fmoc - labeled amino acids and all peptide synthesis reagents were purchased from advanced chem tech , except for n , n - diisopropylethylamine ( diea ; pharmco - aaper ) and acetic anhydride ( aldrich ) . all peptide cleavage reagents were purchased from aldrich , and high - performance liquid chromatography ( hplc ) solvents were purchased from pharmco - aaper . 5,5-dithiobis(2-nitrobenzoic acid ) ( dtnb ) was purchased from acros and d , l - dithiothreitol ( dtt ) and nacn were purchased form aldrich . all peptides were synthesized on an applied biosystems api 443a synthesizer using standard fmoc chemistry with an hbtu / hobt activation cocktail . all branched amino acids were double - coupled . the pal resin was used to furnish a c - terminal carboxamide on cleavage ; treatment of the resin bound peptides with acetic anhydride was used for n - acetylation . purity and identity of peptides was verified by reverse - phase hplc using an analytical - scale dynamax c18 column and matrix - assisted laser desorption / ionization mass spectrometry ( maldi - ms ; performed at the wistar institute , philadelphia , pa ) . since all peptides were nearly ( 95% or greater ) pure following peptide cleavage according to analytical hplc and maldi analysis , preparative - scale hplc was not performed . lyophilized cysteine - containing peptide from cleavage was treated for 20 min in 0.01 m hcl solution and relyophilized to remove residual tfa . the peptide was weighed after lyophilization , dissolved in 50 mm phosphate buffer , ph 7.07.5 , and treated with 100 dtt to ensure the free thiol . residual dtt and any other small impurities were separated from the peptide using a 28 cm column of sephadex g-10 , and the reduced peptide was lyophilized . the peptide was redissolved in 50 mm phosphate buffer ( cm15 peptides ) or 20 mm phosphate ( mbp peptides ) , ph 6.57.0 , and treated with 5 dtnb dissolved in 200 mm phosphate buffer , ph 6.57.0 , to form a mixed disulfide at cysteine . in limited cases , absorbance of the sample was monitored at 412 nm to observe release of the thionitrobenzoate anion and quantify the yield of the mixed disulfide ; yield for all cyanylated peptides was near unity when determined . the dtnbadduct sample was then treated with 50 nacn , and the cyanylated peptide was isolated using the same 28 cm sephadex g-10 column equilibrated with 50 mm ( cm15 peptides ) or 20 mm ( mbp peptides ) sodium phosphate buffer , ph 6.57.0 . peptide - containing fractions were concentrated about 5 using a speed vac centrifugal vacuum device , and the presence of the cn label was verified using infrared spectroscopy . the final concentrated sample concentration was 12 mm peptide in 100200 mm phosphate buffer , ph 6.57.0 . dpc was dried from chloroform by evaporating the solvent under a steady stream of nitrogen . dynamic light scattering using a dynapro ( wyatt technologies , santa barbara , ca ) in a 1.5 mm quartz cuvette ( helma ) indicated a uniform micelle size distribution of 2 nm . to obtain uniform micelle size , samples were extruded 15 times using a mini - extruder ( avanti polar lipids , alabaster , al ) . speed - vaced peptide samples from above were mixed with the dried lipid to yield samples that were approximately 2 mm in peptide and 100 mm in dpc in a 200 mm phosphate buffer . cd spectra were collected from 185 to 260 nm using an aviv model 410 spectropolarimeter . peptide samples at 12 mm peptide concentration ( and 100200 mm sodium phosphate buffer ) were analyzed in a 0.1 mm quartz demountable cell ( starna cells ) . cyanylated peptide samples ( in buffer or 100 mm dpc ) were placed between the windows of a 22 m caf2 biocell ( biotools , jupiter , fl ) placed inside a biojack temperature - circulating jacket held at 25 c . all spectra were collected at 2 cm resolution using a bruker optics vertex 70 ftir spectrometer with a photovoltaic hgcdte detector . a spectrum of buffer solution or 100 mm dpc in buffer solution was subtracted from the raw spectrum , and further baseline correction was accomplished by fitting the baseline ( outside the region from 2145 to 2185 cm ) to a polynomial and subtracting the fit .
cyanylated cysteine , or -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 proteinlipid interactions of membrane proteins .