Distance dependence of long-range electron transfer through helical peptides.

Helical peptides of 8mer, 16mer, and 24mer carrying a disulfide group at the N-terminal and a ferrocene moiety at the C-terminal were synthesized, and they were self-assembled on gold by a sulfur-gold linkage. Infrared reflection-absorption spectroscopy and ellipsometry confirmed that they formed a monolayer with upright orientation. Cyclic voltammetry showed that the electron transfer from the ferrocene moiety to gold occurred even with the longest 24mer peptide. Chronoamperometry and electrochemical impedance spectroscopy were carried out to determine the standard electron transfer rate constants. It was found that the dependence of the electron-transfer rates on the distance was significantly weak with the extension of the chain from 16mer to 24mer (decay constant beta = 0.02-0.04). This dependence on distance cannot be explained by an electron tunneling mechanism even if increased hydrogen-bonding cooperativity or molecular dynamics is considered. It is thus concluded that this long-range electron transfer is operated by an electron hopping mechanism.     

Structures of the glycosylphosphatidylinositol membrane anchors from Aspergillus fumigatus membrane proteins

Glycosylphosphatidylinositol (GPI)-anchored proteins have been identified in all eukaryotes. In fungi, structural and biosynthetic studies of GPIs have been restricted to the yeast Saccharomyces cerevisiae. In this article, four GPI-anchored proteins were purified from a membrane preparation of the human filamentous fungal pathogen Aspergillus fumigatus. Using new methodology applied to western blot protein bands, the GPI structures were characterized by ES-MS, fluorescence labeling, HPLC, and specific enzymatic digestions. The phosphatidylinositol moiety of the A. fumigatus GPI membrane anchors was shown to be an inositol-phosphoceramide containing mainly phytosphingosine and monohydroxylated C24:0 fatty acid. In constrast to yeast, only ceramide was found in the GPI anchor structures of A. fumigatus, even for Gel1p, a homolog of Gas1p in S. cerevisiae that contains diacylglycerol. The A. fumigatus GPI glycan moiety is mainly a linear pentomannose structure linked to a glucosamine residue: Manalpha1-3Manalpha1-2Manalpha1-2Manalpha1-6Manalpha1-4GlcN.     

DNA sequence and complementation analysis of a mutation in the rplX gene from Escherichia coli leading to loss of ribosomal protein L24.

A mutation in Escherichia coli leads to the loss of ribosomal protein L24, severely impaired growth, and a temperature-sensitive phenotype. The mutation was shown to be in rplX, the gene for protein L24, and was due to the alteration of an AAA codon to a TAA stop codon at position 61 in rplX that resulted in a 20-amino acid peptide instead of the 104 amino acids of wild-type L24 protein. rplX genes from three temperature-resistant and fast growing pseudorevertants of the mutant were cloned and sequenced. They were found to have different base substitutions in the TAA codon, resulting in the reappearance of a full-sized protein L24 moiety. Complementation of the slow growth in trans could be achieved with several plasmids containing at least the spc promoter and intact L14 and L24 genes. Plasmids containing genes distal to rplX could further stimulate growth, and the wild type arose when the entire spc operon and the alpha operon were present. In all cases, protein L24 was expressed by the plasmids. Therefore, slow growth could be explained by polarity extending to the alpha operon. However, temperature sensitivity could not be complemented by any of the plasmids in trans, although we found that this phenotype was caused by the mutation in the rplX gene.     

Protein synthesis by mammalian cells treated with C-3-modified analogs of the 12,13-epoxytrichothecenes T-2 and T-2 tetraol.

Modification at the C-3 position of the trichothecenes T-2 and T-2 tetraol affected their ability to inhibit protein synthesis in African green monkey kidney (Vero) and mouse erythroleukemia cells. Replacement of the 3-hydroxyl of T-2 with hydrogen caused a 24-fold decrease in activity, whereas acetylation resulted in a 500-to 1,000-fold decrease. Protection of the 3-hydroxyl with a tetrahydropyranyl moiety gave an analog that was 37-fold more inhibitory to Vero than to mouse erythroleukemia cells; with the other analogs a similar effect on protein synthesis was found for both types of cells. The analogs obtained after alkaline hydrolysis were much less potent than the parent trichothecenes. The 3-tetrahydropyranyl-modified analog was equivalent in potency to T-2 tetraol, while the deoxygenated species was at least threefold less potent. All T-2 analogs caused some degree of polysome "runoff," thereby demonstrating that these species inhibit protein synthesis at the chain initiation stage when added at their 50% infective dose concentrations or lower. From these results, we suggest that the 3-hydroxyl moiety is essential for T-2 to exhibit such high activity on eucaryotic cell protein synthesis and that modification at the C-3 position decreases but does not eliminate this activity.     

Protein synthesis by mammalian cells treated with C-3-modified analogs of the 12,13-epoxytrichothecenes T-2 and T-2 tetraol

Modification at the C-3 position of the trichothecenes T-2 and T-2 tetraol affected their ability to inhibit protein synthesis in African green monkey kidney (Vero) and mouse erythroleukemia cells. Replacement of the 3-hydroxyl of T-2 with hydrogen caused a 24-fold decrease in activity, whereas acetylation resulted in a 500-to 1,000-fold decrease. Protection of the 3-hydroxyl with a tetrahydropyranyl moiety gave an analog that was 37-fold more inhibitory to Vero than to mouse erythroleukemia cells; with the other analogs a similar effect on protein synthesis was found for both types of cells. The analogs obtained after alkaline hydrolysis were much less potent than the parent trichothecenes. The 3-tetrahydropyranyl-modified analog was equivalent in potency to T-2 tetraol, while the deoxygenated species was at least threefold less potent. All T-2 analogs caused some degree of polysome "runoff," thereby demonstrating that these species inhibit protein synthesis at the chain initiation stage when added at their 50% infective dose concentrations or lower. From these results, we suggest that the 3-hydroxyl moiety is essential for T-2 to exhibit such high activity on eucaryotic cell protein synthesis and that modification at the C-3 position decreases but does not eliminate this activity.     

The impact of radiodecay on 125I-Tyr-54-alpha-bungarotoxin

The intramolecular consequences, for the structural and functional integrity of labeled proteins, of the radiodecomposition of covalently bound 125I were analyzed, using the 125I-Tyr-54 derivative of the curarimimetic protein alpha-bungarotoxin (alpha BuTx) as a specific example. Mono- and di-iodinated alpha BuTx derivatives of high specific radioactivity were prepared and monitored for up to ten 125I half-lives. Analysis of the specific activity of a mono-125I-alpha BuTx as a function of storage time was carried out and revealed that at least 95.3% of all radiolabeled alpha BuTx molecules are inactivated by the decay event. Generation of radioactive fragments of low molecular weight was studied in several preparations of di-125I-alpha BuTx; it was found that approximately 89% of all decaying 125I-alpha BuTx molecules undergo fragmentation of the labeled tyrosyl moiety. It has long been known that covalently bound 125I cleaves and inactivates double-stranded DNA with extreme efficiency (see Halpern, A., and St?cklin, G. (1977) Radiat. Environ. Biophys. 14, 167-183; 257-274); the present findings establish that alpha BuTx, a protein, is similarly disabled by 125I decay.     

Characterization of a salt-responsive 24-kilodalton glycoprotein in Mesembryanthemum crystallinum.

A concanavalin A (Con A)-binding polypeptide with a molecular mass of 24 kD (termed "SRgp24") was associated with the intercellular space of Mesembryanthemum crystallinum L. callus. When callus was grown in medium containing between 0 and 100 mM NaCl, SRgp24 was detected by Con A binding. Increasing the NaCl concentration to 200 mM caused a reduction in the amount of SRgp24 within 3 d, and returning the callus to medium without salt resulted in an accumulation of SRgp24. Immunoblot analysis showed that appreciable amounts of SRgp24 accumulated in the leaves when plants were grown under sodium-limiting conditions. Unlike most of the cell-wall Con A-binding proteins in M. crystallinum callus, the carbohydrate moiety of SRgp24 was resistant to endoglycosidase H digestion. After purification of SRgp24, the N terminus was sequenced and found to share 55 to 60% identity with the N terminus of osmotin, a group 5 pathogenesis-related protein (PR-5) that accumulates in salt-adapted tobacco cell suspension. Immunocytochemical assays, with affinity-purified antibodies to SRgp24, indicated that SRgp24 preferentially accumulated in the cell-wall region. We conclude that SRgp24 is a salt-responsive glycoprotein related to the PR-5 family in M. crystallinum.     

Incorporation of isoleucine into protein by a soluble fraction from spermatozoa

A heat-labile, non-dialyzable factor(s) in soluble fractions from porcine, bull, rabbit and cock spermatozoa was found to incorporate the radioactivity of [14C]isoleucine into a 95 degrees C CCl3COOH-insoluble fraction. The incorporation required ATP, Mg2+, casein and 2-mercaptoethanol. Trypsin and alpha-chymotrypsin inhibited the incorporation, while RNase A and DNase I did not. A mixture of 19 amino acids other than isoleucine had no effect on the incorporation. The reaction product was identified as protein. The incorporated moiety was the isoleucyl moiety of isoleucine and it retained a free alpha-amino group in the product protein. Some other characteristics of this incorporation are also described.     

Refined solution structure of a liganded type 2 wheat nonspecific lipid transfer protein

The refined structure of a wheat type 2 nonspecific lipid transfer protein (ns-LTP2) liganded with l-alpha-palmitoylphosphatidylglycerol has been determined by NMR. The (15)N-labeled protein was produced in Pichia pastoris. Physicochemical conditions and ligandation were intensively screened to obtain the best NMR spectra quality. This ns-LTP2 is a 67-residue globular protein with a diameter of about 30 A. The structure is composed of five helices forming a right superhelix. The protein presents an inner cavity, which has been measured at 341 A(3). All of the helices display hydrophobic side chains oriented toward the cavity. The phospholipid is found in this cavity. Its fatty acid chain is completely inserted in the protein, the l-alpha-palmitoylphosphatidylglycerol glycerol moiety being located on a positively charged pocket on the surface of the protein. The superhelix structure of the protein is coiled around the fatty acid chain. The overall structure shows similarities with ns-LTP1. Nevertheless, large three-dimensional structural discrepancies are observed for the H3 and H4 alpha-helices, the C-terminal region, and the last turn of the H2 helix. The lipid is orthogonal to the orientation observed in ns-LTP1. The volume of the hydrophobic cavity appears to be in the same range as the one of ns-LTP1, despite the fact that ns-LTP2 is shorter by 24 residues.     

Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase: physicochemical characteristics of the nucleotide binding site, as deduced from fluorescent spectroscopy measurements

Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase [ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49] is inactivated by the fluorescent sulfhydryl reagent N-(iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-IAEDANS). The inactivation reaction follows pseudo-first-order kinetics with respect to active enzyme to less than 10% remaining enzyme activity, with a second-order inactivation rate constant of 2.6 min-1 mM-1 at pH 7.5 and 30 degrees C. A stoichiometry of 1.05 mol of reagent incorporated per mole of enzyme subunit was found for the completely inactivated enzyme. Almost complete protection of the enzyme activity and of dansyl label incorporation are afforded by MnADP or MnATP, thus suggesting that 1,5-IAEDANS interacts with an enzyme sulfhydryl group at the nucleotide binding site. The fluorescence decay of the AEDANS attached to the protein shows a single-exponential behavior with a lifetime of 18 ns. A comparison of the fluorescence band position and the fluorescence decay with those of the adduct AEDANS-acetylcysteine indicates a reduced polarity for the microenvironment of the substrate binding site. The quenching of the AEDANS moiety in the protein can be described in terms of a collisional and a static component. The rate constant for the collisional component is much lower than that obtained for the adduct in a medium of reduced polarity. These last results indicate that the AEDANS moiety is considerably shielded from the solvent when it is covalently attached to PEPCK.     

The stability of mRNA for eucaryotic elongation factor Tu in Friend erythroleukemia cells varies with growth rate.

The decay rates of eucaryotic elongation factor Tu (eEF-Tu) mRNA and eucaryotic initiation factor 4A (eIF-4A) mRNA in Friend erythroleukemia (FEL) cells were determined under several different growth conditions. In FEL cells which were no longer actively dividing (stationary phase), eEF-Tu mRNA was found to be rather stable, with a t1/2 of about 24 h. In rapidly growing FEL cells eEF-Tu mRNA was considerably less stable, with a t1/2 of about 9 h. In both cases a single rate of mRNA decay was observed. However, when stationary-phase cells resumed growth after treatment with fresh medium, we observed that eEF-Tu mRNA decay followed a biphasic process. The faster of the two decay rates involved approximately 50% of the eEF-Tu mRNA and had a t1/2 of about 1 h. The decay rates for eIF-4A (t1/2 = 2 h) and total poly(A)+ RNA (t1/2 = 3 h) were unaffected by changes in growth conditions. The t1/2 for polysomal eEF-Tu mRNA was found to be about 8 h when stationary FEL cells were treated with fresh medium. Previous work in this laboratory has shown (T. R. Rao and L. I. Slobin, Mol. Cell. Biol. 7:687-697, 1987) that when FEL cells are allowed to grow to stationary phase, approximately 60% of the mRNA for eEF-Tu is found in a nontranslating postpolysomal messenger ribonucleoprotein (mRNP) particle. eEF-Tu mRNP was rapidly cleared from stationary cells after treatment with fresh medium. The data presented in this report indicate that the stability of eEF-Tu mRNP is rapidly altered and the particle is targeted for degradation when stationary FEL cells resume growth.     

Selective cytotoxicity of recombinant STXA1-GM-CSF protein in hematopoetic cancer cells

Chimeric proteins are composed of a cell-targeting moiety and a cell-killing moiety. In this study, a chimeric protein, STXA1-GM-CSF, composed of catalytic domain of Shiga toxin (A1) and granulocyte-macrophage colony-stimulating factor (GM-CSF) was constructed and expressed in E. coli. Cytotoxicity, receptor blocking, and neutralization experiments revealed that the chimeric protein induced cytotoxic effect on different cell lines. This effect was found to be specific, due to the presence of the killing moiety (A1), which exerts its effect through a specific GM-CSF-targeting domain, by binding to its receptor present on those cell lines. These initial investigations indicate that the chimeric protein was functional; further analyses are required for its application.     

Incorporation of glutamic acid into protein by a soluble system

A heat-labile, non-dialyzable factor(s) in soluble fractions from Escherichia coli strains and Bacillus subtilis was found to incorporate the radioactivity of [14C]glutamic acid into 95 degrees C CCl3COOH-insoluble fraction. Incorporation catalyzed by a partially purified factor from E. coli B required ATP, Mg2+, tRNA, casein, carbonate, and 2-mercaptoethanol. A mixture of nineteen amino acids other than glutamic acid had no effect on the incorporation. Heparin, spermine and monovalent cations were inhibitory. Incorporation proceeded via glutamyl-tRNA. The incorporation from [14C]glutamyl-tRNA required Mg2+, casein, carbonate, and 2-mercaptoethanol, and there was no incorporation from [14C]aspartyl-tRNA. The reaction product was identified as protein. The incorporated moiety was the glutamyl moiety of glutamic acid and it retained a free alpha-amino group in the product protein. The incorporating factor of E. coli B was demonstrated to be glutamyl-tRNA synthetase.     

Identification of the cAD1 sex pheromone precursor in Enterococcus faecalis

The Enterococcus faecalis virulence plasmid pAD1 encodes a mating response induced by exposure to an octapeptide sex pheromone, cAD1, secreted by plasmid-free enterococci. The determinant for the pheromone in E. faecalis FA2-2, designated cad, was found to encode a 309-amino-acid lipoprotein precursor with the last 8 residues of its 22-amino acid signal sequence representing the cAD1 moiety. The lipoprotein moiety contained two 77-amino-acid repeats (70% identity) separated by 45 residues. The nonisogenic E. faecalis strain V583 determinant encodes a homologous precursor protein, but it differs at two amino acid positions, both of which are located within the pheromone peptide moiety (positions 2 and 8). Construction of a variant of strain FA2-2 containing the differences present in V583 resulted in cells that did not produce detectable cAD1. The mutant appeared normal under laboratory growth conditions, and while significantly reduced in recipient potential, when carrying pAD1 it exhibited a normal mating response. A mutant of FA2-2 with a truncated lipoprotein moiety appeared normal with respect to recipient potential and, when carrying plasmid DNA, donor potential. A gene encoding a protein designated Eep, believed to be a zinc metalloprotease, had been previously identified as required for pheromone biosynthesis and was believed to be involved in the processing of a pheromone precursor. Our new observation that the pAD1-encoded inhibitor peptide, iAD1, whose precursor is itself a signal sequence, is also dependent on Eep is consistent with the likelihood that such processing occurs at the amino terminus of the cAD1 moiety.     

A photoswitchable ITAM peptidomimetic: synthesis and real time surface plasmon resonance (SPR) analysis of the effects of cis-trans isomerization on binding

The Syk protein plays an important role in immune receptor signaling. The Syk tandem SH2 domain (tSH2)-ITAM interaction is important for recruiting Syk to the receptor complex and for Syk kinase activation. A peptidomimetic ligand for tSH2 was synthesized in which a photoswitchable azobenzene moiety was incorporated. Such a photoswitchable moiety may regulate the distance between the two phosphotyrosine containing ITAM sequences, which bind to tSH2. Different affinities of the cis and trans isomer of the ligand were found by surface plasmon resonance (SPR). By in situ irradiation during SPR measurements the effect of the cis-trans isomerization on binding could be monitored in real time.     

Characterization of SERCA2b Ca2+-Mg2+ ATPase mRNA decay by nuclear proteins

Gene expression is controlled at several levels including mRNA decay. Sarco/endoplasmic reticulum Ca2+-Mg2+-ATPase isoform 2b (SERCA2b) is central to Ca2+ signalling and homeostasis in several tissues. SERCA2b mRNA decay involves interactions between cis-acting elements in its 3'-region and trans-acting nuclear protein factors. In the presence of the protein factors, the synthetic capped and polyadenylated RNA fragment 2b1 (3444-3753) decays faster than other SERCA2b 3'-region fragments. Here we determined the minimum cis-acting destabilizing element in the decay and its interactions with the nuclear protein factors. The in vitro decay required ATP hydrolysis and Mg2+ but not Ca2+. The decay was directional from 3' to 5', and involved a novel 35b GC rich domain designated 2b1-4 corresponding to 3521-3555. The decay of 2b1 RNA was decreased by (a) competition with 2b1-4, (b) mutation of 2b1 to delete 2b1-4, and (c) depleting the extracts of destabilizing trans-acting factors using immobilized 2b1-4. To determine the minimal destabilizing elements 2b1-4 was divided into 7b domains A-E. Deleting AB, BC, CD or DE inactivated the destabilizing cis-acting element but deleting A, B, C, D or E had no effect. In electrophoresis mobility shift assays the nuclear protein extracts retarded the mobility of labeled uncapped 2b1 RNA without a poly A+ tail. A positive co-operativity in the interactions was shown in protein concentration dependence of the shift and in the competition of 2b1-4 in inhibiting the mobility of 2b1 RNA. Based on further experiments, the domain CDE (3535-3555) was sufficient to compete with 2b1 RNA for the protein binding. Consistent with this competition, excess CDE RNA retarded the in vitro decay of 2b1 RNA. Thus the RNA decay required ATP hydrolysis and Mg2+ but not Ca2+, the minimum binding domain was in the sequence 3535-3555, and the decay may involve a multimeric protein complex.     

2-Arylbenzoxazoles as CETP inhibitors: Substitution of the benzoxazole moiety

A series of 2-arylbenzoxazole inhibitors of the cholesterol ester transfer protein (CETP) is described. Structure–activity studies focused on variation of the substitution of the benzoxazole moiety. Substitution at the 5- and 7-positions of the benzoxazole moiety was found to be beneficial for CETP inhibition. Compound 47 was found to be the most potent inhibitor in this series and inhibited CETP with an IC₅₀ of 28 nM.     

Time-resolved extrinsic fluorescence of aromatic-L-amino-acid decarboxylase

The coenzyme-linked fluorescence of aromatic-L-amino-acid decarboxylase decays non-exponentially. The decay of both native and NaBH4 reduced samples can only be fitted by two exponentials each roughly accounting for about half of the total fluorescence. Denaturation of the reduced protein with 8 M urea makes the fluorescence decay mono-exponential, like that observed for the reference compound pyridoxamine-5-phosphate. An extra pyridoxyl moiety can be bound to the enzyme after incubation with excess pyridoxal phosphate and reduction with NaBH4. This sample is almost twice as fluorescent and shows also two lifetimes. After denaturation only one fluorescence lifetime is observed. The presence of two non-equivalent pyridoxal sites in the native enzyme can be postulated. The heterogeneous decay behaviour of the pyridoxyl moiety in the enzyme together with the variability of lifetime shown, makes this fluorophore an even more interesting fluorescent probe for proteins.