Investigation of an unnatural amino acid for use as a resonance Raman probe: detection limits and 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.

Recent advances in linear and non‐linear Raman spectroscopy. Part III

“…Weeks et al [219] exploited ultraviolet resonance Raman spectroscopy and used the incorporation of an unnatural amino acid, 4-cyanophenylalanine, into proteins so they can act as resonance Raman probes to study the protein structure and function. El-Mashtoly et al [220] have also used ultraviolet resonance Raman spectroscopy to study the direct oxygen sensor protein from Escherichia coli.…”

Raman spectroscopy at the beginning of the twenty‐first century II

“…They show that the tryptophan residue(s) serve(s) as an electron donor to excited-state FAD in the photoinduced electron transfer of glucose oxidase. UV resonance Raman spectroscopy is also exploited by Weeks et al, 14 who use the incorporation of an unnatural amino acid, 4-cyanophenylalanine, into proteins so they can act as spectroscopic probes to study protein structure and function. 4-Cyanophenylalanine is a particularly useful probe, since the stretching mode of the nitrile group occurs in a region of the vibrational spectrum that does not contain any modes from the usual components of proteins, and its band position is sensitive to the polarity of its environment.…”

Solvatochromism and the solvation structure of benzophenone

“…Spectroscopic determinations at or near resonance would likely be enhanced 10-to 100-fold in terms of detection limit, indicating that the sensitivity of this Raman probe should surpass that of nitrile based labels under ideal conditions. 26 For the purpose of benchmarking ν(C=O) solvatochromism, 100.0 mM benzophenone were adopted as a standard since this concentration affords an exceptional signal-to-noise ratio while avoiding major electrostatic self-interaction of benzophenone molecules. To quantify the solvatochromic response of benzophenone, it is necessary to adopt a metric for the effective electric field experienced by the molecule in solution.…”

“…The environmental sensitivity of this probe was likewise retained, as practically demonstrated through the detailed characterization of liposome association of a small peptide. 26 In many cases, the use of Raman methods is preferable to infrared spectroscopy due to greater optical penetration of the sample, coupled with the weak contribution of water to this process.…”

“…Furthermore, the potential of resonance Raman spectroscopy to enhance the detection limit of benzophenone suggests that it should surpass existing tools in terms of concentration sensitivity. 26 The primary utility of benzophenone as a vibrational probe would be found in the biological sphere. This chromophore has been incorporated into an unnatural amino acid, p-benzoyl-L-phenylalanine, which has been widely employed as a photoaffinity probe within this context.…”