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

Abstract

β-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≡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 C≡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.

IR Probes of Protein Microenvironments: Utility and Potential for Perturbation

“…Correspondingly, the use of either CN or N 3 probes to study the folding of nSH3 would have erroneously suggested a multistep folding process involving the population of intermediates, as opposed to the simple two-state process revealed by the use of the non-perturbative CÀD probes (Figure 3). The results of this systematic analysis using nSH3 adds to a growing literature [3,13,19,24,35] that suggests that these probes are destabilizing, and combined these results suggest that the destabilization may be general. Thus, in contrast to CÀD labels, which are strictly non-perturbative, the results of studies based on the use of CN and N 3 probes must be interpreted with caution to ensure that they are biologically relevant and not an artifact of the chromophore or its perturbation of the protein.…”

“…Since the H bonds involved are introduced by the probe itself, this renders the data difficult to interpret in terms of the native protein. Boxer and co-workers have noted that it is possible to quantify the contribution of local electrostatics to nitrile peak shifts based on the relationship of IR frequency shifts and 13 C NMR chemical shifts, [5,14] and our data suggest that such corrections are likely to be necessary. Moreover, the data reveal that both extrinsic probes are prone to perturb the protein.…”

“…For example, Londergan and co-workers demonstrated that cyanylation of cysteine destabilized a model helix, and more so if in the middle than near either terminus. [13] Raleigh and co-workers showed that replacing Met1 or Ile4 of the N-terminal domain of the L9 ribosomal protein with azidohomoalanine destabilized the protein to thermal unfolding by 0.8 and 1.9 kcal mol À1 , respectively, [24] while replacing Phe5 with p-cyanophenylalanine, resulted in a slight stabilization to chaotropic denaturation. [35] In addition, Fayer and co-workers demonstrated that the incorporation of two p-cyanophenylalanine probes destabilizes the 35-residue villin headpiece subdomain.…”

Using Infrared Spectroscopy of Cyanylated Cysteine To Map the Membrane Binding Structure and Orientation of the Hybrid Antimicrobial Peptide CM15

“…A lack of complete quantitative agreement between all DPC-bound CD spectra indicates that there is a small perturbative influence of each C* placement on CM15’s DPC-bound structure; this is not particularly surprising in the context of recent measurements of C*’s effect on the helical stability of alanine-repeat peptides. (27) Whether these secondary structural changes affect the orientation of the peptide with respect to the micelle cannot be determined from CD spectra, which report only on the relationships between backbone amide groups of the peptide.…”

Two-dimensional infrared study of 3-azidopyridine as a potential spectroscopic reporter of protonation state

“…Recently, cyanylated cysteine, in which the cysteine thiol has been converted into a thiocyanate, has been the subject of several studies. [37][38][39][40] These studies illustrate that the thiocyanate is very sensitive to its local environment, making it an excellent probe of the local dynamics. Furthermore, because it is prepared by modifying cysteine in the protein, the synthetic methods to incorporate the probe site selectively are straightforward.…”