were 70-80% for HIV-2(ROD) and HIV-1(LAI), while total inhibition occurred at 70 microM. Control peptides consisting of decanoylRVKR and acetylYVADcmk had no effect. In the presence of the alpha1-PDX or the decRVKRcmk at 35 microM, the infectivity of HIV-2(ROD) and HIV-1(LAI) produced was 3-4-fold lower. Both molecules inhibited syncytium formation by HIV-2(ROD) and HIV-1(LAI) to a considerable extent. Finally, the inhibition of viral replication was correlated with the ability of the decRVKRcmk at 35 and 70 microM and of the alpha1-PDX, to inhibit the processing of envelope glycoprotein precursors. The alpha1-PDX protein and the decRVKRcmk peptide at 35 microM inhibited HIV-2 and HIV-1 to a similar level suggesting that identical or closely related endoproteases are involved in the maturation of their envelope glycoprotein precursors into surface and transmembrane glycoproteins. The significant inhibition observed with alpha1-PDX indicates that furin or furin-like endoproteases appear to play a major role in the maturation process.

Solid-phase peptide synthesis in the reverse (N --> C) direction.

[reaction: see text] A new strategy for SPPS in the reverse direction based on the use of 2-Cl-trityl resin, an allyl ester as the temporary protecting group, and Cu(OBt)(2)/DIPCDI or HATU/DIEA as the coupling method is described. These conditions ensure good yields with minimal racemization of the C-terminal residue.

Substituted guanidines: introducing diversity in combinatorial chemistry.

The guanidine moiety is an important motif present in many biologically active compounds. Fully substituted guanidines are of key importance for the development of bioactive molecules. The present paper reports on an efficient procedure for the direct solid-phase conversion of amines to fully substituted guanidines under very mild conditions.

Alkylation of histidine residues of Bothrops jararacussu venom proteins and isolated phospholipases A2: a biotechnological tool to improve the production of antibodies.

Crude venom of Bothrops jararacussu and isolated phospholipases A2 (PLA2) of this toxin (BthTX-I and BthTX-II) were chemically modified (alkylation) by p-bromophenacyl bromide (BPB) in order to study antibody production capacity in function of the structure-function relationship of these substances (crude venom and PLA2 native and alkylated). BthTX-II showed enzymatic activity, while BthTX-I did not. Alkylation reduced BthTX-II activity by 50% while this process abolished the catalytic and myotoxic activities of BthTX-I, while reducing its edema-inducing activity by about 50%. Antibody production against the native and alkylated forms of BthTX-I and -II and the cross-reactivity of antibodies to native and alkylated toxins did not show any apparent differences and these observations were reinforced by surface plasmon resonance (SPR) data. Histopathological analysis of mouse gastrocnemius muscle sections after injection of PBS, BthTX-I, BthTX-II, or both myotoxins previously incubated with neutralizing antibody showed inhibition of the toxin-induced myotoxicity. These results reveal that the chemical modification of the phospholipases A2 (PLA2) diminished their toxicity but did not alter their antigenicity. This observation indicates that the modified PLA2 may provide a biotechnological tool to attenuate the toxicity of the crude venom, by improving the production of antibodies and decreasing the local toxic effects of this poisonous substance in animals used to produce antivenom.

A novel phospholipase A2 (D49) from the venom of the Crotalus oreganus abyssus (North American Grand canyon rattlesnake).

Currently, Crotalus viridis was divided into two species: Crotalus viridis and Crotalus oreganus. The current classification divides "the old" Crotalus viridis into two new and independent species: Crotalus viridis (subspecies: viridis and nuntius) and Crotalus oreganus (subspecies: abyssus, lutosus, concolor, oreganus, helleri, cerberus, and caliginis). The analysis of a product from cDNA (E6d), derived from the gland of a specie Crotalus viridis viridis, was found to produce an acid phospholipase A2. In this study we isolated and characterized a PLA2 (D49) from Crotalus oreganus abyssus venom. Our studies show that the PLA2 produced from the cDNA of Crotalus viridis viridis (named E6d) is exactly the same PLA2 primary sequence of amino acids isolated from the venom of Crotalus oreganus abyssus. Thus, the PLA2 from E6d cDNA is actually the same PLA2 presented in the venom of Crotalus oreganus abyssus and does not correspond to the venom from Crotalus viridis viridis. These facts highlight the importance of performing more studies on subspecies of Crotalus oreganus and Crotalus viridis, since the old classification may have led to mixed results or mistaken data.

Vascular effects and electrolyte homeostasis of the natriuretic peptide isolated from Crotalus oreganus abyssus (North American Grand Canyon rattlesnake) venom.

Crotalus oreganus abyssus is a rattlesnake that is usually found in the Grand Canyon, United States of America. Knowledge regarding the composition of C. o. abyssus venom is scarce. New natriuretic peptides (NPs) have been isolated and characterized from the venoms of members of the Crotalinae family. The NP family comprises three members, ANP (atrial natriuretic peptide), BNP (b-type natriuretic peptide) and CNP (c-type natriuretic peptide), and has an important role in blood pressure regulation and electrolyte homeostasis. The aim of the present study was to characterize a novel natriuretic-like peptide (Coa_NP2), isolated from C. o. abyssus venom. The Coa_NP2 presents an average molecular mass of 3419.88Da (theoretical average molecular mass 3418.94Da, monoisotopic molecular mass 3416.66Da and theoretical PI 7.78) and its amino acid sequence presents the loop region that is characteristic of natriuretic peptides. The peptide has 32 amino acids and its complete sequence is SYGISSGCFGLKLDRIGTMSGLGCWRLLQDSP. Coa_NP2 is a natriuretic peptide of the ANP/BNP-like family, since the carboxyterminal region of CNP has its own NP domain. We demonstrate, herein, that Coa_NP2 produces a dose-dependent decrease in mean arterial pressure in rats, followed by significant increases in concentrations of markers of nitric oxide formation measured in the plasma and vasorelaxation in a thoracic aortic ring bath. The structural and biological aspects confirm Coa_NP2 as a new natriuretic peptide, isolated from snake venom.

Antioxidant and cytotoxic activities of carob tree fruit pulps are strongly influenced by gender and cultivar.

Extracts from fruit pulps of six female cultivars and two hermaphrodite Portuguese carob trees [(Ceratonia siliqua L., Fabaceae)] exhibited strong antioxidant activity and were rich in phenolic compounds. The extracts decreased the viability of different human cancer cell lines on a dose- and time-dependent manner. Gender and cultivar significantly influenced the chemical content and the biological activities of the extracts. Extracts from hermaphrodite trees had a higher content of phenolic compounds, and exhibited higher antioxidant and cytotoxic activities. Among females, cv. Aida had the highest radical scavenging activity and total content of phenolics, Mulata the highest capacity to inhibit lipid oxidation and Gasparinha the strongest cytotoxic activity on HeLa cells. The decrease in cell viability was associated with apoptosis on HeLa and MDA-MB-231 lines. (+)-Catechin and gallic acid (GA) were the main compounds identified in the extracts, and GA contributed to the antioxidant activity. Our results show that the antioxidant and cytotoxic activities of carob tree fruit pulps are strongly influenced by gender and cultivar, and provide new knowledge about the advantages of hermaphrodite trees over female cultivars, namely, as a source of compounds with biological interest, which may represent an increase of their agronomic interest.