Quantifying the Effects of Hydrogen Bonding on Nitrile Frequencies in GFP: Beyond Solvent Exposure

Vibrational spectroscopy is a powerful tool for characterizing the complex noncovalent interactions that arise in biological systems. The nitrile stretching frequency has proven to be a particularly convenient biological probe, but the interpretation of nitrile spectroscopy is complicated by its sensitivity to local hydrogen bonding interactions. This often inhibits the straightforward interpretation of nitrile spectra by requiring knowledge of the molecular-level details of the local environment surrounding the probe. While the effect of hydrogen bonds on nitrile frequencies has been well-documented for small molecules in solution, there have been relatively few studies of these effects in a complex protein system. To address this, we introduced a nitrile probe at nine locations throughout green fluorescent protein (GFP) and compared the mean vibrational frequency of each probe to the specific hydrogen bonding geometries observed in molecular dynamics (MD) simulations. We show that a continuum of hydrogen bonding angles is found depending on the particular location of each nitrile, and that the differences in these angles account for the differences in the measured nitrile frequency. We further observed that the temperature dependence of the nitrile frequencies (measured as a frequency–temperature line slope, FTLS) was a good indicator of the hydrogen bonding interactions of the probe, even in scenarios where the nitrile was involved in complex and restricted hydrogen bonds, both from protein and from water. While constant offsets to the nitrile frequency to all hydrogen bonding environments have been applied before to interpret shifts in nitrile frequency, we show that this is insufficient in systems where the hydrogen bonds may be restricted by the surrounding medium. However, the strength of the observed correlation between nitrile frequency and hydrogen bonding angle suggests that it may be possible to disentangle electrostatic effects and effects of the orientation of hydrogen bonding on the nitrile stretching frequency. Meanwhile, the experimental measurement of the FTLS of the nitrile is an excellent tool to identify changes in the hydrogen bonding interactions for a particular probe