Making ends meet: chemically mediated circularization of recombinant proteins.
Bottom Line: A selective N→S acyl transfer reaction facilitates semi-synthesis of the plant cyclotide kalata B1 from a linear precursor peptide of bacterial origin, through simple appendage of N-terminal cysteine and a thiol-labile C-terminal Gly-Cys motif.This constitutes the first synthesis of a ribosomally derived circular miniprotein, without recourse to protein splicing elements.
Affiliation: Department of Chemistry, University College London, Christopher Ingold Building, 20 Gordon Street, London, WC1H 0AJ, UK.Show MeSH
Mentions: Subsequent in vitro cyclization of the purified linear KB1 peptide was achieved through incubation at 45 °C in the presence of 10 % (w/v) sodium 2-mercaptoethane sulfonate (MESNa). The reaction proceeded for 48 h, after which the linear starting material was almost entirely consumed (Figure S4). The observed drop in molecular mass of approximately 121 Da in the cyclic product was consistent with the excision of the C-terminal cysteine residue and backbone cyclization (Figure 1 D, middle panel). In order to demonstrate that we had produced the correct circular framework, oxidative folding was performed, as previously described, and confirmed by a further loss of six mass units (Figure 1 D, right). We also observed a characteristic increase in HPLC retention time on a reversed-phase chromatography column following cyclization and oxidation (Figure 1 D lower panel), due to the exposure of surface hydrophobic residues in the folded structure, thus indicating the presence of natively folded KB1. Over 1 mg of purified folded material was obtained from 2 L cell culture. Furthermore, we carried out analytical coelution RP-HPLC of KB1 in the presence of native KB1 (nKB1) and observed elution of a single peak, representative of a homogeneous sample (Figure 1 E). Nonetheless, we sought further confirmation of KB1 structural integrity through NMR spectroscopy, including 1H chemical shift assignment (Figures S5 and S6) as previously described. Our KB1 chemical shifts align extremely closely with those of nKB1, including a diagnostic ring current-shifted Hβ in residue P6, at −0.17 ppm (Figure S5), and near-identical deviations of Hα chemical shifts from random coil values, indicative of the native structure (Figure 2). We also identified slow-exchanging backbone amide protons in residues C5, C15, T16, S18, V21, C22, T23, R24, L27 and V29 (Figures 2 and S7), as previously observed for nKB1, which definitively confirmed the native fold and characteristic hydrogen bond network.
Affiliation: Department of Chemistry, University College London, Christopher Ingold Building, 20 Gordon Street, London, WC1H 0AJ, UK.