In situ monitoring of backbone thioester exchange by 19F NMR.

Abstract

backbone thioester exchange; coiled coil; fluorine; NMR spectroscopy; protein foldingBackbone thioester exchange (BTE) is a new technique for assessing the stability of foldingpatterns adopted by polypeptides.[1–5] Implementation of this method requires thereplacement of a single backbone amide group in the polypeptide of interest with a thioester;this replacement is an approximately isostructural substitution.[ 6,7] Combining the backbone-modified polypeptide analogue (a thiodepsipeptide) with a small thiol in pH 7 aqueous bufferinitiates a thiol–thioester exchange reaction.[8] The equilibrium constant for this exchange isstrongly influenced by the stability of the folded conformation adopted by the full-lengththiodepsipeptide. We have shown that BTE enables examination of sequence–stabilityrelationships for a variety of small folding motifs under native conditions including β-hairpins,coiled coils and a small protein, the chicken villin headpiece subdomain.[1–5] BTE analysisis complementary to traditional methods of assessing protein conformational stability, in whichthe folding pattern is disrupted with heat or chemical additives.[ 9] The traditional approachesrequire extrapolation to obtain folding parameters that apply under native conditions, whileBTE enables direct measurement under native conditions. BTE has provided new insights oncommon protein folding motifs[ 1,3–5] and on contributions of unnatural side chains to tertiarystructural stability,[2] but continued development is necessary to maximize the utility of thisnew tool.Here we report that quantitative BTE analysis can be conducted in situ by using