Hydrogen Bonding\nand Noncovalent Electric Field Effects\nin the Photoconversion of a Phytochrome

A profound understanding of protein structure and mechanism\nrequires\ndedicated experimental and theoretical tools to elucidate electrostatic\nand hydrogen bonding interactions in proteins. In this work, we employed\nan approach to disentangle noncovalent and hydrogen-bonding electric\nfield changes during the reaction cascade of a multidomain protein,\ni.e., the phytochrome Agp2. The approach exploits the spectroscopic\nproperties of nitrile probes commonly used as reporter groups of the\nvibrational Stark effect. These probes were introduced into the protein\nthrough site-specific incorporation of noncanonical amino acids resulting\nin four variants with different positions and orientations of the\nnitrile groups. All substitutions left structures and the reaction\nmechanism unchanged. Structural models of the dark states (Pfr) were\nused to evaluate the total electric field at the nitrile label and\nits transition dipole moment. These quantities served as an internal\nstandard to calculate the respective properties of the photoinduced\nproducts (Lumi-F, Meta-F, and Pr) based on the relative intensities\nof the nitrile stretching bands. In most cases, the spectral analysis\nrevealed two substates with a nitrile in a hydrogen-bonded or hydrophobic\nenvironment. Using frequencies and intensities, we managed to extract\nthe noncovalent contribution of the electric field from the individual\nsubstates. This analysis resulted in profiles of the noncovalent and\nhydrogen-bond-related electric fields during the photoinduced reaction\ncascade of Agp2. These profiles, which vary significantly among the\nfour variants due to the different positions and orientations of the\nnitrile probes, were discussed in the context of the molecular events\nalong the Pfr → Pr reaction cascade.