Structure-based design of a fluorimetric redox active peptide probe

Structure-based iterative design was used to prepare a disulfide-containing nonapeptide as a fluorimetric probe for chemical and biochemical disulfide forming and breaking reactions. The peptide is composed entirely of natural amino acids and exhibits a marked (42%) change in fluorescence between its oxidized and its reduced states. The probe is easily synthesized and highly water soluble and exhibits well-behaved kinetics on reduction with the reductant tris-carboxyethylphosphine. The reduced peptide is an excellent substrate of the enzyme quiescin-sulfhydryl oxidase and may find utility in the characterization of other disulfide oxidoreductases.     

Enzymatic synthesis of delta sleep-inducing peptide

The delta sleep-inducing peptide was assembled enzymatically from three tripeptide fragments. All the peptide bonds were prepared by either papain- or alpha-chymotrypsin-mediated synthesis. Secondary hydrolysis was suppressed by introducing N alpha-protected amino acid or peptide esters as carboxyl components and using an alkaline pH. The protected nonapeptide was oxidized with ferric chloride to deprotect the C-terminal phenylhydrazide and then hydrogenated. The homogeneous peptide was obtained by reversed phase high-performance liquid chromatography. Comparison of enzymatic and chemical preparations showed no obvious differences.     

Covalent attachment of an Arg-Gly-Asp sequence peptide to derivatizable polyacrylamide surfaces: support of fibroblast adhesion and long-term growth

A synthetic nonapeptide (Tyr-Ala-Val-Thr-Gly-Arg-Gly-Asp-Ser), which includes the adhesive Arg-Gly-Asp (RGD) sequence, was covalently immobilized on chemically well-defined polyacrylamide gel surfaces utilizing N-succinimidyl active esters. The amount of peptide immobilized varied linearly with the concentration added to the gels. Immobilization was approximately 80% efficient (based on peptide added), resulting in up to 17.5 nmol peptide/cm2 gel surface. Balb/c 3T3 mouse fibroblast cells adhered readily to peptide-derivatized surfaces, even in the absence of serum. Furthermore, surfaces derivatized with 2 nmol peptide/cm2 gel supported long-term fibroblast growth at a rate and to an extent comparable to that on tissue culture plastic. Surfaces derivatized with a control nonapeptide having no RGD sequence were nonsupportive of cell attachment or growth. The immobilization technology used to derivatize the gel surfaces with adhesive nonapeptide can be modified to allow coderivatization with proteins, glycoproteins, glycosides, or other amine-containing compounds to test their effects on long-term cell behaviors.     

Characterization of the amino acids essential for the photo- and radioprotective effects of a Bowman-Birk protease inhibitor-derived nonapeptide

The Bowman-Birk protease inhibitor has been reported to exert photo- and radioprotective activity. This effect was assigned to a cyclic nonapeptide sequence which is known to contain the amino acids responsible for the anti-chymotryptic activity of the BBI. The present study indicated that linearization of the nonapeptide resulted in a significant loss of anti-proteolytic activity, whereas the photo- and radioprotective capacity persisted. Substitution of the amino acids Leu or Ser of the nonapeptide, essential for the anti-proteolytic activity, with different amino acids, indicated that rather the hydrophobic features of the amino acids in this position than charge are critical to retain the photo- and radioprotective effect. These results suggest the existence of a bifunctional peptide sequence with anti-proteolytic and photo-/radioprotective capacity. However, the lack of correlation between the photo-/radioprotective activity and the anti-proteolytic activity within the peptides generated by modification of the linear nonapeptide argues for the existence of two closely colocalized domains within the nonapeptide responsible for photo-/radioprotection and protease inhibition.     

Differential peptide dynamics is linked to major histocompatibility complex polymorphism

Peptide presentation by major histocompatibility complex (MHC) molecules is of central importance for immune responses, which are triggered through recognition of peptide-loaded MHC molecules (pMHC) by cellular ligands such as T-cell receptors (TCR). However, a unifying link between structural features of pMHC and cellular responses has not been established. Instead, pMHC/TCR binding studies suggest conformational and/or flexibility changes of the binding partners as a possible cause of differential T-cell stimulation, but information on real-time dynamics is lacking. We therefore probed the real-time dynamics of a MHC-bound nonapeptide (m9), by combining time-resolved fluorescence depolarization and molecular dynamics simulations. Here we show that the nanosecond dynamics of this peptide presented by two human MHC class I subtypes (HLA-B*2705 and HLA-B*2709) with differential autoimmune disease association varies dramatically, despite virtually identical crystal structures. The peptide dynamics is linked to the single, buried polymorphic residue 116 in the peptide binding groove. Pronounced peptide flexibility is seen only for the non-disease-associated subtype HLA-B*2709, suggesting an entropic control of peptide recognition. Thermodynamic data obtained for two additional peptides support this hypothesis.     

Chiral interaction in peptide molecules: effects of chiral peptide species on helix-sense induction in an N-terminal-free achiral peptide

Noncovalent chiral domino effect (NCDE) has been proposed as terminal-specific interaction upon a 3(10)-helical peptide chain, of which the helix sense is manipulated by an external chiral stimulus (mainly amino acid derivatives) operating on the N-terminus (Inai, Y., et al. J Am Chem Soc 2000, 122, 11731-11732; ibid., 2002, 124, 2466-2473; ibid., 2003, 125, 8151-8162). We have investigated here a helix-sense induction in an optically inactive N-terminal-free nonapeptide (1) through the screening of several peptide species that differ in chiral sequence, chain length, and C-terminal group. Helix-sense induction in peptide 1 depends largely on both the C-terminal chirality and carboxyl group in the external peptide, in which N-carbonyl-blocked amino acids, "monopeptide acids," should be the minimum requirement for effective induction. N-Protected mono- to tetrapeptides of L-Leu residue commonly induce a right-handed helix. NMR study and theoretical computation reveal that the N-terminal segment of peptide 1 binds the N-protected dipeptide molecule through multipoint coordination to induce a right-handed helix preferentially. The present findings not only will improve our understanding of the chiral roles in peptide or protein helical termini, but also might demonstrate potential applications to chirality-responsive materials based on peptide helical fragments.     

Deprotonation of hydrochloride salts of amino acid esters and peptide esters using commercial zinc dust.

The deprotonation of hydrochloride salts of ethyl and methyl esters of amino acids and peptides is accomplished using activated zinc dust. The reaction is neat and quantitative. Thus, the free amino acid esters and peptide esters have been isolated in good yield and purity.     

Effects of secondary interactions on the kinetics of peptide and peptide ester hydrolysis by tissue kallikrein and trypsin

Kinetic constants for the hydrolysis by porcine tissue beta-kallikrein B and by bovine trypsin of a number of peptides related to the sequence of kininogen (also one containing a P2 glycine residue instead of phenylalanine) and of a series of corresponding arginyl peptide esters with various apolar P2 residues have been determined under strictly comparative conditions. kcat and kcat/Km values for the hydrolysis of the Arg-Ser bonds of the peptides by trypsin are conspicuously high. kcat for the best of the peptide substrates, Ac-Phe-Arg-Ser-Val-NH2, even reaches kcat for the corresponding methyl ester, indicating rate-limiting deacylation also in the hydrolysis of a peptide bond by this enzyme. kcat/Km for the hydrolysis of the peptide esters with different nonpolar L-amino acids in P2 is remarkably constant (range 1.7), as it is for the pair of the above pentapeptides with P2 glycine or phenylalanine. kcat for the ester substrates varies fivefold, however, being greatest for the P2 glycine compounds. Obviously, an increased potential of a P2 residue for interactions with the enzyme lowers the rate of deacylation. In contrast to results obtained with chymotrypsin and pancreatic elastase, trypsin is well able to tolerate a P3 proline residue. In the hydrolysis of peptide esters, tissue kallikrein is definitely superior to trypsin. Conversely, peptide bonds are hydrolyzed less efficiently by tissue kallikrein and the acylation reaction is rate-limiting. The influence of the length of peptide substrates is similar in both enzymes and indicates an extension of the substrate recognition site from subsite S3 to at least S'3 of tissue kallikrein and the importance of a hydrogen bond between the P3 carbonyl group and Gly-216 of the enzymes. Tissue kallikrein also tolerates a P3 proline residue well. In sharp contrast to the behaviour of trypsin is the very strong influence of the P2 residue in tissue-kallikrein-catalyzed reactions. kcat/Km varies 75-fold in the series of the dipeptide esters with nonpolar L-amino acid residues in P2, a P2 glycine residue furnishing the worst and phenylalanine the best substrate, whereas this exchange in the pentapeptides changes kcat/Km as much as 730-fold. This behaviour, together with the high value of kcat/Km for Ac-Phe-Arg-OMe of 3.75 X 10(7) M-1 s-1, suggests rate-limiting binding (k1) in the hydrolysis of the best ester substrates.(ABSTRACT TRUNCATED AT 400 WORDS)     

The applicability of subtilisin Carlsberg in peptide synthesis.

The synthesis of peptide bonds catalysed by subtilisin Carlsberg was studied in different hydrophilic organic solvents with variable H2O concentration. Z-Val-Trp-OMe and Z-Ala-Phe-OMe were used as acyl donors, and a series of amino acid derivatives, di- and tripeptides of the general structure Xaa-Gly, Gly-Xaa, Gly-Gly-Xaa (Xaa represents all natural L-amino acids except cysteine) and other peptides were used as nucleophiles. A comparative study of the enzymatic synthesis in aqueous DMF (50%, v/v) and acetonitrile containing 10% (v/v) of H2O demonstrated that the yields of peptide products were higher in most cases when acetonitrile with low H2O concentration was used. The acylation of weak nucleophiles was improved in organic solvents with very low H2O concentration (2%). The reactions in anhydrous Bu(t)-OH proceeded with substantially lower velocity. Generally, the restricted nucleophile specificity of the enzyme for glycine and hydrophilic amino acid residues in P1' position, as well as numerous side reactions, limit the utilization of subtilisin in peptide synthesis, especially in the case of the segment condensations. Contrary to the published data, we have proved that proline derivatives were not acylated in any media with the help of subtilisin Carlsberg. Effective ester hydrolysis of a protected nonapeptide corresponding to the N-terminal sequence of dicarba-eel-calcitonin catalysed by subtilisin was achieved.     

A short peptide eluted from the H-2Kb molecule of a polyomavirus-positive tumor corresponds to polyomavirus large T antigen peptide at amino acids 578 to 585 and induces polyomavirus-specific immunity.

A short peptide in complex with the H-2Kb molecule on PyRMA, a polyomavirus transfectant of the mouse lymphoma cell line RMA, was identified as a polyomavirus tumor-specific transplantation antigen. The peptide was obtained by affinity chromatography, acidic extraction, and reverse-phase high-pressure liquid chromatography (HPLC). In one HPLC fraction, a peptide sequence in which 5 of 8 amino acids, GKxGLxxA, corresponded to residues 578 to 585 of polyomavirus large T antigen was identified. In tumor rejection assays, we therefore tested three related synthetic peptides, corresponding to the octapeptide LT 578-585, GKTGLAAA; the nonapeptide LT 578-586, GKTGLAAAL; and the decapeptide LT 578-587, GKTGLAAALI. The octapeptide was found to give the most effective immunization against the outgrowth of the polyomavirus DNA-positive PyRMA tumor. However, none of the three peptides immunized against the original polyoma-virus-negative RMA line.     

Alamethicin and synthetic peptide fragments as uncouplers of mitochondrial oxidative phosphorylation. Effect of chain length and change

Alamethicin, its derivatives and some synthetic fragments have been shown to be uncouplers of oxidative phosphorylation in rat liver mitochondria. A minimum peptide chain length of 13 residues is necessary for this activity. Peptide esters are more efficient uncouplers than the corresponding peptide acids. Esterification of the Glu(18) γ-COOH group in alamethicin does not diminish uncoupling activity. The structural requirements for uncoupling activity parallel those determined for ionophoretic action in small, unilamellar liposomes.     

alpha-Chymotrypsin as the catalyst for peptide synthesis.

alpha-Chymotrypsin (EC 3.4.21.1)-catalysed syntheses of peptides were performed with various N-acylated amino acid or peptide esters as donors, and amino acid derivatives, peptides or their derivatives as acceptors. Under optimal conditions the synthesis was almost quantitative. As acceptor nucleophiles, free amino acids or the ester derivatives were inadequate, but amino acid amides or hydrazides, di- or tri-peptides, or the amides, hydrazides and esters of the peptides were useful. The nucleophile specificity for synthesis was markedly similar to the leaving-group specificity in hydrolysis; hydrophobic or bulky amino acid residues were most effecient at both P1' and P2' positions [notation of Schechter & Berger (1967) Biochem. Biophys. Res. Commun. 27, 157-162], but L-proline as well as D-amino acid residues were the worst choices. The synthesis was further dependent on the solubility of the products synthesized; a higher yield of products was expected with lower solubility. As donor esters, good substrates were all useful. Accordingly, fragment condensation was possible by using N-acylated peptide esters and various peptides. The present study suggested that alpha-chymotrypsin may become a useful tool for peptide synthesis.     

Inhibition of smooth muscle contraction and platelet aggregation by peptide 204-212 of lipocortin 5: an attempt to define some structure requirements

Peptide 204-212 of lipocortin (LC) 5 inhibited porcine pancreatic phospholipase A(2) (PLA(2)) induced rat stomach strip contractions and ADP induced rabbit platelet aggregation in a concentration dependent manner (IC(30) of 10 muM and 400 muM, respectively). The first two amino acids are not necessary since the eptapeptide 206-212 was equipotent in both assays (IC(30) of 12.5 muM and 420 muM). Of the two pentapeptides 204-208 and 208-212 only the latter showed inhibitory activity in both models although the potency was much reduced (IC(30) of 170 muM and 630 muM) compared with that of the parent nonapeptide. Comparison of peptide 204-212 effects with those of its analogues on LC1 and LC2 indicate that lysine 208 and aspartic acid 211 are essential in order to maintain a fully active nonapeptide.     

A novel arginine vasopressin-binding peptide that blocks arginine vasopressin modulation of immune function

We report on a novel peptide that blocks the neuroendocrine hormone arginine vasopressin (AVP) helper signal for IFN-gamma production by direct interaction with the hormone. The AVP-binding nonapeptide has the sequence Thr-Met-Lys-Val-Leu-Thr-Gly-Ser-Pro (binding peptide). AVP and its 6-amino acid N-terminus cyclic ring pressinoic acid (PA) are both capable of replacing the IL-2 requirement for IFN-gamma production by mouse splenic lymphocytes. We show that the AVP-binding peptide specifically and reversibly blocks AVP help in IFN-gamma production, but fails to block the helper signal of PA. Thus the intact AVP molecule and not just the N-terminal cyclic ring is important for interaction with the binding peptide. AVP interacts with the binding peptide with an apparent KD of approximately 50 nM. The AVP-binding peptide does not inhibit AVP interaction with its receptor on lymphocytes. Interestingly, whereas the AVP-binding peptide does not block the PA helper signal for IFN-gamma induction, the complex of AVP and binding peptide does reversibly block the PA signal. The AVP family of hormones requires conformational flexibility for signal transduction. Thus, we hypothesize that the AVP-binding peptide restricts this flexibility and converts AVP into an antagonist of its own action.     

Characterization of the carboxyl terminal flanking peptide of rat progastrin

A peptide identical in structure to the carboxyl-terminal flanking nonapeptide of rat progastrin, predicted by cDNA sequence, was synthesized. The synthetic peptide was used for production of a rabbit antiserum. This antiserum was used to develop a radioimmunoassay specific for rat carboxyl terminal flanking peptide. This assay was used to monitor the purification of immunoreactivity from rat antral extracts. Gel permeation, anion exchange and reverse phase chromatography steps resulted in a single absorbance peak associated with the carboxyl terminal flanking peptide immunoreactivity. The purified peptide eluted in the same position as the synthetic peptide during all three types of chromatography. This material was shown to be identical in mass to Ser-Ala-Glu-Glu-Glu-Asp-Gln-Tyr-Asn, the predicted sequence of the carboxyl terminal nonapeptide of rat progastrin.     

Methods and strategies of peptide ligation.

This review focuses on the concept, methods, and strategies of orthogonal peptide ligation. It updates our previous review in 1999 on the same subject matter in Biopolymers (Peptide Science, 1999, Vol. 51, p. 311). Orthogonal peptide ligation is an amino terminal specific method to couple chemically unprotected peptides or proteins derived from synthetic or biosynthetic sources. Unlike conventional chemical methods, peptide ligation methods do not require coupling reagents or protection schemes, but are achieved through a variable chemoselective capture step and then an invariable intramolecular acyl transfer reaction. It is also a convergent method with the fewest steps. More than a dozen orthogonal ligation methods have been developed based on captures by either imine or thioester chemistries to afford native and unusual amino acids at ligation sites of linear, branched, or cyclic peptides. The ligation strategies for multiple segments including sequential and tandem ligations are also discussed.     

Polymer-bound alkyltriazenes for mild racemization-free esterification of amino acid and peptide derivatives.

A novel tool for polymer-assisted solution phase (PASP) esterification of amino acid and peptide derivatives has been developed. When treated with carboxylic acids, polymer-bound alkyltriazenes react with a loss of nitrogen and transfer of the alkyl moiety to the carboxylate anion to form the corresponding alkyl esters. There are no limitations with regard to either the protecting groups or the nature of the amino acid. Furthermore no racemization occurs at the chiral centers of the amino acids as demonstrated by chiral GC-MS analyses. Alkyltriazene-resins were also applied successfully to the esterification of peptide acids and other peptidic structures, such as tripalmitoyl-S-glyceryl-cysteine (Pam3Cys). The triazene-mediated esterification reaction is exceptionally mild, and there is no need for prior activation of the carboxy groups. This method is therefore particularly suitable for the alkylation of complex peptidomimetic structures prone to racemization and for acid-sensitive structures.     

Comparison of the fatty acids in Baltic herring and available plankton feed

Triglycerides and phospholipids dominate in Baltic herring flesh lipids throughout the plankton growth season though at the same time plankton species and their lipids vary considerably. Wax esters and fatty alcohols do not exist in significant amounts in Baltic herring flesh or plankton. Saturated fatty acids exist in equal proportions in herring and plankton lipids whereas monoenoic acids are more dominant in herring and polyunsaturated acids in plankton lipids, respectively. The component alkyl chains are similar in both fish and plankton fatty acids, thus suggesting the planktonic origin. During the period of abundant availability of plankton the changes in the fatty acids of the herring depot fat are related to the changes in the plankton lipid composition.     

Identification of peptide epitopes of MAGE-1, -2, -3 that demonstrate HLA-A3-specific binding

 The MAGE gene family of tumour antigens are expressed in a wide variety of human cancers. We have identified 43 nonamer peptide sequences, from MAGE-1, -2 and -3 proteins that contain binding motifs for HLA-A3 MHC class I molecules. The T2 cell line, transfected with the cDNA for the HLA-A3 gene, was used in a MHC class I stabilisation assay performed at 37°C and 26°C. At 37°C, 2 peptides were identified that stabilised HLA-A3 with high affinity (fluorescence ratio, FR >1.5), 4 peptides with low affinity (FR 1.11 – 1.49) and 31 peptides that did not stabilise this HLA haplotype (FR <1.1). At 26°C, 12 peptides were identified that stabilised HLA-A3 with high affinity, 8 peptides with low affinity and 17 peptides that did not stabilise this HLA haplotype. Two peptides stabilised HLA-A3 at both temperatures. Small changes in one to three amino acids at positions distinct from the anchor residues altered peptide affinity. Data were compared to a similar study in which a peptide competition assay was used to investigate MAGE-1 peptide binding to several HLA haplotypes. This study demonstrates that anchor residues do not accurately predict peptide binding to specific HLA haplotypes, changes in one to three amino acids at positions distinct from anchor residues influence peptide binding and alternative methods of determining peptide binding yield different results. We are currently investigating the ability of these peptides to induce antitumour cytotoxic T lymphocyte activity as they may be of potential therapeutic value. Received: 4 January 1996 / Accepted: 20 March 1996     

Catalysis in peptide synthesis

Enzyme catalyzed formation of peptide bonds reached practical significance in partial syntheses such as the conversion of pork insulin to human insulin. Catalysis is important also in coupling with active esters. The earlier applied acid-base catalysis was replaced by the use of bifunctional catalysts, 1-hydroxybenzotriazole being an outstanding example: it brought about major improvements in peptide bond formation. Intramolecular catalysis can be discerned in the rapid acylation by certain active esters, e.g., esters of catechol, 4-nitroguaiacol or 8-hydroxyquinoline. The ready formation of symmetrical anhydrides in the reaction of acylamino acids with carbodiimides is best explained by intramolecular catalysis within the O-acylisourea intermediates. Catalysis plays important roles both in introduction and in removal of blocking groups. Preparation of alkyl esters through base-catalyzed transesterification of active aryl esters and the application of this reaction for the anchoring of peptides to polymeric supports are described. Enzyme catalyzed hydrolysis of alkyl esters and hydrolytic fission of the phenylacetyl group from lysine side chains with aid of penicillin amidohydrolase are characteristic examples of the application of catalysis for the removal of blocking groups. Acidolysis of the benzyl groups including the benzyloxcarbonyl group is catalyzed by thioanisole or by 4-methylthiophenol. The catalytic effect of solvents is demonstrated with the cleavage of triphenylmethyl and biphenylyl-isopropyloxycarbonyl groups by 1-hydroxybenzotriazole in trifluoroethanol. The increasing role of catalysis in peptide synthesis and its future application for the solution of fundamental problems, such as amine activation, are discussed.