Peptide research最新文献

An Epstein-Barr virus-specific cytotoxic T cell (CTL) clone was previously shown to recognize the epitope FLRGRAYGL. The ability of substituted peptides to sensitize target cells for lysis by this clone was investigated using two peptide replacement sets, one synthesized with free carboxyl termini and another made with a carboxyl terminal beta-alanine-diketopiperazine (beta A-DKP) group. Curiously, the effect of certain substitutions differed for the two peptide sets. For example, NH2-FLRGRAYGL-beta A-DKP was 15-fold more active than NH2-FLRGRAYGI-beta A-DKR, but NH2-FLRGRAY-GL-COOH had the same activity as NH2-FLRGRAYGI-COOH. Evidence presented here illustrates that the activity of beta A-DKP peptide preparations was entirely due to contaminating peptides with free-COOH termini. The amount of active-COOH contaminants within a -beta A-DKP preparation thus appeared to vary depending on the substitution, resulting in some anomalous results from replacement set analysis using -beta A-DKP peptides.

The cleavage of a peptide resin attached to a phenylacetomidomethyl (PAM) resin was investigated using bromotrimethylsilane (TMSBr) with thioanisole in trifluoroacetic acid (TFA), and by chlorotrimethylsilane (TMSCl) in the same reagents with lithium bromide. Both TMSBr and TMSCl cleaved the peptide from the resin, but TMSCl required elevated temperature (50 degrees C) to effect the cleavage. Procedures were investigated for the deprotection and cleavage of either N(g)-tosyl (Tos)- or mesitylenesulfonyl (MTS)-arginine residues attached to 4-methylbenzylhydrylamine (MBHA) resin, and of a peptide containing an MTS-arginine residue attached to MBHA resin, using either TMSCl or TMSBr as cleavage reagents. The MTS group is cleanly removed from arginine using TMSBr with thioanisole in TFA and by TMSCl in the same reagents with lithium bromide.

The first x-ray diffraction structure analysis of a C alpha-ethyl, C alpha-benzylglycine [(alpha Et)Phe]-containing peptide, N alpha-benzyloxycarbonyl-alpha-aminoisobutyryl-alpha-amino-isobutyr yl-(S)- C alpha-benzylglycyl-alpha-aminoisobutyric acid (methanol solvate), has been performed. In the crystal state the N alpha-protected tetrapeptide is folded in an incipient, left-handed 3(10)-helical structure. This finding confirms that the relationship between (alpha Et)Phe alpha-carbon chirality and screw sense of the helix that is formed is opposite to that exhibited by protein amino acids, including Phe.

A synthetic peptide strategy was used to study structure-function relationships between residues 32 to 46 of the glycoprotein hormone alpha subunit (GPH alpha) and the testicular follicle-stimulating hormone (FSH) and luteinizing hormone (LH/hCG) receptors. A peptide amide corresponding to this region [GPH-alpha-(32-46)] inhibited both 125I-hFSH and 125I-hCG binding to their respective calf testis membrane receptors. The concentration at which GPH-alpha-(32-46) peptide amide inhibited FSH binding by 50% (IC50) was 36 microM, and for hCG it was 54 microM. GPH-alpha-(32-46) peptide amide also inhibited FSH-stimulated estradiol biosynthesis in cultured rat Sertoli cells. In order to determine the involvement of individual residues within this region of the glycoprotein hormone alpha subunit in receptor binding inhibitory activity, truncated and alanine-substituted peptide analogs were synthesized and tested in both FSH and hCG radioligand receptor competition assays. Based on the relative potency of each peptide, we conclude that Phe-33, Arg-35, Arg-42, Ser-43 and Lys-44 may be important, and Cys-32 is required, for inhibition of FSH and hCG binding to their respective receptor. Our results demonstrate involvement of the glycoprotein hormone alpha-subunit in receptor binding, identify residues 32 to 46 as a receptor binding domain, and define the relative importance of specific residues within this region of the alpha subunit for hormone-receptor interaction.

Two cytosolic regions of the rat testicular FSH receptor (FSHR), residues 533-555 and 645-653, have been identified as G protein-coupling domains. We localized the activity in these domains to their C-terminal sequences, residues 551-555 (KIAKR, net charge +3) and 650-653 (RKSH, net charge +3), and examined the effects of charge on G protein activation by the C-terminal peptides, using synthetic analogs containing additions, through alanine (A) linkages, of arginine (R, +), histidine (H, +) or both. RA-KIAKR (net charge +4) mimicked the effect of FSHR-(551-555) on guanine nucleotide exchange in rat testis membranes, but reduced its ability to inhibit FSH-stimulated estradiol biosynthesis in cultured rat Sertoli cells. Further increasing net charge by the addition of H (HARA-KIAKR, net charge +5) increased guanosine 5'-triphosphate (GTP) binding, but eliminated FSHR-(551-555) effects on FSH-stimulated steroidogenesis. HA-RKSH (net charge +4) significantly inhibited guanine nucleotide exchange in rat testis membranes, but stimulated basal and potentiated FSH-induced estradiol biosynthesis in cultured rat Sertoli cells. Addition of two H residues (HAHA-RKSH, net charge +5) restored GTP binding and further potentiated basal and FSH-stimulated steroidogenesis. These results suggest that positive charges in G protein-coupling domains of the FSHR play a role in modulating G protein activation and postbinding effects of FSH, such as steroidogenesis.

Glycosaminoglycans are complex sulfated polysaccharides with a diverse range of biological functions. Three glycosaminoglycan standards--chondroitin sulfate, dermatan sulfate and heparin--were characterized during this study. The interaction of the heparin binding site of protein C inhibitor, represented by the peptide sequence 264-283, in solution with the above glycosaminoglycan standards was studied. Circular dichroism spectroscopy was used to determine the dominant secondary structure induced in the peptide upon binding the relevant glycosaminoglycans. The various glycosaminoglycans induced different secondary structures. The level of induced secondary structure by dermatan sulfate and heparin was approximately twice that induced by chondroitin sulfate. For chondroitin sulfate and heparin, alpha-helix was the dominant ordered secondary structure, whereas for dermatan sulfate the beta-strand conformation dominated. The order of secondary structure induction of the protein C inhibitor peptide by the glycosaminoglycans paralleled the reported biological activities of these glycosaminoglycans for mediation of the biological activity in the intact protein. The strength of the interaction of dermatan sulfate and heparin with the protein C inhibitor peptide was measured by determining the concentration of salt required to inhibit 50% of the interaction. The values determined were 0.1 and 0.3 M salt for dermatan sulfate and heparin, respectively. These results show that different glycosaminoglycans can support different secondary structures in the protein C inhibitor peptide.

PLTX II, a presynaptic calcium channel blocker in Drosophila isolated from the plectreurys spider venom, is a 44-residue peptide containing ten Cys residues and an O-palmitoylated threonine amide at the carboxy-terminus. In this study, the palmitoylated peptide was synthesized in solution by applying our maximum protection strategy using the HF method at the final deprotecting step. Before designing the synthesis, we examined the stability of the palmitoyl moiety under the conditions for the synthesis of the peptide using several model peptides. The O-palmitoyl group was confirmed to be stable during elongation of the peptide bonds, but was partially removable during the deprotection reaction in HF. The depalmitoylation reaction in HF was temperature- and time-dependent. Therefore, the decision was made to protect the Asp residues with benzyl ester, since it is more susceptible to HF than cyclohexyl ester, which is now commonly used in the Boc-based, solid-phase synthesis. Thus, the HF reaction was carried out at -10 degrees or -15 degrees C for 1 h in order to reduce the extent of the depalmitoylation reaction. The resulting palmitoylated and depalmitoylated products were separated, the remaining Acm groups were removed using Hg(OAc)2, and then the completely deprotected peptides were folded to their native forms. The final palmitoylated peptide was proven to be identical with the natural one using various HPLC systems and by bioassay.(ABSTRACT TRUNCATED AT 250 WORDS)

The detection of secondary amino groups in building blocks attached to solid phase using tetrachlorobenzoquinone (chloranil) was re-examined. A new procedure, about ten times more sensitive than the original method, has been developed. Except for N(p-tolyl)glycine, all secondary amino groups that were examined were detected reliably in a substitution range down to 2.8 micro eq/g. Protected imidazole in His(Trt) did not interfere with the detection method.

A synthetic library that presents potential pharmacophores in a linear fashion with variable spacing was designed (alpha, beta, gamma-library). To prove the concept, we synthesized a number of individual compounds as well as a model library. Diamino acids connected by amide bonds via their alpha- or side-chain amino groups were used to form the backbone (scaffold) of this library. The remaining amino group of the diamino acids were acylated by a variety of carboxylic acids, generating an appreciable diversity of compounds in this library. The compositions of compounds in the library were identified by reading a peptide tag synthesized concurrently with the library structures. This code contained the information regarding the carboxylic acid coupled, and the diamino acid and amino group to which the acid was coupled.

The solution structures of human calcitonin gene-related peptide (hCGRP, 37 residues) and of its antagonistic fragment hCGRP 8-37 have been determined by two-dimensional 1H nuclear magnetic resonance (NMR) spectroscopy and molecular modeling. Analysis of the double quantum filtered correlation spectroscopy, total correlation spectroscopy and nuclear Overhauser enhancement spectroscopy spectra led to a complete assignment and to the identification of more than 350 intra- and interresidue connectivities for each peptide. Molecular models were calculated by molecular dynamics and energy minimization using distance constraints. The structure of hCGRP is characterized by a rigid N-terminal disulfide-bonded loop followed by helix segments (Val8-Leu16), a gamma-turn (Ser19-Gly21) and several local hydrogen-bonded patterns. The structure of hCGRP 8-37 is less defined than the structure of hCGRP and no helix structure is present. Molecular models of both peptides are consistent with the NH temperature coefficients and secondary chemical shifts of the alpha-protons. Hydrogen bonding with the disulfide-bonded ring appears to be critical for helix formation, both structural elements being essential for agonistic activity.