Monitoring structural transitions in IDPS by vibrational spectroscopy of cyanylated cysteine

Abstract

The fast intrinsic time scale of infrared absorption and the sensitivity of molecular vibrational frequencies to their environments can be applied with site-specificity by introducing the artificial amino acid β- thiocyanatoalanine, or cyanylated cysteine, into chosen sites within intrinsically disordered proteins. This amino acid can be inserted through native chemical ligation at single cysteines introduced via site-directed mutagenesis. The CN stretching band of cyanylated cysteine is sensitive to local changes in both structural content and solvent exposure. This dual sensitivity makes cyanylated cysteine an especially useful probe of binding-induced structural transitions in IDPs. The general strategy of creating single-site cysteine mutations and chemically modifying them to create the vibrational chromophore, as well as observation, processing and analysis of the CN stretching band, is presented. © 2012 Springer Science+Business Media, LLC.