The Effects of α-Helical Structure and Cyanylated Cysteine on Each Other

Beta-thiocyanatoalanine, or cyanylated cysteine, is an artificial amino acid that can be introduced at solvent-exposed cysteine residues in proteins via chemical modification. Its facile post-translational synthesis means that it may find broad use in large protein systems as a probe of site-specific structure and dynamics. The C[triple bond]N stretching vibration of this artificial side chain provides an isolated infrared chromophore. To test both the perturbative effect of this side chain on local secondary structure and its sensitivity to structural changes, three variants of a model water-soluble alanine-repeat helix were synthesized containing cyanylated cysteine at different sites. The cyanylated cysteine side chain is shown to destabilize, but not completely disrupt, the helical structure of the folded peptide when substituted for alanine. In addition, the [triple bond]N stretching bandwidth of the artificial side chain is sensitive to the helix-coil structural transition. These model system results indicate that cyanylated cysteine can be placed into protein sequences with a native helical propensity without destroying the helix, and further that the CN probe may be able to report local helix formation events even when it is water-exposed in both the ordered and disordered conformational states. These results indicate that cyanylated cysteine could be a widely useful probe of structure-forming events in proteins with large in vitro structural distributions.