Cyanophenylalanine structural reconfiguration induced by ion binding: Magnesium-specific nitrile chelation motif in M²⁺ complexes

Noncoded amino acids are widely used as spectroscopic probes of complex biological systems. Para-cyanophenylalanine has found particularly broad use as a fluorescence, FRET, and infrared probe of protein structure and function. For example, its nitrile stretching absorption provides a readout of the electrostatic environments important to enzyme catalysis in a region of the infrared spectrum free from interference from other transitions. However, in complex systems it is often challenging to separate the spectral contributions from effects of interest, such as active pocket environment, from other factors like probe isomerization.

We characterize the spectral and structural response of para-cyanophenylalanine to well-controlled changes in electrostatic environment by collecting infrared spectra of the cold (~10 K), isolated ion alone and in complex with different divalent cations. This is accomplished by isolating the mass-selected, gas-phase cation in a temperature-controlled, radiofrequency ion trap and monitoring the laser-induced photodissociation of weakly bound “mass tags,” typically H₂ or He, as a function of photon energy. The linear absorption spectra so collected are interpreted using electronic structure calculations, showing that the Mg²⁺ complex adopts a dominant configuration - characterized by direct interaction between the nitrile group and bound ion - that is distinct from the dominant configurations of the complexes with other divalent cations (Ca²⁺, Sr²⁺, Ba²⁺).