Wild-type and mutant hemagglutinin fusion peptides alter bilayer structure as well as kinetics and activation thermodynamics of stalk and pore formation differently: mechanistic implications.

Viral fusion peptides are short N-terminal regions of type-1 viral fusion proteins that are critical for virus entry. Although the importance of viral fusion peptides in virus-cell membrane fusion is established, little is known about how they function. We report the effects of wild-type (WT) hemagglutinin (HA) fusion peptide and its G1S, G1V, and W14A mutants on the kinetics of poly(ethylene glycol)(PEG)-mediated fusion of small unilamellar vesicles composed of dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, sphingomyelin, and cholesterol (molar ratio of 35:30:15:20). Time courses of lipid mixing, content mixing, and content leakage were obtained using fluorescence assays at multiple temperatures and analyzed globally using either a two-step or three-step sequential ensemble model of the fusion process to obtain the rate constant and activation thermodynamics of each step. We also monitored the influence of peptides on bilayer interfacial order, acyl chain order, bilayer free volume, and water penetration. All these data were considered in terms of a recently published mechanistic model for the thermodynamic transition states for each step of the fusion process. We propose that WT peptide catalyzes Step 1 by occupying bilayer regions vacated by acyl chains that protrude into interbilayer space to form the Step 1 transition state. It also uniquely contributes a positive intrinsic curvature to hemi-fused leaflets to eliminate Step 2 and catalyzes Step 3 by destabilizing the highly stressed edges of the hemi-fused microstructures that dominate the ensemble of the intermediate state directly preceding fusion pore formation. Similar arguments explain the catalytic and inhibitory properties of the mutant peptides and support the hypothesis that the membrane-contacting fusion peptide of HA fusion protein is key to its catalytic activity.

Arginine catabolic mobile element encoded speG abrogates the unique hypersensitivity of Staphylococcus aureus to exogenous polyamines.

Polyamines, including spermine (Spm) and spermidine (Spd), are aliphatic cations that are reportedly synthesized by all living organisms. They exert pleiotropic effects on cells and are required for efficient nucleic acid and protein synthesis. Here, we report that the human pathogen Staphylococcus aureus lacks identifiable polyamine biosynthetic genes, and consequently produces no Spm/Spd or their precursor compounds putrescine and agmatine. Moreover, while supplementing defined medium with polyamines generally enhances bacterial growth, Spm and Spd exert bactericidal effects on S. aureus at physiological concentrations. Small colony variants specifically lacking menaquinone biosynthesis arose after prolonged Spm exposure and exhibited reduced polyamine sensitivity. However, other respiratory-defective mutants were no less susceptible to Spm implying menaquinone itself rather than general respiration is required for full Spm toxicity. Polyamine hypersensitivity distinguishes S. aureus from other bacteria and is exhibited by all tested strains save those belonging to the USA-300 group of community-associated methicillin-resistant S. aureus (CA-MRSA). We identified one gene within the USA-300-specific arginine catabolic mobile element (ACME) encoding a Spm/Spd N-acetyltransferase that is necessary and sufficient for polyamine resistance. S. aureus encounters significant polyamine levels during infection; however, the acquisition of ACME encoded speG allows USA-300 clones to circumvent polyamine hypersensitivity, a peculiar trait of S. aureus.

Accurate quantitation of standard peptides used for quantitative proteomics.

MS-based proteomics has become an indispensable tool in system biology generating a need for accurate and precise quantitation of peptide standards. The presented method utilizes ultra performance LC-MS/MS (UPLC-MS/MS) to accurately quantify peptide standards at concentrations of 0.1-10 microM. The ability for accurate quantitation of micro-molar concentrations has the advantages that quantitation can be performed routinely with high precision and the high sensitivity of the method minimizes the amounts required.

Purification of a murine IgM monoclonal antibody.

An IgM monoclonal antibody, S11-23.4, raised against the 47-62 amino acid sequence in bovine prothrombin fragment 1 (F-1, the amino-terminal 156 residues of prothrombin), was purified from tissue culture supernatants and ascites using different purification schemes to determine the best method. There are many different purification schemes for the purification of IgG antibodies, which are generally easier to purify than IgM antibodies. Several different methods and schemes were tried to purify S11-23.4, and it was determined that the best purification schemes are ion exchange chromatography for cell culture IgM antibodies, and a G-100 gel filtration column, in conjunction with precipitation, reduction, and alkylation, for the same IgM antibody in ascites.

Production of metal ion-dependent monoclonal antibodies against peptides in bovine prothrombin fragment 1.

Bovine prothrombin fragment 1 (F-1: the amino-terminal 156 residues of prothrombin) is used as a model to study the Ca(II) and phospholipid binding of prothrombin. The 35-46 segment in F-1 posses an alpha-helical region and three aromatic residues, conserved in several vitamin K-dependent blood coagulation factors. These residues are believed to have a specific function and to be important in the phospholipid binding of F-1. The 47-62 region, a disulfide loop, is believed to stabilize the gamma-carboxyglutamic acid domain of the protein. Goals of this research were to produce monoclonal antibodies against the above two sequences, for later functional studies. Antibodies S9-32.8 and S9-5.5 were produced against the 35-46 sequence; antibody S11-23.4 was raised against the 47-62 region. Both S9-32.8 and S9-5.5 bound to F-1 immobilized on ELISA plates in the presence of 10 mM Ca(II) with higher affinity than to F-1 coated in the presence of 10 mM Mg(II) or in the absence of metal ions. S11-23.4 showed greatest binding to F-1 coated in the presence of 10 mM Mg(II). Thus, the epitopes of the antibodies are metal ion-dependent and are developed by Ca(II) binding to F-1.

S-arylcysteine-keratin adducts as biomarkers of human dermal exposure to aromatic hydrocarbons.

To measure biomarkers of skin exposure to ubiquitous industrial and environmental aromatic hydrocarbons, we sought to develop an ELISA to quantitate protein adducts of metabolites of benzene and naphthalene in the skin of exposed individuals. We hypothesized that electrophilic arene oxides formed by CYP isoforms expressed in the human skin react with nucleophilic sites on keratin, the most abundant protein in the stratum corneum that is synthesized de novo during keratinocyte maturation and differentiation. The sulfhydryl groups of cysteines in the head region of the keratin proteins 1 (K1) and 10 (K10) are likely targets. The following synthetic S-arylcysteines were incorporated into 10-mer head sequences of K1 [GGGRFSS( S-aryl-C)GG] and K10 [GGGG( S-aryl-C)GGGGG] to form the predicted immunogenic epitopes for antibody production for ELISA: S-phenylcysteine-K1 (SPK1), S-phenylcysteine-K10 (SPK10), S-(1-naphthyl)cysteine-K1 (1NK1), S-(1-naphthyl)cysteine-K10 (1NK10), S-(2-naphthyl)cysteine-K1 (2NK1), and S-(2-naphthyl)cysteine-K10 (2NK10). Analysis by ELISA was chosen based on its high throughput and sensitivity, and low cost. The synthetic modified oligopeptides, available in quantity, served both as immunogens and as chemical standards for quantitative ELISA. Polyclonal rabbit antibodies produced against the naphthyl-modified keratins reacted with their respective antigens with threshold sensitivities of 15-31 ng/mL and high specificity over a linear range up to 500 ng/mL. Anti- S-phenylcysteine antibodies were not sufficiently specific or sensitive toward the target antigens for use in ELISA under our experimental conditions. In dermal tape-strip samples collected from 13 individuals exposed to naphthalene-containing jet fuel, naphthyl-conjugated peptides were detected at levels from 0.343 +/- 0.274 to 2.34 +/- 1.61 pmol adduct/microg keratin but were undetectable in unexposed volunteers. This is the first report of adducts of naphthalene (or of any polycyclic aromatic hydrocarbon) detected in the exposed intact human skin. Quantitation of naphthyl-keratin adducts in the skin of exposed individuals will allow us to investigate the importance of dermal penetration, metabolism, and adduction to keratin and to predict more accurately the contribution of dermal exposure to systemic dose for use in exposure and risk-assessment models.

Antibodies as drug carriers III: design of oligonucleotides with enhanced binding affinity for immunoglobulin G.

PURPOSE: To understand the structural requirements in designing epitope-bearing oligonucleotides with high antibody-binding affinity. METHODS: Binding affinity (KA) and stoichiometry (n) of dinitrophenyl (DNP)-derivatized model 27-mer oligonucleotides (ODNs), GGG(AAA)7GGG, to monoclonal anti-trinitrophenyl (TNP) antibodies were determined using isothermal titration calorimetry (ITC). Structural variations were made in the ODNs to assess the effects of antigenic valence, epitope density, inter-epitope linker length, and linker flexibility. Binding isotherms were fitted with a single binding-site model to obtain K(A) and n, from which changes in Gibbs free energy (deltaG(0)), entropy (deltaS(0)), and enthalpy (deltaH(0)) were derived. RESULTS: As expected, ligands displaying increased epitope density showed increases in K(A): for example, K(A) for (DNP)2-Cys is 3.3-fold greater than that for DNP-Lys. Introduction of multiple DNP groups via long and flexible linkers to one end of the 27-mer ODN resulted in a bivalent behavior with n value of 1. A bivalent ligand, derivatized at both ends with a long and flexible linker, failed to form an immune complex when hybridized to its antisense strand, presumably due to intercalation of the DNP moiety to the double strand. ODNs derivatized with flexible linkers exhibited a higher K(A) than those with a rigid linker. Ligands with flexible inter-epitope linkers measuring distances of 110, 60, and 40 angstroms yielded 13-, 30-, and 13-fold increases in K(A), respectively. The combination of these factors; namely, bivalence, flexible inter-epitope linkers, and optimal inter-epitope distance, resulted in an overall 66-fold increase in K(A). Thermodynamic analysis of binding indicates that the formation of high-affinity ODN-IgG complexes was a spontaneous and exothermic event, characterized by large negative deltaS degrees, deltaH degrees, and deltaG degrees values. CONCLUSIONS: All four strategies tested during this investigation, namely bivalence, epitope density, inter-epitope linker flexibility, and optimal inter-epitope distance, proved to be useful in improving the binding affinity of DNP-labeled ODNs to anti-TNP IgG. The final ODN design incorporating these strategies will be used in testing the systemic pharmacokinetic advantage gained from complexing such ODNs to IgG.

Synthesis and characterization of peptides containing a cyclic Val adduct of diepoxybutane, a possible biomarker of human exposure to butadiene.

1,3-Butadiene, a potential human carcinogen widely used in industry, is oxidized by cytochrome P450 to diepoxybutane (DEB), which is the most mutagenic of the known butadiene metabolites. Assessment of the toxicological significance of DEB formation in humans and animals requires identification of a biomarker uniquely associated with DEB for use in molecular dosimetry studies. We wished to develop a specific and sensitive assay for one such suitable marker, the cyclic adduct 2-(3,4-dihydroxypyrrolidin-1-yl)-3-methylbutyramide (pyr-V), which is formed from addition of DEB to the terminal Val of the alpha- and beta-chains of hemoglobin. We needed to prepare a pure, rigorously characterized DEB-modified N-terminal oligopeptide for raising antibodies both to use in an immunoaffinity purification step and to standardize the assay. In addition, we needed a pure isotopomer to serve as an internal standard for quantitation by LC-MS. Direct modification of the globin sequences by reaction with DEB in vitro proved to be unproductive. We therefore opted to synthesize the cyclic Val adduct and incorporate it by FMOC chemistry into the appropriate oligopeptide sequences. In vitro and in vivo, butadiene is oxidized to enantiomeric and meso forms of DEB. A priori, all three DEB isomers are expected to form pyr-V adducts, resulting in three diastereomeric N-terminal peptides. We therefore synthesized a mixture of the cyclic Val diastereomers as their methyl esters by reaction of DEB with l-Val methyl ester hydrochloride. After protection as the di-O-tert-butyl derivatives, the mixture of 2-(3,4-di-t-butoxypyrrolidin-1-yl)-3-methylbutyric acid diastereomers was incorporated as the N-terminal residue into the 1-11 human globin alpha-chain sequence VLSPADKTNVK. The presence of the three diastereomers was confirmed by two-dimensional correlation NMR spectroscopy and temperature-dependent (1)H NMR. This strategy enabled us to obtain pure, rigorously characterized haptens in quantity for the preparation of polyclonal antibodies. Use of FMOC-protected (2)H(3)-Leu in the automated oligopeptide synthesis provided the required isotopomers for use as internal standard.

Expression and self-assembly of norwalk virus capsid protein from venezuelan equine encephalitis virus replicons.

The Norwalk virus (NV) capsid protein was expressed using Venezuelan equine encephalitis virus replicon particles (VRP-NV1). VRP-NV1 infection resulted in large numbers of recombinant NV-like particles that were primarily cell associated and were indistinguishable from NV particles produced from baculoviruses. Mutations located in the N-terminal and P1 domains of the NV capsid protein ablated capsid self-assembly in mammalian cells.