Photochemistry of Receptor-Bound Flavin Resolved in Living Human Cells by Infrared Spectroscopy.

In-cell experiments on proteins have revealed that the cellular environment can exert a considerable influence on protein mechanism and structure. Here, we introduce in-cell infrared difference spectroscopy (ICIRD) as a method to study soluble receptors in living human embryonic kidney cells by applying the attenuated total reflection approach. We demonstrate on the sensory domains of plant cryptochrome and aureochrome1a, a light, oxygen, or voltage (LOV) protein, that experiments can be performed using stable and transient transfection. Cells were cultivated and transfected on an internal reflection element directly inside the spectrometer, while their viability and growth were monitored by infrared spectroscopy. Using ICIRD, we then resolved the photoreactions of oxidized flavin to the flavin neutral radical in cryptochrome and to the flavin-cysteine adduct in LOV inside eukaryotic cells, to our knowledge for the first time, and thus confirmed their photochemical mechanisms in living human cells. However, we observed for LOV a significant upshift in signals of the carbonyl stretching modes of oxidized flavin and cysteine adduct compared to measurements, which could not be rationalized by effects of molecular crowding, dehydration, or temperature. Accordingly, we identified a strong impact of the eukaryotic cellular environment on the hydrogen bonding network and structure of flavin in LOV, which needs to be considered in physiology and optogenetic applications. In conclusion, we introduce ICIRD as a noninvasive, label-free approach to study soluble photoactivatable receptors in mammalian cells and provide insight into the in-cell mechanisms of two photoreceptors.