Synthesis of peptides catalyzed by papain in organic solvents with minimal water content

Synthesis of a model peptide, Z-Ala-Val-OBut, catalyzed by papain in acetonitrile containing 0-5% water was studied. Z-Ala-OCH2CONH2 was used as a carboxyl component. The optimal concentration of water was found to be 0.3-0.5%. Under these conditions the reaction rate is 50 to 100 times lower than in water but the yield of the peptide can be as high as 99.5%. Effect of reagent concentrations and temperature on the reaction was also studied.     

SPR and NMR characterization of the molecular interaction between A9 peptide and a model system of HER2 receptor: A fragment approach for selecting peptide structures specific for their target

The binding process of A9 peptide toward HER2‐DIVMP, a synthetic model of the receptor domain IV, was studied by using the surface plasmon resonance (SPR) technique, with the aim of validating it as a fast and reliable screening method for selecting peptide ligands specifically targeting a domain of their target. To investigate the structural basis of A9 binding to the model of HER2‐DIVMP, multiple ligand‐based nuclear magnetic resonance (NMR) methods were applied. The use of saturation transfer difference (STD) and WaterLOGSY NMR experiments identified key residues in the peptide for the receptor binding. Moreover, the bound conformation of the A9 peptide was obtained using transferred nuclear Overhauser effect spectroscopy (trNOESY) experiments. The NMR data revealed an extended binding surface that confirms an in silico model previously reported. These structural findings could provide good starting points for future lead structures optimization specific for the receptor target.     

High-performance liquid chromatography of peptide bioregulators, their fragments and derivatives. I. Chromatographic pattern of corticotropin fragments of Zorbax ODS sorbent

The influence of acetonitrile concentration in the eluent and of the peptide hydrophobicity on the capacity factors has been studied. The equation is proposed that describes retention as a function of the eluent characteristics and the peptide composition. The hydrophobicity increments for -COOH, -NH2, and greater than CHCONH-fragments in the studied chromatographic system have been determined. The proposed model of peptide retention is useful for a prior evaluation of the eluent composition that is necessary to elute a compound at a given capacity factor. It can be also used for the qualitative interpretation of peptide chromatograms.     

Structure and membrane interaction of the internal fusion peptide of avian sarcoma leukosis virus.

The structure and membrane interaction of the internal fusion peptide (IFP) fragment of the avian sarcoma and leucosis virus (ASLV) envelope glycoprotein was studied by an array of biophysical methods. The peptide was found to induce lipid mixing of vesicles more strongly than the fusion peptide derived from the N-terminal fusion peptide of influenza virus (HA2-FP). It was observed that the helical structure was enhanced in association with the model membranes, particularly in the N-terminal portion of the peptide. According to the infrared study, the peptide inserted into the membrane in an oblique orientation, but less deeply than the influenza HA2-FP. Analysis of NMR data in sodium dodecyl sulfate micelle suspension revealed that Pro13 of the peptide was located near the micelle-water interface. A type II beta-turn was deduced from NMR data for the peptide in aqueous medium, demonstrating a conformational flexibility of the IFP in analogy to the N-terminal FP such as that of gp41. A loose and multimodal self-assembly was deduced from the rhodamine fluorescence self-quenching experiments for the peptide bound to the membrane bilayer. Oligomerization of the peptide and its variants can also be observed in the electrophoretic experiments, suggesting a property in common with other N-terminal FP of class I fusion proteins.     

Incorporation of a synthetic mitochondrial signal peptide into charged and uncharged phospholipid monolayers

The interaction of the chemically synthesized 25-residue signal peptide of subunit IV of yeast cytochrome c oxidase with synthetic and natural phospholipids was studied by using a monolayer technique. Incorporation of the peptide into phospholipid monolayers was measured as surface area increase at constant surface pressure. The peptide was readily soluble in aqueous buffer, yet spontaneously inserted from an aqueous subphase into phospholipid monolayers up to limiting pressures of 30-40 mN/m. The incorporation of the positively charged peptide was strongly enhanced by the presence of negatively charged phospholipids. The molecular area of the signal peptide in monolayers was determined with a 14C-labeled signal peptide and was 560 +/- 170 A2. This is consistent with a 25-residue alpha-helical peptide incorporating with its long axis parallel to the plane of the monolayer. Incorporation isotherms into synthetic phosphatidylcholine and phosphatidylglycerol monolayers at different charge densities were analyzed in terms of a simple incorporation/binding model, involving partitioning of the peptide into the monolayer and an in-plane binding reaction of the negatively charged phospholipids to the partitioned peptide.     

Deuterium NMR relaxation studies of peptide-lipid interactions.

A unique model membrane system composed of a synthetic amphiphilic peptide (Lys2-Gly-Leu16-Lys2-Ala-amide) and a specifically labeled phospholipid (1,2-[7,7-2H2]dipalmitoyl-sn-glycero-3-phosphocholine) has been studied by 2H NMR, using inversion recovery, quadrupolar echo, and modified Jeener-Broekaert sequences, from 213 to 333 K, at molar peptide concentrations of 0, 2, 4, and 6%. Analysis of the experiments, employing a density matrix treatment based on the stochastic Liouville equation, revealed information about the dynamic organization of the lipid in the model membrane system, whose phase behavior has been determined previously [Huschilt et al. (1985) Biochemistry 24, 1377-1386]. The dynamic organization is described in terms of segmental and molecular order parameters and in terms of correlation times corresponding to both internal and overall lipid motions. In the liquid crystalline phase, the molecular order parameter, SZZ, was observed to decrease slightly upon addition of peptide while the conformational order parameter corresponding to the seventh segment, SZ'Z', did not change for any concentration of peptide. In general, the gauche-trans isomerization rate in the middle of the chain was not observed to change upon peptide addition, whereas the whole body reorientational correlation times (tau R parallel and tau R perpendicular) increased by nearly an order of magnitude. The anisotropy ratio (tau R perpendicular/tau R parallel) decreased with peptide added. An additional motion which involves a jump about the axis of the sn-2 chain is also observed to be slowed down significantly in the presence of peptide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heterodetic bicyclic decapeptide cyclo (Glu1-Leu2-Pro3-Gly4-Ser5-Ile6-Pro7- Ala8) cyclo-(1 gamma-5 beta) Phe9-Gly10. Synthesis and 2-D NMR conformational analysis

Bicyclic peptides are useful model molecules that can mimic the constrained local folding of a great number of natural peptides and proteins, such as ionophoric peptides, enzyme active site, and ligand-receptor active site. The synthesis of the bicyclic title compound with the liquid phase method is described with experimental details. Of particular interest is the heterodetic closure of the second ring. The peptide showed a complexing activity with metal cations like Ba2+, Ca2+, and Mg2+. The free bicyclic peptide conformation in solution has been studied by means of NMR spectroscopy and a plausible structure model worked out with model building on NMR constraints is proposed.     

Oxidative fragmentation of collagen and prolyl peptide by Cu(II)/H2O2. Conversion of proline residue to 2-pyrrolidone.

Oxidative degradation of collagen and the model peptides by Cu(II)/H2O2 has been studied. The depolymerization of collagen was predominantly observed by use of gel filtration chromatography. Polyproline was used as a model for collagen, and the oxidative modification was examined by amino acid analysis. Glutamic acid and gamma-aminobutyric acid were identified in the hydrolysates of oxidized polyproline. The formation of glutamic acid was reduced by treatment with NaBH4. The model peptide, (Pro-Pro-Gly)10, was also degraded by Cu(II)/H2O2, and a new N-terminal glycine was generated in proportion to the reaction time. Hydroxyl radical scavengers show only partial inhibition of the degradation of (Pro-Pro-Gly)10. In order to estimate the fragmentation mechanism, we used N-tert-butoxycarbonyl (Boc)-L-prolylglycine as a model for collagen and (Pro-Pro-Gly)10. The degradation products were isolated and characterized. Then N-tert-Boc-2-pyrrolidone, which provides gamma-aminobutyric acid by acid hydrolysis, was identified. The formation of a 2-pyrrolidone compound from oxidized Boc-L-prolylglycine is direct evidence for the scission of the peptide bond. The time-dependent formation of N-tert-Boc-2-pyrrolidone and liberation of glycine from N-tert-Boc-L-prolylglycine exposed to Cu(II)/H2O2 was observed. These results suggest that the cleavage of the peptide bond (Pro-Gly) was caused by oxidation of the proline residue, which led to the formation of the 2-pyrrolidone compound. We confirmed that proline oxidation leads to the fragmentation of proteins, accompanied by the formation of a 2-pyrrolidone structure.     

A new signal peptide useful for secretion of heterologous proteins from yeast and its application for synthesis of hirudin.

The BGL2 gene from Saccharomyces cerevisiae encodes a beta-glucanase which is localized to the yeast cell wall. The ability of a 23-amino acid (aa) signal peptide derived from the BGL2 gene to direct a heterologous protein to the secretory pathway of yeast has been compared to that of the MF alpha 1-encoded signal peptide in a series of gene fusions. As a model protein, the leech anticoagulant, recombinant hirudin variant 2-Lys47 (HIR) has been studied. From a multicopy plasmid chimaeric proteins were produced which carry the BGL2 signal peptide (or the artificial BGL2 pre-Val7 variant) (i) in front of the MF alpha 1 pro sequence (or modified versions of MF alpha 1 pro), i.e., a prepro signal, or (ii) joined directly to the heterologous protein. Accumulation of active HIR in yeast culture supernatants was observed when the BGL2 (or the BGL2 pre-Val7) signal peptide were used in combination with either of three versions of the MF alpha 1 pro peptide: the authentic MF alpha 1 pro, a partially deleted MF alpha 1 pro-delta 22-61, or a pro bearing an aa change (MF alpha 1 pro-Gly22). In each case the BGL2 signal peptide (or its variant) has proven equally productive to the corresponding MF alpha 1 peptide. Four times more active HIR was detected in the culture supernatant when either signal peptide was fused directly to the recombinant protein, as compared to a prepro protein version. Correct signal peptide cleavage was obtained when HIR was produced as a BGL2 pre-Val7::fusion protein.     

Peptide secondary structure induced by a micellar phospholipidic interface: proton NMR conformational study of a lipopeptide.

The conformational change of the model peptide Ac-K-G-R-G-D-G-amide induced by a phospholipidic interface was investigated by proton nuclear magnetic resonance (1H NMR). In aqueous solution, the free peptide is highly flexible and disordered, even in the presence of deuterated dodecyl-phosphocholine (DPC-d38) micelles which mimic a membrane interface. The lipopeptide, obtained by grafting a lipid anchor [2,3-dipalmitoyl-D-(+)-glyceric acid] to the lysine side chain of the peptide, was studied by standard 2D 1H NMR spectroscopy combined with distance geometry and simulated annealing calculations. When anchored to a micelle interface, the peptide acquires a definite turn (II/I') conformation. We were also able to describe precisely the conformation of the diacylglyceric fragment of the lipopeptide in a lipid environment and to establish the average orientation of the peptide segment with respect to the micelle surface.     

Cyanamide mediated syntheses of peptides containing histidine and hydrophobic amino acids

Summary Using the model of a primitive earth evaporation pond, the synthesis of three histidyl peptides in yields of up to 11% was demonstrated when aqueous solutions of histidine, leucine, ATP, cyanamide, and MgCl₂ were evaporated and heated for 24 h at 80°C. In addition, peptides were formed in yields of up to 56%, 35%, and 21%, respectively for phenylalanine, leucine, and alanine when aqueous solutions of the appropriate amino acid were evaporated and heated with cyanamide and one or more of the following components: ATP, AMP, 4-amino-5-imidazole carboxamide, or MgCl₂. The greatest peptide yield occurred at pH 3. But peptide formation was demonstrated for a system of Leu, cyanamide, and MgCl₂ adjusted to pH 7 with NH₄OH.Peptide synthesis was also studied in the presence of CaCl₂, ZnCl₂, different adenosine nucleotides, and UTP to compare their effects on peptide synthesis. The optimum conditions for cyanamide mediated peptide synthesis were also studied in terms of pH, reaction time, reaction temperature, and cyanamide concentration. The major side product in nearly all reactions studied appears to be an amino acid-cyanamide adduct. Peptides were analyzed and identified by thin layer chromatography, acid hydrolysis, and enzymatic degradation.     

Rotational orientation of monomers within a designed homo-oligomer transmembrane helical bundle

A peptide designed to form a homo-oligomeric transmembrane helical bundle was reconstituted into lipid bilayers and studied by using (2)H NMR (nuclear magnetic resonance) with magic angle spinning to confirm that the helical interface corresponds to the interface intended in the design. The peptide belongs to a family of model peptides derived from a membrane-solubilized version of the water-soluble coiled-coil GCN4-P1. The variant studied here contains two asparagines thought to engage in interhelical hydrogen bonding critical to the formation of a stable trimer. For the NMR studies, three different peptides were synthesized, each with one of three consecutive leucines in the transmembrane region deuterium labeled. Prior to NMR data collection, polarized infrared spectroscopy was used to establish that the peptides were reconstituted in lipid bilayers in a transmembrane helical conformation. The (2)H NMR line shapes of the three different peptides are consistent with a trimer structure formed by the designed peptide that is stabilized by inter-helical hydrogen bonding of asparagines at positions 7 and 14.     

The amino-terminal region of the fusion peptide of influenza virus hemagglutinin HA2 inserts into sodium dodecyl sulfate micelle with residues 16-18 at the aqueous boundary at acidic pH. Oligomerization and the conformational flexibility

The conformation and interactions with membrane mimics of the NH(2)-terminal fragment 1-25 of HA2, HA2-(1-25), of influenza virus were studied by spectroscopic methods. Secondary structure analysis of circular dichroism data revealed 45% helix for the peptide at pH 5.0. Tryptophan fluorescence quenching by acrylamide and NMR experiments established that the Trp(14) is inside the vesicular interior and residues 16-18 are at the micellar aqueous boundary. NBD fluorescence enhancement of the NH(2)-terminal labeled fluorophore on the vesicle-bound peptide indicated that the NH(2) terminus of the fusion peptide was located in the hydrophobic region of the lipid bilayer. No significant change in insertion depth was observed between pH 5.0 and 7.4. Collectively, these spectroscopic measurements pointed to an equilibrium between helix and non-helix conformations, with helix being the dominant form, for the segment in the micellar interior. The conformational transition may be facilitated by the high content of glycine, a conformationally flexible amino acid, within the fusion peptide sequence. Self-association of the 25-mer peptide was observed in the N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine SDS-gel electrophoresis experiments. Incorporating the NMR signal attenuation, fluorescence, and gel electrophoresis data, a working model for the organization of the fusion peptide in membrane bilayers was proposed.     

Aggregation of cateslytin beta-sheets on negatively charged lipids promotes rigid membrane domains. A new mode of action for antimicrobial peptides?

Cateslytin, a positively charged (5+) arginine-rich antimicrobial peptide (bCgA, RSMRLSFRARGYGFR), was chemically synthesized and studied against membranes that mimic bacterial or mammalian systems. Circular dichroism, polarized attenuated total reflection infrared spectroscopy, (1)H high-resolution MAS NMR, and (2)H and (31)P solid state NMR were used to follow the interaction from peptide and membrane points of view. Cateslytin, which is unstructured in solution, is converted into antiparallel beta-sheets that aggregate mainly flat at the surface of negatively charged bacterial mimetic membranes. Arginine residues are involved in the binding to negatively charged lipids. Following the interaction of the cateslytin peptide, rigid and thicker membrane domains enriched in negatively charged lipids are found. Much less interaction is detected with neutral mammalian model membranes, as reflected by only minor percentages of beta-sheets or helices in the peptide secondary structure. No membrane destruction was detected for both bacterial and mammalian model membranes. A molecular model is proposed in which zones of different rigidity and thickness bring about phase boundary defects that ultimately lead to permeability induction and peptide crossing through bacterial membranes.     

Chymotrypsin-catalyzed peptide synthesis. Kinetic analysis of the kinetically controlled peptide-bond formation

The kinetics of peptide-bond formation catalyzed by delta-chymotrypsin has been studied for a number of peptide products of different length using fixed concentrations of the acyl component (Ac-Phe-OMe, Ac-Ala-Ala-Phe-OMe, or Ac-Ala-Ala-Tyr-OMe) and varying concentration of the amino component (H-Ala-NH2 or H-Ala-Ala-NH2). The time course of the reactions was followed by monitoring ester consumption and peptide product formation by analytical HPLC. On the basis of a plausible four-centre mechanistic model, the theoretical time course of these reactions was calculated using rate and equilibrium constants determined by separate kinetic experiments. The excellent agreement observed between the theoretical and the experimental time courses supports the proposed mechanism and provides evidence for the validity of the present kinetic approach. By focusing attention on the rate constants which are critical for efficient synthesis, this mechanistic information constitutes a valuable basis for the use of the enzymatic peptide synthesis in preparative applications.     

Theoretical structure of the polar regions of the tropocollagen molecule

The theoretical conformations of poly (Gly-Ala-Glu) have been studied. This peptide was chosen as a model for the glycine led triads of the polar regions in collagen. The most favorable conformations are found to be based on the extended and folded forms of the 2₇ helix (2_7a and 2_7b). It is suggested that triple-strand structures of folded 2₇ helices exist in the polar collagen regions, and a structural model is presented which is in accord with recent ultrastructural deformation studies. It is a necessary condition for this structure that glycine occur in the lead of the peptide triads. In regions of the collagen molecule where the primary sequence does not contain triads (e.g., in the telopeptide region), random structures based on energy minimization of peptide neighbors are considered briefly. It seems likely that such regions contain an admixture of left-hand α, polyproline II, and 2₇ helix structures.     

Mechanism of unfolding of a model helical peptide

Synthetic model helical peptides, Acetyl-W(EAAAR)(5)A-amide with (13)C=O specifically labeled alanine segments in repeats n = 1,2 or 4,5 were studied in aqueous D(2)O solution as a function of temperature using Fourier transform infrared spectroscopy and two-dimensional correlation analysis. The (13)C==O provided a probe which was sensitive to the carbonyl stretch in the peptide bonds of the alanine residues at the amino terminal end in one peptide as compared to the probe in the carboxy terminal end of the other peptide during thermal perturbation. The relative stability of each terminal end was examined; the more stable terminal was determined to be the amino terminal end. Also studied were the glutamate and arginine side-chain modes involved in the salt bridging interaction. Two-dimensional correlation analysis enabled enhanced resolution in the spectral region of 1520--1700 cm(-1), and thus, the order in which these vibrational modes were perturbed as a function of increasing temperature were established.     

Structure of the smooth muscle myosin light-chain kinase calmodulin-binding domain peptide bound to calmodulin

The interaction between the peptide corresponding to the calmodulin-binding domain of the smooth muscle myosin light-chain kinase and (Ca2+)4-calmodulin has been studied by multinuclear and multidimensional nuclear magnetic resonance methods. The study was facilitated by the use of 15N-labeled peptide in conjunction with 15N-edited and 15N-correlated 1H spectroscopy. The peptide forms a 1:1 complex with calcium-saturated calmodulin which is in slow exchange with free peptide. The 1H and 15N resonances of the bound have been assigned. An extensive set of structural constraints for the bound peptide has been assembled from the analysis of nuclear Overhauser effects and three-bond coupling constants. The backbone conformation of the bound peptide has been determined using these constraints by use of distance geometry and related computational methods. The backbone conformation of the peptide has been determined to high precision and is generally indicative of helical secondary structure. Nonhelical backbone conformations are seen in the middle and at the C-terminal end of the bound peptide. These studies provide the first direct confirmation of the amphiphilic helix model for the structure of peptides bound to calcium-saturated calmodulin.     

Interaction of the lantibiotic nisin with mixed lipid bilayers: a 31P and 2H NMR study

Nisin is a positively charged antibacterial peptide which binds to the negatively charged membranes of Gram-positive bacteria. The initial interaction of the peptide with model membranes of neutral (phosphatidylcholine) and negatively charged (phosphatidylcholine/phosphatidylglycerol) model lipid membranes was studied using nonperturbing solid state magic angle spinning (MAS) (31)P NMR and (2)H wide-line NMR. In the presence of nisin, the coexistence of two bilayer lipid environments was observed both in charged and in neutral membranes. One lipid environment was found to be associated with lipid directly interacting with nisin and one with noninteracting lipid. Solid state (31)P MAS NMR results show that the acidic membrane lipid component partitions preferentially into the nisin-associated environment. Deuterium NMR ((2)H NMR) of the selectively headgroup-labeled acidic lipid provides further evidence of a strong interaction between the charged lipid component and the peptide. The segregation of acidic lipid into the nisin-bound environment was quantified from (2)H NMR measurements of selectively headgroup-deuterated neutral lipid. It is suggested that the observed lipid partitioning in the presence of nisin is driven, at least initially, by electrostatic interactions. (2)H NMR measurements from chain-perdeuterated neutral lipids indicate that nisin perturbs the hydrophobic region of both charged and neutral bilayers.     

The sulfhydryl groups involved in the active site of myosin B adenosinetriphosphatase. IV. Structure around the Sa thiol group.

The structure of a tryptic peptide containing one specific sulfhydryl group (Sa), which is responsible for the activation of Mg2+-ATPase of myosin B and is present in the light meromyosin region of the myosin molecule, was studied. The amino acid sequence was deduced to be Thr (or Ser)-Asn-Ala-Ala-Cys-Ala-Ala-Leu-Asp-Lys-Lys. In addition, a space-filling model around Sa was built up by comparing Sa-peptide with the amino acid sequence around Cys 190 of alpha-tropomyosin, and the high reactivity of Sa with N-ethylmaleimide is considered based on this model.