Terminal Phenylalanine Research Articles

N,N'-[1,4-Phenyl-enebis(imino-carbon-yl)]bis-(l-phenyl-alanine) tetra-hydro-furan disolvate.

The title compound, C26H26N4O6·2C4H8O, representing a bis-urea with terminal phenyl-alanine units, crystallized with two tetra-hydro-furan (THF) mol-ecules. The main mol-ecule is located on a crystallographic twofold axis, while the solvent mol-ecule is disordered over two positions, with occupancies of 0.571 (15) and 0.429 (15). The host mol-ecules are linked by N-H⋯O=C hydrogen bonds and C-H⋯O contacts with R 2 1(6) and R 2 1(7) ring motifs. The THF mol-ecules enclosed in the crystal are connected to the bis-urea compound via O-H⋯O and C-H⋯O inter-actions.

An Artificial Single Molecular Channel Showing High Chloride Transport Selectivity and pH-Responsive Conductance.

Inspired by the unique structure and function of the natural chloride channel (ClC) selectivity filter, we present herein the design of a ClC-type single channel molecule. This channel displays high ion transport activity with half-maximal effective concentration, EC50 , of 0.10 μM, or 0.075 mol % (channel molecule to lipid ratio), as determined by fluorescent analysis using lucigenin-encapsulated vesicles. Planar bilayer lipid membrane conductance measurements indicated an excellent Cl- /K+ selectivity with a permeability ratio P /P up to 12.31, which is comparable with the chloride selectivity of natural ClC proteins. Moreover, high anion/anion selectivity (P /P =66.21) and pH-dependent conductance and ion selectivity of the channel molecule were revealed. The ClC-like transport behavior is contributed by the cooperation of hydrogen bonding and anion-π interactions in the central macrocyclic skeleton, and by the existence of pH-responsive terminal phenylalanine residues.

An Artificial Single Molecular Channel Showing High Chloride Transport Selectivity and pH‐Responsive Conductance

AbstractInspired by the unique structure and function of the natural chloride channel (ClC) selectivity filter, we present herein the design of a ClC‐type single channel molecule. This channel displays high ion transport activity with half‐maximal effective concentration, EC50, of 0.10 μM, or 0.075 mol % (channel molecule to lipid ratio), as determined by fluorescent analysis using lucigenin‐encapsulated vesicles. Planar bilayer lipid membrane conductance measurements indicated an excellent Cl−/K+ selectivity with a permeability ratio P /P up to 12.31, which is comparable with the chloride selectivity of natural ClC proteins. Moreover, high anion/anion selectivity (P /P =66.21) and pH‐dependent conductance and ion selectivity of the channel molecule were revealed. The ClC‐like transport behavior is contributed by the cooperation of hydrogen bonding and anion–π interactions in the central macrocyclic skeleton, and by the existence of pH‐responsive terminal phenylalanine residues.

Re-Examining Interaction between Antimicrobial Peptide Aurein 1.2 and Model Cell Membranes via SFG.

Aurein 1.2 (Aur), a highly efficient 13-residue antimicrobial peptide (AMP) with a broad-spectrum antibiotic activity originally derived from the Australian frog skin secretions, can nonspecifically disrupt bacterial membranes. To deeply understand the molecular-level detail of the antimicrobial mechanism, here, we artificially established comparative experimental models to investigate the interfacial interaction process between Aur and negatively charged model cell membranes via sum frequency generation vibrational spectroscopy. Sequencing the vibrational signals of phenyl, C-H, and amide groups from Aur has characteristically helped us differentiate between the initial adsorption and subsequent insertion steps upon mutual interaction between Aur and the charged lipids. The phenyl group at the terminal phenylalanine residue can act as an anchor in the adsorption process. The time-dependent signal intensity of α-helices showed a sharp rise once the Aur molecules came into contact with the negatively charged lipids, indicating that the adsorption process was ongoing. Insertion of Aur into the charged lipids then offered the detectable interfacial C-H signals from Aur. The achiral and chiral amide I signals suggest that Aur had formed β-folding-like aggregates after interacting with the charged lipids, along with the subsequent descending α-helical amide I signals. The above-mentioned experimental results provide the molecular-level detail on how the Aur molecules interact with the cell membranes, and such a mechanism study can offer the necessary support for the AMP design and later application.

Reflection Absorption Infrared Spectroscopy Characterization of SAM Formation from 8-Mercapto-N-(phenethyl)octanamide Thiols with Phe Ring and Amide Groups.

Multifunctional amide-containing self-assembled monolayers (SAMs) provide prospects for the construction of interfaces with required physicochemical properties and distinctive stability. In this study, we report the synthesis of amide-containing thiols with terminal phenylalanine (Phe) ring functionality (HS(CH2)7CONH(CH2)2C6H5) and the characterization of the formation of SAMs from these thiols on gold by reflection absorption infrared spectroscopy (RAIRS). For reliable assignments of vibrational bands, ring deuterated analogs were synthesized and studied as well. Adsorption time induced changes in Amide-II band frequency and relative intensity of Amide-II/Amide-I bands revealed two-state sigmoidal form dependence with a transition inflection points at 2.2 ± 0.5 and 4.7 ± 0.5 min, respectively. The transition from initial (disordered) to final (hydrogen-bonded, ordered) structure resulted in increased Amide-II frequency from 1548 to 1557 cm−1, which is diagnostic for a strongly hydrogen-bonded amide network in trans conformation. However, the lateral interactions between the alkyl chains were found to be somewhat reduced when compared with well-ordered alkane thiol monolayers.

Concentric microring structures containing gold nanoparticles for SERS-based applications

Concentric microring structures containing gold nanoparticles were fabricated using a Bessel-like beam array with the 30 μm and 60 μm periods. The fabricated structures coated with 5 nm thick gold layer demonstrate enhancement of Raman signals due to their induced localized plasmon resonance. The measured Raman signal intensity of self-assembled monolayers from thiols with terminal phenylalanine ring group with using various Raman spectrometers exhibit that concentric gold nanoparticles structures enhance the Raman signal from different size of analyzing area. The demonstrated method provides a facile fabrication of microring structures containing gold nanoparticles and shows their potential to exploit them for surface-enhanced Raman scattering (SERS) based applications.

Sum frequency generation spectroscopy probing of formation of self-assembled monolayers from thiols with terminal phenylalanine ring and intrachain amide groups

Formation of a self-assembled monolayer onto the polycrystalline gold substrate from thiols containing a terminal phenylalanine (Phe) ring and intrachain amide groups (HS-(CH2)7-CONH-(CH2)2-C6H5) was characterized by vibrational sum frequency generation (SFG) spectroscopy. The temporal evolution of the characteristic Phe ring stretching vibration ν(=C–H) near 3069 cm–1 and the Amide A mode were monitored by nonlinear vibrational spectroscopy. The SFG data revealed the complete ordering of the terminal Phe rings after about 60 min of adsorption. Formation of a strong hydrogen bonding between the adjacent chains of adsorbed molecules was evident by appearance of the Amide A mode at the relative low frequency (3280–3308 cm–1). The well-ordered and strongly hydrogen bonded SAM with the terminal Phe ring functionality is a promising platform for the analysis of interactions and the function of aromatic rings in biomolecular processes.

Unraveling the Interplay between Single-Stranded DNA-Binding Protein, DNA Polymerase and Single-Stranded DNA

Bacteriophage T7 gene 2.5 protein (gp2.5), a single-stranded DNA binding (SSB) protein, contains two main structural elements which confer it essential functions. First, an oligosaccharide/oligonucleotide binding fold which can interact with ssDNA and thus provide a protective role while removing secondary structure impediments. Second, an acidic C-terminal tail capable to interact with other gp2.5 units and with other replisome proteins, such as the T7 DNA primase-helicase (gp4) and polymerase (gp5), allowing for organizational roles within the replisome. The impact of SSBs on the replisome dynamics is hard to study using conventional biochemistry tools. Here, we present data obtained from a unique combination of optical tweezers and confocal fluorescence microscopy, which offers real-time high spatial and temporal resolution to study protein-DNA interactions. Our results show that the SSB gp2.5 binds efficiently to ssDNA, forming highly static protein-DNA complexes. We also observed a decrease in the ssDNA end-to-end length indicating that the protein binds by bending or wrapping ssDNA, resulting in a highly tension-sensitive binding mode. Next, we studied to what degree the presence of wild type (wt) gp2.5 or a mutant lacking the terminal phenylalanine (ΔF), the interaction motif with DNA polymerase, affects replication activity. We show that wt gp2.5 alters the rate, duration and probability of occurrence of replicative and proofreading polymerase events in contrast to the ΔF mutant or the absence of the protein. Thus, our findings indicate that the presence of gp2.5 strongly stimulates the function of DNA polymerase within the replisome.

Analogs of Eu 3+ DOTAM-Gly-Phe-OH and Tm 3+ DOTAM-Gly-Lys-OH: Synthesis and magnetic properties of potential PARACEST MRI contrast agents

Chelated lanthanide ions, especially gadolinium, have found wide use as contrast agents in magnetic resonance imaging. A new paradigm for generating contrast, termed PARACEST, was recently described that requires the slow exchange of water or other exchangeable protons present in the ligand framework. In previous work, we have described a synthetic method for the preparation of dipeptide conjugates of DOTAM for use as PARACEST agents. Two compounds possessed interesting magnetic properties: the Eu 3+ complex of DOTAM-Gly-Phe-OH and the Tm 3+ complex of DOTAM-Gly-Lys-OH. To understand the relationship between the structure of these complexes and their magnetic properties, we have expanded our synthetic methodology and prepared several new complexes. Ligands have been prepared in which the terminal phenylalanine moieties have been replaced with tryptophan or tyrosine, the distance to the amino acid residue possessing an α-substituent has been changed, or phenylalanine and lysine have been combined in the peptide sequence. The preparation of lanthanide(III) complexes of these ligands has been achieved and their PARACEST properties have been determined.

Transfection Activity of Polyamidoamine Dendrimers Having Hydrophobic Amino Acid Residues in the Periphery

We designed poly(amidoamine) dendrimers with phenylalanine or leucine residues at their chain ends. Thereby, we achieved efficient gene transfection of cells through synergy of the proton sponge effect, which is induced by the internal tertiary amines of the dendrimer, and hydrophobic interaction by the hydrophobic amino acid residues in the dendrimer periphery. Dendrimers having 16, 29, 46, and 64 terminal phenylalanine residues were prepared by the reaction of the amine-terminated poly(amidoamine) G4 dendrimer and L-phenylalanine using condensing reagent 1,3-dicyclohexylcarbodiimide. Transfection activity of these phenylalanine-modified dendrimers for CV1 cells, an African green monkey kidney cell line, increased concomitant with the increasing number of the terminal phenylalanine residues, except for the dendrimer with 64 phenylalanine residues, which showed poor water solubility and hardly formed a complex with DNA at neutral pH. However, under weakly acidic conditions, the dendrimer with 64 phenylalanine residues formed a complex with DNA, thereby achieving highly efficient transfection. In contrast, the attachment of L-leucine residues was unable to improve the transfection activity of the parent dendrimer, probably because of the relatively lower hydrophobicity of this amino acid. The phenylalanine-modified dendrimer exhibited a higher transfection activity and a lower cytotoxicity than some widely used transfection reagents. For that reason, the phenylalanine-modified dendrimers are considered to be promising gene carriers.

Membrane localization of the S1 subunit of pertussis toxin in Bordetella pertussis and implications for pertussis toxin secretion.

Pertussis toxin is secreted from Bordetella pertussis with the assistance of the Ptl transport system, a member of the type IV family of macromolecular transporters. The S1 subunit and the B oligomer combine to form the holotoxin prior to export from the bacterial cell, although the site of assembly is not known. To better understand the pathway of pertussis toxin assembly and secretion, we examined the subcellular location of the S1 subunit, expressed with or without the B oligomer and the Ptl proteins. In wild-type B. pertussis, the majority of the S1 subunit that remained cell associated localized to the bacterial membranes. In mutants of B. pertussis that do not express pertussis toxin and/or the Ptl proteins, full-length S1, expressed from a plasmid, partitioned almost entirely to the bacterial membranes. Several lines of evidence strongly suggest that the S1 subunit localizes to the outer membrane of B. pertussis. First, we found that membrane-bound full-length S1 was almost completely insoluble in Triton X-100. Second, recombinant S1 previously has been shown to localize to the outer membrane of Escherichia coli (J. T. Barbieri, M. Pizza, G. Cortina, and R. Rappuoli, Infect. Immun. 58:999-1003, 1990). Third, the S1 subunit possesses a distinctive amino acid motif at its carboxy terminus, including a terminal phenylalanine, which is highly conserved among bacterial outer membrane proteins. By using site-directed mutagenesis, we determined that the terminal phenylalanine is critical for stable expression of the S1 subunit. Our findings provide evidence that prior to assembly with the B oligomer and independent of the Ptl proteins, the S1 subunit localizes to the outer membrane of B. pertussis. Thus, outer membrane-bound S1 may serve as a nucleation site for assembly with the B oligomer and for interactions with the Ptl transport system.

Porphyrin-Based Peptide Receptors: Syntheses and NMR Analysis

The synthesis and purification of a water-soluble host compound that contains three pyridinium units and one spacer-connected benzocrown ether unit in the meso-positions of porphyrin and of its Zn(II) or Cu(II) complexes is described. Metalation leads to small (compared to the apo-derivative) changes of selectivities with different peptides, with complexation constants in water of above 10(5)M(-1). One complex containing the tripeptide Gly-Gly-Phe is analyzed in detail by COSY, HSQC, HMBC, and NOESY NMR experiments. Temperature-dependent spectra show activation energies for a intramolecular hydrogen exchange of amide protons with valence isomerization of the porphyrin ring, in accordance with the literature. Sharp signals for the spin system are only found at elevated temperature. Vicinal coupling constants within the crown ether moiety indicate stronger puckering than that reported for benzocrowns. All NMR signals of the complexed peptide are shielded, in particular those of the terminal phenylalanine unit, in line with its stacking on the porphyrin surface. A corresponding structural model, obtained by CHARMm simulation, is also in line with the observed intermolecular NOE cross peaks.

A Bacteroides thetaiotaomicron outer membrane protein that is essential for utilization of maltooligosaccharides and starch

Previous studies suggested that the first step in utilization of starch by Bacteroides thetaiotaomicron was binding of the polysaccharide to the cell surface, followed by translocation of the polysaccharide across the outer membrane into the periplasm. In this study, we report the molecular characterization of a gene that encodes an outer membrane protein that is essential for utilization of both maltooligosaccharides and starch. The gene, susC, encoded a protein of 115.3 kDa. Antibodies were raised against SusC, and the outer membrane location of SusC could be confirmed by Western blot (immunoblot) analysis. SusC had a possible signal sequence of between 20 and 39 amino acids, depending on which N-terminal methionine initiates the start of the protein. It also had some features typical of well-characterized outer membrane proteins from members of the family Enterobacteriaceae, such as a terminal phenylalanine residue and a region in the amino portion of the protein thought to be involved in stabilizing the protein in the outer membrane. The amino acid sequence, together with results of gene disruption experiments, suggested that SusC was not an amylolytic enzyme. Transcriptional fusion experiments, using beta-glucuronidase as a reporter group, showed that expression of susC was maltose regulated at the transcriptional level. This is the first molecular characterization of a B. thetaiotaomicron outer membrane protein involved in maltooligosaccharide and starch utilization.

Evidence for cholecystokinin receptor subtype in endocrine pancreas

Cholecystokinin (CCK) is a gut hormone that regulates pancreatic endocrine functions via CCK A receptors. CCK 4 (Trp-Met-Asp-Phe-NH 2) has an insulinotropic effect, but is 1000-fold less potent than CCK 8. The in vitro potencies and selectivity of newly synthesized CCK 4 analogs were investigated. Exchanging various a amino acids, for example Met by Nle and modifying Phe and/or Trp, led to compounds that were much more effective than CCK 4 itself and show insulinotropic effects comparable with those of CCK 8. Compounds that possess electron withdrawing groups on the C- terminal phenylalanine were especially effective; compounds with electron-donating groups had no effect. In contrast to CCK 8 the synthetic CCK 4 compounds were selective for the endocrine pancreas: they had no agonistic or antagonistic effect on the contraction of the guinea pig ileum, amylase release from isolated acini, and no major effect on the feeding behavior of mice being supplied with either compound by an implantable Alzet R pump for 8 days. The data indicate that some of the synthetic tetrapeptides exhibit a high affinity for the CCK receptor of the endocrine pancreas and that they are highly selective for this (peripheral) CCK A receptor subtype. The β-cell CCK A receptors are different from those in exocrine pancreas, smooth muscle, and those for regulating appetite; these peripheral receptor subtypes can be discriminated for the first time.

Monoclonal Antibodies Against Myelin Proteolipid Protein: Identification and Characterization of Two Major Determinants

This report describes the preparation and characterization of a panel of monoclonal antibodies (mAbs) against the myelin proteolipid protein (PLP). A Lewis rat was immunized with bovine proteolipid apoprotein and 27 mAbs were selected based on their reactivity against bovine PLP on enzyme-linked immunosorbent assays. Eleven mAbs recognized the PLP carboxyl-terminal sequence when tested against a panel of synthetic peptides in a solid-phase assay. A carboxyl-terminal pentapeptide (residues 272-276) was sufficient for antibody binding and the terminal phenylalanine residue was found particularly important. Deletion, modification, or replacement of this residue markedly reduced or obliterated antigen-antibody interaction. Nine mAbs reacted with a second antigenic determinant, residues 209-217, but these could be identified only by competitive immunoassays. This peptide was a more effective inhibitor than the longer peptides 202-217 and 205-221, suggesting that flanking residues may interfere with peptide-antibody interaction. Seven antibodies did not react with any of the synthetic peptides tested and their determinants remain unidentified. Immunoblot analysis showed that the mAbs reacted with both the PLP and the DM-20 isoforms. Twenty-three of the mAbs were of the immunoglobulin G2a or b isotype; the remaining antibodies were immunoglobulin M and all of these were specific for residues 209-217. Cultured murine oligodendrocytes were stained by most of the mAbs tested, but the most intense reactivity was observed with the carboxyl-terminus-specific mAbs. The immunocytochemical analyses demonstrate that the mAbs react with the native PLP in situ and show their potential usefulness for studies of the cell biology of myelin and oligodendrocytes.

Determination of Various Molecular Forms of Cholecystokinin from Canine Mucosa by Radioimmunoassay and Bioassay

Bioassays using amylase release from isolated pancreatic acini measure only cholecystokinin (CCK) forms with an intact carboxyl terminus ending with phenylalanine amide, but it cannot be excluded that peptides not structurally related to CCK are also responsible for CCK-like bioactivity. CCK exists in several molecular forms in intestinal mucosa which are released into the circulating blood. We studied the molecular forms of CCK in canine intestinal extracts after separation by high performance liquid chromatography by bioassay and compared them with those detected by radioimmunoassay. For the radioimmunoassay, an antibody was used which needs the carboxyl terminal phenylalanine amide for recognition. Three immunoreactive peaks were reproducibly seen in HPLC eluates which eluted in the regions of synthetic CCK-8, purified porcine CCK-33-39 (which co-elute using this gradient) and purified canine CCK-58. All these peaks were bioactive for amylase release from isolated pancreatic acini. No further bioactive peaks were detected in the HPLC eluates. When an antibody was used which recognizes the midregion of CCK-58, an additional peak was detected which eluted between CCK-33-39 and CCK-58. This form presumably represents an amino terminal fragment of CCK lacking the carboxyl terminus. It can be concluded that bioassays of CCK measure only CCK bioactivity in intestinal mucosal extracts, whereas radioimmunoassays may detect biologically inactive forms depending on the antibody recognition site.

The repressing and enhancing functions of the herpes simplex virus regulatory protein ICP27 map to C-terminal regions and are required to modulate viral gene expression very early in infection

The phenotypic properties of ICP27 temperature-sensitive and deletion mutants and the results of transient expression assays have demonstrated that ICP27 has a modulatory effect on viral gene expression induced by ICPs 0 and 4. In order to identify the regions of the ICP27 molecule that are responsible for its enhancing and repressing activities, 10 nonsense and 3 in-frame deletion mutations were introduced into the coding sequence of the cloned ICP27 gene. These mutant genes were tested in transient expression assays for their ability to complement an ICP27 null mutant and to enhance and repress expression from a spectrum of herpes simplex virus type 1 promoters in reporter CAT genes when expression was induced by ICP0 or ICP4. The results of assays with cloned mutant genes demonstrate that the ICP27 polypeptide contains two regions, located between amino acid residues 327 and 407 and residues 465 and 511, that contribute to its repressing activity. The amino acid region located between the two repressing regions (residues 407 to 465) is able to interfere with ICP27 repressing activity. None of the mutant genes exhibited efficient enhancing activity for any of the herpes simplex type 1 promoters tested, demonstrating that amino acids comprising the carboxy-terminal half of the ICP27 molecule, including the terminal phenylalanine residue, are required for wild-type enhancement as well as for efficient complementation of an ICP27 null mutant. Phenotypic characterization of an in-frame deletion mutant, vd3, and a previously isolated null mutant, 5dl 1.2 (A. M. McCarthy, L. and P. A. Schaffer, J. Virol. 63:18-27, 1989), demonstrated that ICP27 is required to induce the expression of all classes of viral genes very early in infection and confirmed the requirement for ICP27 later in infection (i) to repress early gene expression, (ii) to induce wild-type levels of delayed-early or gamma 1 gene expression, and (iii) to induce true late or gamma 2 gene expression. The vd3 mutant, which specifies an ICP27 peptide lacking the repressing region between residues 327 and 407, is able to (i) repress early gene expression, consistent with the repressing ability of the d3 mutation in transient expression assays, (ii) induce the synthesis of significant but reduced levels of delayed-early (gamma 1) proteins and no gamma 2 proteins (thus vd3 exhibits a late protein phenotype intermediate between that of the wild-type virus and 5dl 1.2), and (iii) confer altered electrophoretic mobility on ICP4, demonstrating a role for ICP27 in the posttranslational modification of this essential regulatory protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Experimental phenylketonuria: replacement of carboxyl terminal tyrosine by phenylalanine in infant rat brain tubulin.

In the brains of newborn rats, about half of the tubulin molecules are modified posttranslationally by the addition of an aromatic amino acid at the carboxyl terminus of the alpha chain. Of the added residues, 96 percent are tyrosine and 4 percent are phenylalanine. After induction of hyperphenylalaninemia, the proportion of tubulin molecules containing carboxyl terminal phenylalanine increases up to eightfold and the pool of tyrosine-containing molecules decreases by an equivalent amount.

E.S.R. of Spin-trapped Radicals in Aqueous Solutions of Peptides

SummaryThe reactions of hydroxyl radicals with 30 dipeptides and several larger peptides were studied in aqueous solutions. The OH radicals were generated by U.V. photolysis of H2O2. The short-lived peptide radicals were spin-trapped using t-nitrosobutane and identified by e.s.r. For dipeptides containing the amino terminal residues glycine, alanine and phenylalanine, abstraction of the hydrogen from the carbon adjacent to the peptide nitrogen was the major process leading to the spin-adducts. Such radicals will be referred to as backbone radicals. Dipeptides with a carbonyl terminal serine residue and also glycylglutamic acid form both backbone and side-chain radicals, with the latter being formed in larger quantities. For dipeptides, side-chain radicals were detected on either the carboxyl or amino terminal residues or both. The effect of pD on the e.s.r. spectrum of the spin-adducts of glycylglycine was studied and the pK of the carboxyl group of this radical was determined to be 2·5. For (Ala)3 and (Ala)n, with an average value of n = 1800, backbone and minor side-chain radicals were observed. For ribonucleases-S-peptide, containing 20 amino acid residues, both backbone and side-chain radicals were detected.

Die Enzyme der Biosynthese aromatischer Aminosäuren bei Hansenula henricii : Chorismat-Mutase

32-fold enriched chorismate mutase (EC 5.4.99.5) of Hansenula henricii with a molecular weight of 120 000 dalton (determined by gel filtration) catalyzes the transformation of chorismate to prephenate, the reaction of the first step of the terminal tyrosine and phenylalanine synthetic pathway, only in the presence of L-tryptophan. The activation by tryptophan is competitive with respect to the substrate. If the concentration of tryptophan is low, a positive cooperativity of at least two substrate binding sites occures. Tyrosine and phenylalanine have no detectable effect on chorismate mutase activity.