Crystal structure of a catalytically active, non-toxic endopeptidase derivative of Clostridium botulinum toxin A.

Botulinum neurotoxins (BoNTs) modulate cholinergic nerve terminals to result in neurotransmitter blockade. BoNTs consists of catalytic (LC), translocation (Hn) and cell-binding domains (Hc). The binding function of the Hc domain is essential for BoNTs to bind the neuronal cell membrane, therefore, removal of the Hc domain results in a product that retains the endopeptidase activity of the LC but is non-toxic.

Effects of a hairpin polyamide on DNA melting: comparison with distamycin and Hoechst 33258.

We have used DNase I footprinting and fluorescence melting studies to study the interaction of the hairpin polyamide Im-Py-Py-Py-(R)H2Ngamma-Im-Py-Py-Py-beta-Dp with its preferred binding sites (5'-WGWWCW; W=A or T) and other sequences. DNase I footprinting confirmed that the ligand binds to the sequence AGAACA at nanomolar concentrations and that changing the terminal A to G causes a dramatic decrease in affinity, while there was no interaction with the reverse sequence WCWWGW. Fluorescence melting studies with 11-mer duplexes showed that the polyamide had very different effects on the forward (TGWWCT) and reverse (TCTAGT) sequences.

DNA sequence recognition by an isopropyl substituted thiazole polyamide.

We have used DNA footprinting and fluorescence melting experiments to study the sequence-specific binding of a novel minor groove binding ligand (thiazotropsin A), containing an isopropyl substituted thiazole polyamide, to DNA. In one fragment, which contains every tetranucleotide sequence, sub-micromolar concentrations of the ligand generate a single footprint at the sequence ACTAGT. This sequence preference is confirmed in melting experiments with fluorescently labelled oligonucleotides.

DNA sequence specificity of triplex-binding ligands.

We have examined the ability of naphthylquinoline, a 2,7-disubstituted anthraquinone and BePI, a benzo[e]pyridoindole derivative, to stabilize parallel DNA triplexes of different base composition. Fluorescence melting studies, with both inter- and intramolecular triplexes, show that all three ligands stabilize triplexes that contain blocks of TAT triplets. Naphthylquinoline has no effect on triplexes formed with third strands composed of (TC)n or (CCT)n, but stabilizes triplexes that contain (TTC)n.

Thermodynamic and kinetic stability of intermolecular triple helices containing different proportions of C+*GC and T*AT triplets.

We have used oligonucleotides containing appropriately placed fluorophores and quenchers to measure the stability of 15mer intermolecular triplexes with third strands consisting of repeats of TTT, TTC, TCC and TCTC. In the presence of 200 mM sodium (pH 5.0) triplexes that contain only T.

The metal ion in the active site of the membrane glucose dehydrogenase of Escherichia coli.

All pyrroloquinoline quinone (PQQ)-containing dehydrogenases whose structures are known contain Ca(2+) bonded to the PQQ at the active site. However, membrane glucose dehydrogenase (GDH) requires reconstitution with PQQ and Mg(2+) ions (but not Ca(2+)) for activity. To address the question of whether the Mg(2+) replaces the usual active site Ca(2+) in this enzyme, mutant GDHs were produced in which residues proposed to be involved in binding metal ion were modified (D354N-GDH and N355D-GDH and D354N-GDH/N355D-GDH).