PMC

(a). (D)-ShK polypeptide folding (Upper) Reduced polypeptide; (Lower) After air oxidation for 42h. *Folded product
(b). HPLC purified D-ShK toxin Mobs.= 4054.3±0.4Da

Proposed reaction pathway for formation of α- and β-linked peptide products during native chemical ligation at an -Asp-Cys- ligation site. R` = CH₂CH₂CO-Phe-COOH

(a). (L)-ShK polypeptide folding. (Upper) Reduced polypeptide; (Lower) After air oxidation for 63h. *Folded product
(b). HPLC purified L-ShK toxin Mobs.= 4054.2±0.4Da Mcalc. (av. isotope composition)=4054.8

(a). (D)-ShK polypeptide ligation T=0, Peak 1’ =D-[Cys¹⁷-Cys³⁵]; Peak 2’ = D- [ Arg¹ -Gln¹⁶]-α-thioester.
T=19h, * Ligation product.
Early eluting broad peaks are non-peptidic.
(b). Purified D-ShK Polypeptide Mobsd.= 4060.1±0.4Da

(a). (L)-ShK polypeptide ligation T=0, Peak 1 = [Cys¹⁷-Cys³⁵]; Peak 2 =[Arg¹-Gln¹⁶]-α-thioester.
T=19h, *Ligation product.
Early eluting broad peaks are non-peptidic.
(b). Purified L-ShK Polypeptide Mobsd.= 4060.3±0.4Da Mcalc.(av. isotope composition)=4060.8 Da

Convergent synthesis of ShK toxin by native chemical ligation of two unprotected peptide segments, followed by folding and formation of disulfides.

Native chemical ligations between peptide-Glx-^αCOSR` and Cys-peptide: * correct product. (Left) −Gln- COSR reaction products; [Inset: close-up of ligation products – the vertical bar shows the elution position of the γ-linked product] (Right) −Glu- COSR reaction products – the vertical bar shows the elution position of the γ-linked product. Reaction conditions: (Left) pH 7.0, 20 mM MPAA, 6 M Gu·HCl, 200 mM Na2HPO4, 10 mM TCEP·HCl, RT; (Right) pH 6.7, 20 mM MPAA, 6 M Gu·HCl, 200 mM Na2HPO4, 10 mM TCEP·HCl, RT.

Native chemical ligations between peptide-Asx-^αCOSR` and Cys-peptide: * correct product. (Left) −Asn-<sup>α</sup>COSR` reaction products [Inset: close-up of ligation products – the vertical bar shows the elution position of the β-linked product]; (Right) −Asp-^αCOSR` reaction products – the vertical bar shows the elution position of β-linked product. Reaction conditions: pH 7.0, 20 mM MPAA, 6 M Gu ·HCl, 200 mM Na₂HPO₄, 10 mM TCEP·HCl, room temperature (RT).

Close up view of the interfaces between the enantiomeric L-ShK (Green) and D-ShK (Cyan) molecules in the DL-protein racemate crystal. (a), Hydrogen bonding between (D)-Thr⁶ and (L)-Arg¹¹, (L)-Thr⁶ and (D)-Arg¹¹; (b), Hydrogen bonding between side chain of (D)-His¹⁹ and backbone of (L)-Cys³², also hydrogen bonding connected by water molecules at multiple sites, Water molecules are shown as small red spheres; (c), Hydrogen bonding between side chain of (L)-Ser² and main chain of (D)-Lys⁹, side chain (D)-Ser² and main chain of (L)-Lys⁹, also hydrogen bonding connected by water molecules at multiple sites; (d), Weak hydrophobic interaction between Leu²⁵ and Met²¹.

Crystal structure of ShK DL-protein racemate. (a), Cartoon representation of the packing of D-ShK (Cyan) and L-ShK (Green) toxin in unit cell in P2₁/c, unit cell composed of 4 molecules, 2 L-ShK and 2 D-ShK. (b), SigmaA-weighted 2Fo - Fc electron density map at a σ level of 2, showing Tyr that derived from the best SHELXS solution (CFOM = 0.14).

(a) Cartoon representation of the L-ShK toxin. The structure was solved as the racemate at 0.97 Å X-ray resolution by direct methods. The protein fold is shown as ribbons with three S-S bonds shown in stick mode. (b) Superposition of the NMR structure (PDB ID: 1ROO) of ShK toxin (in red) and the present L-enantiomer from the X-ray racemic crystal structure (in green); the selected NMR model (#10) displays the minimal C_α r.m.s. deviation of 1.8 Å. (c) Superposition of the L-ShK crystal structure (in blue) and L-ShK enantiomer structure from the DL-protein racemate (Green).

The amino acid sequence of the ShK toxin protein is shown in . Synthesis of ShK toxin by Fmoc chemistry SPPS has previously been reported.^,[ In our hands, stepwise synthesis of the 35 residue ShK polypeptide using highly optimized Boc chemistry SPPS gave an HPLC yield of only ~5% of the desired polypeptide accompanied by significant amounts of byproducts. For that reason, we decided to establish a more effective total chemical synthesis of the ShK polypeptide chain using modern chemical ligation methods. The most effective ligation chemistry is thioester-mediated amideforming native chemical ligation at −Xaa-Cys− sites. There are six Cys residues (3 disulfides) in the native ShK toxin protein.

Functional blockade of hKv1.3 currents by L- and D- ShK synthetic toxins using the cut-open oocyte voltage clamp method to measure potassium ionic currents from Xenopus laevis oocytes that contained expressed human Kv1.3 (hKv1.3) channels. Peak currents were recorded during a 150ms step to + 30 mV from a holding voltage of −90 mV (upper inset). The fractions of current blockade are plotted for test concentration of L-ShK (filled squares, n = 3–5 cells) and D-ShK (open squares, n = 3–6 cells). Dotted lines indicate dose-response curves fitted by the Hill equation. Error bars in figure and uncertainties in table indicate standard error of the mean.