Needles in a haystack: H-bonding in an optogenetic protein observed with isotope labeling and 2D-IR spectroscopy

Recently, re-purposing of cyanobacterial photoreceptors as optogentic actuators enabled light-regulated protein expression in different host systems. These new bi-stable optogenetic tools enable interesting new applications, but their light-driven working mechanism remains largely elusive on a molecular level. Here, we study the optogenetic cyanobacteriochrome Am1-c0023g2 with isotope labeling and two dimensional infrared (2D-IR) spectroscopy. Isotope labeling allows us to isolate two site-specific carbonyl marker modes from the overwhelming mid-IR signal of the peptide backbone vibrations. Unlike conventional difference-FTIR spectroscopy, 2D-IR is sensitive to homogeneous and inhomogeneous broadening mechanisms of these two vibrational probes in the different photostates of the protein. We analyse the 2D-IR line shapes in the context of available structural models and find that they reflect the hydrogen-bonding environment of these two marker groups.