Cyanylated Cysteine: A Covalently Attached Vibrational Probe of Protein-Lipid Contacts

Abstract

Cyanylated cysteine, or beta-thiocyanatoalanine, is an artificial amino acid that can be introduced into peptides and proteins by post-translational chemical modification of solvent-exposed cysteine side chains, and thus it can be used in any protein with a suitable expression and mutagenesis system. In this study, cyanylated cysteine is introduced at selected sites in two model peptides that have been shown to bind to membrane interfaces: a membrane-binding sequence of the human myelin basic protein and the antimicrobial peptide CM15. Far-UV circular dichroism indicates that the secondary structures of the bound peptides are not influenced by introduction of the artificial side chain. Infrared spectra of both systems in buffer and exposed to dodecylphosphocholine micelles indicate that the CN stretching absorption band of cyanylated cysteine can clearly distinguish between membrane burial and solvent exposure of the artificial side chain. Since infrared spectroscopy can be applied in a wide variety of lipid systems, and since cyanylated cysteine can be introduced into proteins of arbitrary size via mutagenesis and post-translational modification, this new probe could see wide use in characterizing the protein-lipid interactions of membrane proteins.

Figure 1

Far-UV CD spectra of all peptides in phosphate buffer and DPC-bound conditions: (a) native-sequence MBP 81−95, (b) MBP N89C*, (c) MBP F87C*, (d) unlabeled CM15, (e) CM15 A10C*, (f) CM15 I8C*. Note: since the MBP sequence does not contain a good UV chromophore for concentration determination, scaled raw ellipticities of samples are presented, and shape comparisons are most useful.

Figure 2

Solvent dependence of the CN stretching infrared absorption band of methyl thiocyanate in solvents relevant to membrane binding studies.

Figure 3

CN stretching absorption bands for cyanylated peptides in aqueous solution and DPC micelles: (a) MBP N89C*, (b) MBP F87C*, (c) CM15 A10C*, (d) CM15 I8C*. Intensities are scaled to the peak intensity for comparison; all optical densities are between 200 and 400 μOD.