ja4046795_si_001.pdf (1.47 MB)
Native Chemical Ligation at Asx-Cys, Glx-Cys: Chemical Synthesis and High-Resolution X‑ray Structure of ShK Toxin by Racemic Protein Crystallography
journal contribution
posted on 2013-08-14, 00:00 authored by Bobo Dang, Tomoya Kubota, Kalyaneswar Mandal, Francisco Bezanilla, Stephen B. H. KentWe
have re-examined the utility of native chemical ligation at
-Gln/Glu-Cys- [Glx-Cys] and -Asn/Asp-Cys- [Asx-Cys] sites. Using the
improved thioaryl catalyst 4-mercaptophenylacetic acid (MPAA), native
chemical ligation could be performed at -Gln-Cys- and Asn-Cys- sites
without side reactions. After optimization, ligation at a -Glu-Cys-
site could also be used as a ligation site, with minimal levels of
byproduct formation. However, -Asp-Cys- is not appropriate for use
as a site for native chemical ligation because of formation of significant
amounts of β-linked byproduct. The feasibility of native chemical
ligation at -Gln-Cys- enabled a convergent total chemical synthesis
of the enantiomeric forms of the ShK toxin protein molecule. The d-ShK protein molecule was ∼50,000-fold less active in
blocking the Kv1.3 channel than the l-ShK protein molecule.
Racemic protein crystallography was used to obtain high-resolution
X-ray diffraction data for ShK toxin. The structure was solved by
direct methods and showed significant differences from the previously
reported NMR structures in some regions of the ShK protein molecule.