Peptide research最新文献

The formylpeptides formyl-L-methionyl-L-leucyl-L-N-methylphenylalanine methyl ester 1, formyl-L-methionyl-L-leucyl-L-2-oxy-3-phenylpropionic acid methyl ester 2 and formyl-L-methionyl-L-leucyl-L-2-aminoxy-3-phenylpropionic acid methyl ester 3 were synthesized to investigate the role of the amide bond at position 3 in biological activity in human neutrophiles. Our results indicate that this amide bond is required for optimal chemotactic activity, but not for superoxide anion production.

Multiple cyclic antigen peptides (McAPs) are dendrimers that have branched, multiple closed-chain architectures. We describe an approach for their stepwise, solid-phase synthesis that permits a self-assembly of cyclization reactions of a McAP with four copies of cyclic peptides in solution after their cleavage from the resin with all protecting groups removed. The conceptual framework of our approach is the development of a method favoring intrachain cyclization based on ring-chain tautomerism between an N-terminal Cys and an aldehyde attached to the side chain of Lys to form a loop linked by a thiazolidine ring. The McAP precursor contains an amino Cys(St-Bu) and an internal Lys(Ser). A trialklyphosphine is used to deblock Cys(St-Bu) on the amino terminus and to effect the concomitant thiazolidine formation with the glyoxyl moiety obtained from an oxidative conversion of the Ser on the Lys side chain. Two McAPs, each containing cyclic peptides of 17 and 24 amino acid residues, have been prepared. To evaluate intrachain cyclization yields, a cleavage site as Asp-Pro incorporated at the COOH terminus of each monomeric loop and subsequently released after completion of the cyclization by treatment with formic acid at an elevated temperature. Reversed-phase high-performance liquid chromatography analyses of the liberated cyclic peptide monomer with synthetic standards support the theory that intrachain cyclization is the predominant cyclization pathway and validate the usefulness of this ring-chain tautomerization concept in the self-assembly of cyclic peptides on a branched peptide dendrimer.

Phase clones with affinity for polystyrene/polyurethane magnetic particles were isolated from a 10-men peptide display library. Sequence analysis revealed that 40 out of 80 clones contained the consensus WXXWXXXW. Some of the selected phages showed high surface activity and adsorbed to plastic surfaces even in the presence of blocking agents or surfactants. Covalent attachment of a synthetic peptide (KG), carrying one of the selected sequences to alkaline phosphatase (AP) or bovine serum albumin (BSA) enhanced binding of AP to a wide range of materials and improved the ability of BSA to prevent binding of antibodies and phages to polystyrene. Interestingly, the WXXW/XXXW motif occurs in the beta- and gamma-chains of the natural "adhesive" protein fibrinogen, and a synthetic peptide carrying the gamma-chain 369-376 sequence turned out to have essentially the same binding properties as the KG peptide. Furthermore, adsorption in different types of polystyrene was similar for AP carrying either the KG or gamma-chain peptide intact fibrinogen and plasmin-generated fragment D1. The latter fragment contains two copies of the WXXWXXXW motif but lacks the alpha-chain: protuberances previously implicated in fibrinogen adsorption. Thus, our study may have revealed a hitherto unknown structural determinant for fibrinogen's adsorptivity, located in the 13-kDa C terminal region of the gamma-chain.

We have synthesized a hydrophilic crosslinked aminoalkyl polydimethylacrylamide-beaded support upon which peptides have been assembled using standard Fmoc chemistry in automated batch-wise equipment. The resin was prepared by the free radical-initiated co-polymerization of N,N-dimethylacryl-amide, N,N'-bisacrylyl-1,3-diaminopropane and a functional monomer N-methacrylyl-1,3-diaminopropane hydrochlorid. After coupling of N-alpha-tert-butyloxycarbonyl-glycine (Boc-glycine), amino acid analyses gave resin loading capacities of 0.66 mmol/g. The resulting polymer was highly swollen by polar solvents including aqueous buffers and had an exclusion limit of 50 kDa for soluble proteins. This resin was found to be an excellent support for peptide synthesis using Fmoc chemistry. Typical purities of crude peptides were 80%-95%, including sequences that failed on conventional polystyrene resins.

A variety of thiol-containing compounds, in combination with m-cresol, were tested as scavengers in hydrogen fluoride (HF) cocktails that are used to deprotect haloacetylated peptidyl resins. Our results indicate that brome and chloroacetyl moieties on a synthetic peptide remain intact following HF treatment when the HF cocktail contains m-cresol along with either thiophenol, m-thiocresol or 1,2-ethanedithiol. The free thiols prevent the formation of a number of impurities in the preparation of bromo- and chloroacetylated peptides that contain amino acids that could be oxidized in a nonreducing HF environment. Ethvimethylsulfide, however, could not be used with bromoacetylated peptides, but it could be used with chloroacetylated peptides.

Human statherin, at low molecular weight (M 5380 Da. 43 amino acid residues) acidic tyrosine-rich phosphoprotein secreted mainly by salivary glands, has been synthesized successfully for the first time following standard solid-phase Fmoc chemistry. Synthesis of this phosphoprotein was accomplished using preformed phosphoserin building blocks. The phosphorylated protein thus synthesized was analyzed and compared with the native molecule and was found to have identical characteristics in its entirety, is evidenced by various analytical methods including mass spectral analysis. Analysis of both the synthetic and native statherin by circular dichroism spectroscopy showed an increase in helicity upon the addition of an organic cosolvent, trifluoroethanol (50%, vol/vol), indicating the presence of potentially amphipathic helical regions. Circular dichroism studies and hydrophobic moment calculations on this synthetic phosphoprotein revealed that the molecule adopts an amphipathic helical conformation at the N-terminus connected to a long poly-L-proline type II segment, which, in turn, is linked to an extended beta-strand. In correlation with previous studies. It appears that the strong binding affinity of statherin for hydroxyapatite can be attributed primarily to the N-terminal sequence, which prefers to adopted helical conformation and provides both electrostatic and hydrogen bonding interactions, thereby inhibiting its mineralization. Production of this highly homogenous synthetic statherin by chemical means may circumvent the prevailing obstacles encountered in conducting its tertiary structural investigations under various physiological conditions.

Plant defensins are a class of cysteine-rich peptides of which several members have been shown to be potent inhibitors of fungal growth. A series of overlapping 15-mer peptides based on the amino acid sequence of the radish antifungal protein Rs-AFP2 have been synthesized. Peptides 6, 7, 8 and 9, comprising the region from cysteine 27 to cysteine 47 of Rs-AFP2 showed substantial antifungal activity against several fungal species (minimal inhibitory concentrations of 30-60 micrograms/mL), but no activity towards bacteria (except peptide 6 at 100 micrograms/mL). The active peptides were shown to be sensitive to the presence of cations in the medium and to the composition and pH of the medium. When present at a subinhibitory concentration (20 micrograms/mL), peptides 1, 7, 8 and 10 potentiated the activity of Rs-AFP2 from 2.3-fold to 2.8-fold. By mapping the characteristics of the active peptide on the structure of Rs-AFP2 as determined by nuclear magnetic resonance, the active region of the antifungal protein appears to involve beta-strands 2 and 3 in combination with the loop connecting those strands. A cyclized synthetic mimic of the loop, cysteine 36 to cysteine 45, was shown to have antifungal activity. Substitution of tyrosine 38 by alanine in the cyclic peptide substantially reduced the antifungal activity, indicating the importance of this residue for the activity of Rs-AFP2 as demonstrated carrier by mutational analysis.

Five peptides related to the N-terminal sequence of the vasoactive intestinal peptide (VIP) have been synthesized. Two-dimensional nuclear magnetic resonance (2D-NMR) experiments (i.e., correlated spectroscopy [COSY]) and low temperature coefficient measurements for particular NH chemical shifts suggest the presence of hydrogen bondings in both VIP (1-7, and VIP (1-11) fragments. Nuclear Over-hauser enhancement spectroscopy (NOESY) show that aromatic interactions stabilize a preferred conformation. The crucial role of the first histidine residue, which is a determinant for the biological activity, is explained by specific interactions with the aromatic protons of Phe6 and Tyr10.

The molecular and crystal structures of four peptides containing one L-Trp guest residue in Aib (alpha-aminoisobutyric acid or C alpha-methyl alanine) host oligopeptide chains have been determined by X-ray diffraction. The peptides are Z-Aib-L-Trp-Aib-OMe, Z-(Aib)2-L-Trp-Aib-OMe, Z-(Aib)3-L-Trp-Aib-OtBu and Boc-(Aib)3-L-Trp-Aib-OMe. Right-handed beta-turns and incipient and fully developed 3(10)-helices are formed in the crystal state by the tri-, tetra- and pentapeptides, respectively. The Trp residue is easily accommodated in these folded structures. The average geometry and preferred conformation for the Trp indolyl side chain are also discussed.

The sites of the disulfide bonds in a napin protein isolated from Brassica napus have been identified by proteolytic cleavage and subsequent peptide mapping by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Napins consist of two polypeptide chains containing two and six cysteine residues, respectively, that are held together by disulfide bonds. Upon initial cleavage of native napin by Endo-Lys-C, a disulfide-linked core complex of four peptides was obtained. This core peptide was isolated by reversed-phase HPLC and further digested by thermolysin, and the resulting fragments were identified by MALDI-MS. In a separate set of experiments, intact napin was subjected to proteolysis by thermolysin, and an isolated disulfide-linked peptide of interest was subdigested again using thermolysin. The combined data resulting from these experiments allowed the assignment of the disulfide linkages in a relatively abundant napin isoform, BngNAP1, apart from an ambiguity concerning the adjacent cysteines at positions 14' and 15' of the long chain. Two intermolecular disulfide bonds link Cys10 (short chain) with Cys25' (long chain) and Cys23 with Cys14' (or Cys15'), respectively. The long chain of napin contains two intramolecular disulfide bonds connecting Cys27' with Cys80' and Cys14' (or Cys15') with Cys72'.