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Investigation of an unnatural amino acid for use as a resonance Raman probe: detection limits and solvent and temperature dependence of the νCN band of 4‐cyanophenylalanine
Author(s) -
Weeks Colin L.,
Polishchuk Alexei,
Getahun Zelleka,
DeGrado William F.,
Spiro Thomas G.
Publication year - 2008
Publication title -
journal of raman spectroscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.748
H-Index - 110
eISSN - 1097-4555
pISSN - 0377-0486
DOI - 10.1002/jrs.2067
Subject(s) - raman spectroscopy , chemistry , resonance (particle physics) , resonance raman spectroscopy , analytical chemistry (journal) , intermolecular force , molecule , chromatography , organic chemistry , optics , physics , particle physics
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 whose 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 νCN 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 µ M , approximately 100‐fold lower than the concentrations used in infrared (IR) studies, which increases the potential applications of PheCN as a vibrational probe. The wavenumber of the PheCN νCN band was strongly dependent on the polarity of its environment; when the solvent was changed from H 2 O to tetrahydrofuran (THF) it decreased by 8 cm −1 . The presence of liposomes caused a similar though smaller decrease in νCN 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. Copyright © 2008 John Wiley & Sons, Ltd.