No abstract

A unique peptide toxin, named double-knot toxin (DkTx), was recently purified from the venom of the tarantula Ornithoctonus huwena and was found to stably activate TRPV1 channels by targeting the outer pore domain. DkTx has been shown to consist of two inhibitory cysteine-knot (ICK) motifs, referred to as K1 and K2, each containing six cysteine residues. Beyond this initial characterization, however, the structural and functional details about DkTx remains elusive in large part due to the lack of a high yielding methodology for the synthesis and folding of this cysteine-rich peptide. Here, we overcome this obstacle by generating pure DkTx in quantities sufficient for structural and functional analyses. Our methodology entails expression of DkTx in E. coli followed by oxidative folding of the isolated linear peptide. Upon screening of various oxidative conditions for optimizing the folding yield of the toxin, we observed that detergents were required for efficient folding of the linear peptide. Our synthetic DkTx co-eluted with the native toxin on HPLC, and irreversibly activated TRPV1 in a manner identical to native DkTx. Interestingly, we find that DkTx has two interconvertible conformations present in a 1∶6 ratio at equilibrium. Kinetic analysis of DkTx folding suggests that the K1 and K2 domains influence each other during the folding process. Moreover, the CD spectra of the toxins shows that the secondary structures of K1 and K2 remains intact even after separating the two knots. These findings provide a starting point for detailed studies on the structural and functional characterization of DkTx and utilization of this toxin as a tool to explore the elusive mechanisms underlying the polymodal gating of TRPV1.

The purpose of this study was to develop a biobetter version of recombinant human interferon-β 1a (rhIFN-β 1a) to improve its biophysical properties, such as aggregation, production and stability, and pharmacokinetic properties without jeopardizing its activity. To achieve this, we introduced additional glycosylation into rhIFN-β 1a via site-directed mutagenesis. Glycoengineering of rhIFN-β 1a resulted in a new molecular entity, termed R27T, which was defined as a rhIFN-β mutein with two N-glycosylation sites at 80th (original site) and at an additional 25th amino acid due to a mutation of Thr for Arg at position 27th of rhIFN-β 1a. Glycoengineering had no effect on rhIFN-β ligand-receptor binding, as no loss of specific activity was observed. R27T showed improved stability and had a reduced propensity for aggregation and an increased half-life. Therefore, hyperglycosylated rhIFN-β could be a biobetter version of rhIFN-β 1a with a potential for use as a drug against multiple sclerosis.

No abstract

Amphipathic protein toxins from tarantula venom inhibit voltage-activated potassium (Kv) channels by binding to a critical helix-turn-helix motif termed the voltage sensor paddle. Although these toxins partition into membranes to bind the paddle motif, their structure and orientation within the membrane are unknown. We investigated the interaction of a tarantula toxin named SGTx with membranes using both fluorescence and NMR spectroscopy. Depth-dependent fluorescence-quenching experiments with brominated lipids suggest that Trp30 in SGTx is positioned approximately 9 A from the center of the bilayer. NMR spectra reveal that the inhibitor cystine knot structure of the toxin does not radically change upon membrane partitioning. Transferred cross-saturation NMR experiments indicate that the toxin's hydrophobic protrusion contacts the hydrophobic core of the membrane, whereas most surrounding polar residues remain at interfacial regions of the bilayer. The inferred orientation of the toxin reveals a twofold symmetry in the arrangement of basic and hydrophobic residues, a feature that is conserved among tarantula toxins. These results have important implications for regions of the toxin involved in recognizing membranes and voltage-sensor paddles, and for the mechanisms by which tarantula toxins alter the activity of different types of ion channels.