Identification, chemical synthesis, structure, and function of a new K_V1 channel blocking peptide from Oulactis sp.

Abstract

Rapid progress in transcriptomic and proteomic studies of sea anemones has led to the identification of a large number of new peptide sequences. Some of these peptides have high sequence similarity and identical cysteine frameworks to those of previously reported sequences. One such peptide we have identified from a transcriptomic study of Oulactis sp is OspTx2a, which has a cysteine framework similar to that of ShK (from Stichodactyla helianthus) and BgK (from Bunodosoma granulifera). This peptide was made using solid‐phase peptide synthesis, but, upon oxidative folding, it generated two peptides with identical masses (OspTx2a‐p1 and OspTx2a‐p2) that were distinguishable by high‐performance liquid chromatography. The structures of OspTx2a‐p1 and OspTx2a‐p2 were determined using nuclear magnetic resonance spectroscopy, and voltage‐clamp electrophysiology assays were performed in order to assess the activity against a range of potassium channels. The structures of the two peptides were very similar to each other and to BgK, with the same disulfide bond connectivities, and both had an all‐trans backbone conformation. In functional assays, both OspTx2a‐p1 and OspTx2a‐p2 inhibited KV1.2 and KV1.6 channel currents at low µM concentrations, with similar but not identical IC50 values. Peptides containing a C‐terminal Cys residue are particularly sensitive to racemization at this residue, and the two products obtained for OspTx2a could be a consequence of racemization of the Cys residue at its C‐terminus during synthesis. NMR chemical shift differences between the two products and their structural preferences for a d‐Cys residue were consistent with this interpretation.

Modulation of Lymphocyte Potassium Channel KV1.3 by Membrane-Penetrating, Joint-Targeting Immunomodulatory Plant Defensin

“…Slight peak broadening was observed in the spectrum of VvK1 peak 1 compared to VvK1 peak 2 (Figure S2). In addition, the chemical shifts of a few resonances in the VvK1 peak 1 spectrum were quite distinct from those in the VvK1 peak 2 spectrum (Figures S4, S5); such differences between two peptides with the same sequences and masses may have arisen from racemization of the C-terminal cysteine residue during peptide synthesis . Hydrodynamic radii calculated from translational diffusion measurements made by NMR (Table S2) confirmed that the defensins are monomeric in solution at pH 5 and 20 °C, in contrast to their dimeric structures in crystals.…”

Aggregation and the Intrinsic Structural Disorder of Dipeptide Repeat Peptides of C9orf72-Related Amyotrophic Lateral Sclerosis and Frontotemporal Dementia Characterized by NMR

“…The new cross-peaks observed in the region from 5.5 to 5.7 ppm (F2-dimension) and from ∼1.5 to ∼4 ppm (F1-dimension) in the 2D TOCSY spectra (i.e., within the box for two different temperatures) are uncommon; their pattern represents whole spin systems of Arg and Gly residues (Figure B). Such peaks have been observed for the residues in the well-folded peptides and proteins with a large hydrophobic core region, most probably due to aromatic ring-current effects. − In (GR) 6 , the upfield chemical shifts were not due to ring-current effects, as the peptide does not contain any aromatic residues. Therefore, it is likely that these resonances indicate the formation of new structures over time.…”

Identification, Synthesis, Conformation and Activity of an Insulin-like Peptide from a Sea Anemone

“…Based on the sequence similarity to insulin and previously characterized ILPs, we were unable to predict the function of IlO1_i1. It is becoming increasingly evident that sequence similarity alone is not a sufficient indicator of peptide functionality [ 62 , 63 , 64 ]. As more invertebrate ILPs are characterized with a range of functions relating to tissue-specific regions, it may become possible to more accurately predict which sequences are likely to have a particular functionality.…”