Investigation of an unnatural amino acid for use as a resonance Raman probe: Detection limits, solvent and temperature dependence of the νC≡N band of 4-cyanophenylalanine

Abstract

The incorporation of unnatural amino acids into proteins that act as spectroscopic probes can be used to study protein structure and function. One such probe is 4-cyanophenylalanine (PheCN), the nitrile group of which has a stretching mode that occurs in a region of the vibrational spectrum that does not contain any modes from the usual components of proteins and the wavenumber is sensitive to the polarity of its environment. In this work we evaluate the potential of UV resonance Raman spectroscopy for monitoring the sensitivity of the νC≡N band of PheCN incorporated into proteins to the protein environment. Measurement of the Raman excitation profile of PheCN showed that considerable resonance enhancement of the Raman signal was obtained using UV excitation and the best signal-to-noise ratios were obtained with excitation wavelengths of 229 and 244 nm. The detection limit for PheCN in proteins was ~10 μM, approximately a hundred-fold lower than the concentrations used in IR studies, which increases the potential applications of PheCN as a vibrational probe. The wavenumber of the PheCN νC≡N band was strongly dependent on the polarity of its environment, when the solvent was changed from H(2)O to THF it decreased by 8 cm(-1). The presence of liposomes caused a similar though smaller decrease in νC≡N for a peptide, mastoparan X, modified to contain PheCN. The selectivity and sensitivity of resonance Raman spectroscopy of PheCN mean that it can be a useful probe of intra- and intermolecular interactions in proteins and opens the door to its application in the study of protein dynamics using time-resolved resonance Raman spectroscopy.

Figure 1

UV-vis absorption spectrum and Raman excitation profiles of the νC≡N and phenyl ring ν_8a bands of PheCN in aqueous solution.

Figure 2

Raman spectra of aqueous solutions of PheCN (concentrations as labeled) with NaCN (100 mM) as the internal standard. The spectra were obtained using 229 nm excitation and 10 min acquisition time per spectrum.

Figure 3

Raman spectra of equimolar solutions of PheCN and cytochrome c (concentrations as labeled) in pH 7.6 phosphate buffer at 20 °C for solutions. The spectra were obtained using 229 nm excitation and the intensities normalized using the 2238 cm⁻¹ band from N₂ in the air on the front surface of the sample.

Figure 4

Effect of solvent polarity on the νC≡N band. Raman spectra of PheCN (100 μM) in H₂O/THF solutions (%v/v as labeled). The spectra were obtained using 244 nm excitation and a 10 min acquisition time per spectrum.

Figure 5

Deconvolution of the νC≡N band from 244 nm excitation Raman spectra of PheCN (100 μM) in H₂O/THF solutions.

Figure 6

Speciation diagram from fitting of the PheCN νC≡N band in the 244 nm excitation Raman spectra of the H₂O/THF solutions.

Figure 7

Temperature dependence of the νC≡N wavenumber of PheCN in aqueous solution from fitting a single Lorentzian peak to the spectrum.

Figure 8

The νC≡N band from the 229 nm excitation Raman spectra of MP_X5 (a) 20 μM in pH 7.1 buffer (b) 33 μM in POPC liposomes in pH 7.1 buffer, MP_X5:POPC = 1:40.