Cysteine-linked aromatic nitriles as UV resonance Raman probes of protein structure Colin L. Weeks, a Hyunil Jo, b Brandon Kier, c William F. DeGrado b and Thomas G. Spiro c * Nitriles introduced into peptides and proteins can serve as useful vibrational spectroscopic probes, because the nitrile C  N stretch is well isolated from backbone and sidechain vibrational bands. Aromatic nitriles offer large nC  N absorption inten- sity in infrared spectra and resonance enhancement in Raman spectra with ultraviolet excitation. We report the ultraviolet res- onance Raman spectra of cyanophenylalanine attached to cysteine, through linkage reactions that are applicable to cysteine residues in proteins. Excitation profiles are reported, and the nC  N detection limit is estimated to be 5 mM. The band position is sensitive to solvent polarity and especially to strong H-bonding. The derivatization of mastoparan X peptide at introduced cysteine residues demonstrated the effectiveness of a cyanophenylcysteine probe in reporting the lowered environmental polarity when the peptide was incorporated into liposomes. For an asymmetrical cyanophenyl derivative, 2-CBCys, the intensity ratio of asymmetric and symmetric ring modes, n8 b and n8 a , was found to respond to solvent polarity and not to H-bonding. Copyright © 2012 John Wiley & Sons, Ltd. Supporting information can be found in the online version of this article. Keywords: peptides; unnatural amino acids; proteins; UV resonance Raman; cyanophenylalanine Introduction Vibrational spectroscopy provides a powerful approach to moni- tor structure and dynamics in proteins because vibrational modes are sensitive to local conformation and molecular environment. However, the method is often limited by spectral crowding and the overlap of bands arising from multiple copies of the same structural elements, the peptide links [1,2] and the amino acid sidechains. [3] Introduction of additional probes, with unique vibrational signatures, can provide a valuable adjunct to protein studies. Nitrile derivatives are particularly attractive, because the nC  N band, at ~2200 cm 1 , is well removed from other bands in the protein spectrum. Because of the polarizability of the C–N bond, the nC  N wavenumber responds to the polarity of the nitrile environ- ment [4–7] and to the proximity of H-bond donors. Boxer and coworkers have shown that the dipolar effect is less pronounced than that of specific H-bonding and that the two effects can be distinguished by correlating nC  N with 13 C nuclear magnetic resonance (NMR) chemical shifts. [8] The nC  N band can be measured using infrared (IR) or Raman spectroscopy. The C  N stretch produces a strong induced IR dipole, particularly if the C  N group is attached to an aromatic ring. The environmental sensitivity of nC  N (Stark tuning rate) is also larger for aromatic than for aliphatic nitriles. [4,5] Neverthe- less, millimolar concentrations of protein are required for IR spec- troscopy. For Raman spectroscopy, however, detection limits are lowered into the micromolar range with ultraviolet (UV) excita- tion of the cyanophenylalanine (PheCN) Raman spectrum, thanks to resonance enhancement. Cyanophenylalanine can be incorporated into chemically synthe- sized polypetides. Incorporation into recombinant proteins via the difficult method of orthogonal tRNA synthetase pairs is also possible. [9,10] Labeling of proteins by a more facile method is desir- able, and the use of arylation or alkylation reactions to attach phenyl or benzyl nitriles to cysteine residues for IR studies was recently reported in a preliminary communication. [11] These methods are described more fully in the present paper, and UV resonance Raman (UVRR) characteristics of the aromatic nitrile adducts are reported. Boxer and coworkers have incorporated the thiocyanate (SCN) label into proteins, for IR [12] and NMR studies. To determine whether UVRR spectroscopy could be applied to these labels, we investi- gated the UVRR spectra of ethylthiocyanate (EtSCN). Unfortunately, resonance enhancement was found to be weak, not greater than that of cyanide ion itself, even with deep UV excitation. Thus, UVRR spectroscopy is useful only for aromatic nitriles (Fig. 1). Methods Raman spectra were obtained on samples sealed in 5-mm quartz NMR tubes, which were spun to ensure mixing of the solution, using 5  2 min of acquisition times to check for sample degradation (decrease in peak intensity vs NaClO 4 or NaCN standard and/or peak deformation) and averaged. The scattered light was collected at * Correspondence to: Thomas G. Spiro, Department of Chemistry, University of Washington, Box 351700, Seattle, WA, 98195, USA. E-mail: spiro@chem. washington.edu a Department of Chemistry and Biochemistry, University of Northern Iowa, Cedar Falls, IA, 50614, USA b Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA, 19104-6059, USA c Department of Chemistry, University of Washington, Seattle, WA, 98195, USA J. Raman Spectrosc. (2012) Copyright © 2012 John Wiley & Sons, Ltd. Research Article Received: 3 November 2011 Revised: 15 December 2011 Accepted: 21 December 2011 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/jrs.3167