First chemical synthesis of a scorpion alpha-toxin affecting sodium channels: the Aah I toxin of Androctonus australis hector.

Aah I is a 63-residue alpha-toxin isolated from the venom of the Buthidae scorpion Androctonus australis hector, which is considered to be the most dangerous species. We report here the first chemical synthesis of Aah I by the solid-phase method, using a Fmoc strategy. The synthetic toxin I (sAah I) was renatured in DMSO-Tris buffer, purified and subjected to thorough analysis and comparison with the natural toxin. The sAah I showed physico-chemical (CD spectrum, molecular mass, HPLC elution), biochemical (amino-acid composition, sequence), immunochemical and pharmacological properties similar to those of the natural toxin. The synthetic toxin was recognized by a conformation-dependent monoclonal anti-Aah I antibody, with an IC50 value close to that for the natural toxin. Following intracerebroventricular injection, the synthetic and the natural toxins were similarly lethal to mice. In voltage-clamp experiments, Na(v) 1.2 sodium channel inactivation was inhibited by the application of sAah I or of the natural toxin in a similar way. This work describes a simple protocol for the chemical synthesis of a scorpion alpha-toxin, making it possible to produce structural analogues in time.     

Tackling the plant proteome: practical approaches, hurdles and experimental tools

The study of complex biological questions through comparative proteomics is becoming increasingly attractive to plant biologists as the rapidly expanding plant genomic and expressed sequence tag databases provide improved opportunities for protein identification. This review focuses on practical issues associated with comparative proteomic analysis, including the challenges of effective protein extraction and separation from plant tissues, the pros and cons of two-dimensional gel-based analysis and the problems of identifying proteins from species that are not recognized models for functional genomic studies. Specific points are illustrated using data from an ongoing study of the tomato and pepper fruit proteomes.     

Production of fully assembled and active Aquifex aeolicus F1FO ATP synthase in Escherichia coli

Background

F₁F_O ATP synthases catalyze the synthesis of ATP from ADP and inorganic phosphate driven by ion motive forces across the membrane. A number of ATP synthases have been characterized to date. The one from the hyperthermophilic bacterium Aquifex aeolicus presents unique features, i.e. a putative heterodimeric stalk. To complement previous work on the native form of this enzyme, we produced it heterologously in Escherichia coli.

Methods

We designed an artificial operon combining the nine genes of A. aeolicus ATP synthase, which are split into four clusters in the A. aeolicus genome. We expressed the genes and purified the enzyme complex by affinity and size-exclusion chromatography. We characterized the complex by native gel electrophoresis, Western blot, and mass spectrometry. We studied its activity by enzymatic assays and we visualized its structure by single-particle electron microscopy.

Results

We show that the heterologously produced complex has the same enzymatic activity and the same structure as the native ATP synthase complex extracted from A. aeolicus cells. We used our expression system to confirm that A. aeolicus ATP synthase possesses a heterodimeric peripheral stalk unique among non-photosynthetic bacterial F₁F_O ATP synthases.

Conclusions

Our system now allows performing previously impossible structural and functional studies on A. aeolicus F₁F_O ATP synthase.

General significance

More broadly, our work provides a valuable platform to characterize many other membrane protein complexes with complicated stoichiometry, i.e. other respiratory complexes, the nuclear pore complex, or transporter systems.     

Random coil chemical shifts in acidic 8 M urea: Implementation of random coil shift data in NMRView

Studies of proteins unfolded in acid or chemical denaturant can help in unraveling events during the earliest phases of protein folding. In order for meaningful comparisons to be made of residual structure in unfolded states, it is necessary to use random coil chemical shifts that are valid for the experimental system under study. We present a set of random coil chemical shifts obtained for model peptides under experimental conditions used in studies of denatured proteins. This new set, together with previously published data sets, has been incorporated into a software interface for NMRView, allowing selection of the random coil data set that fits the experimental conditions best.     

Sequence diversity of the peptaibol antibiotic suzukacillin-A from the mold Trichoderma viride.

From the culture broth of the mold Trichoderma viride, strain 63 C-I, the polypeptide antibiotic suzukacillin (SZ) was isolated. A peptide mixture named SZ-A was obtained by crystallization from crude SZ. Individual peptides from SZ-A were isolated by semipreparative HPLC and sequences were determined by HPLC-ESI-MS. The data confirm a general sequence of SZ-A published previously and in addition establish the individual sequences of 15 acetylated eicosa peptides with C-terminal alcohols. The major peptide SZ-A4 (21% of all peptides) shows the sequence:Ac-Aib-Ala-Aib-Ala-Aib-Ala(6)-Gln-Aib-Lx(9)-Aib-Gly-Aib(12)-Aib-Pro-Vx(15)-Aib-Vx(17)-Gln-Gln-Fol. Amino acid exchanges of the peptaibol are located in position 6 (Ala/Aib), 9 (Vx/Lx), 12 (Aib/Lx), 17 (Aib/Vx) and possibly at position15 (Val/Iva) (uncommon abbreviations: Aib (alpha-aminoisobutyric acid); Iva (D-isovaline); Lx (L-leucine or L-isoleucine); Vx (L-valine or D-isovaline); Fol (L-phenylalaninol)).     

Homologous protein import machineries in chloroplasts and cyanelles

The cyanelles of the glaucocystophyte alga Cyanophora paradoxa resemble endosymbiotic cyanobacteria, especially in the presence of a peptidoglycan wall between the inner and outer envelope membranes. However, it is now clear that cyanelles are in fact primitive plastids. Phylogenetic analyses of plastid, nuclear and mitochondrial genes support a single primary endosymbiotic event. In this scenario, cyanelles and all other plastid types are derived from an ancestral photosynthetic organelle combining the high gene content of rhodoplasts and the peptidoglycan wall of cyanelles. This means that the import apparatuses of all primary plastids, i.e. those from glaucocystophytes, red algae, green algae and higher plants, should be homologous. If this is the case, then transit sequences should be similar and heterologous import experiments feasible. Thus far, heterologous in vitro import has been shown in one direction only: precursors from C. paradoxa were imported into isolated pea or spinach chloroplasts. Cyanelle transit sequences differ from chloroplast stroma targeting peptides in containing in their N-terminal domain an invariant phenylalanine residue which is shown here to be crucial for import. In addition, we now demonstrate that heterologous precursors are readily imported into isolated cyanelles, provided that the essential phenylalanine residue is engineered into the N-terminal part of chloroplast transit peptides. The cyanelle and likely also the rhodoplast import apparatus can be envisaged as prototypes with a single receptor/channel showing this requirement for N-terminal phenylalanine. In chloroplasts, multiple receptors with overlapping and less stringent specificities have evolved, explaining the efficient heterologous import of native precursors from C. paradoxa.     

Amino Acid Residues 226–240 of τ Which Encompass the First Lys-Ser-Pro Site of τ, Are Partially Phosphorylated in Alzheimer Paired Helical Filament-τ

Abstract: A synthetic peptide corresponding to residues 226–240 (E9 peptide) of human τ, which contains an Lys-Ser-Pro motif, was used to raise a polyclonal antibody. The antibody, E9, was 10-fold less reactive with phospho-E9 peptide than with native E9 peptide. E9 antibody was used to study the extent of phosphorylation in a modified form of τ (PHF-τ) that is found in Alzheimer's disease brain and is incorporated into paired helical filaments (PHFs). E9 immunolabeled Alzheimer's disease neurofibrillary tangles and abnormal neurites in brain sections intensely, with increased immunoreactivity detected after pretreatment of sections with phosphatase. On immunoblots and ELISA, E9 reacted with PHF-τ and recombinant human τ but not with the high and middle molecular weight neurofilament proteins. Phosphatase treatment of PHF-τ improved the E9 immunoreactivity by 30–50%. Dephosphorylated high but not middle molecular weight neurofilament protein became reactive with E9. These results indicate that <50% of the PHF-T is phosphorylated in the subregion corresponding to residues 226–240 of τ and suggest that the phosphorylation of this region may not be essential for PHF formation.