Resolving Structural Changes of Photoreceptors in Living via In-cell Infrared Difference Spectroscopy.

Several in-cell spectroscopic techniques have been developed recently to investigate the structure and mechanism of proteins in their native environment. Conditions differ dramatically from those selected for experiments. Accordingly, the cellular environment can affect the protein mechanism for example by molecular crowding or binding of small molecules. Fourier transform infrared (FTIR) difference spectroscopy is a well-suited method to study the light-induced structural responses of photoreceptors including changes in cofactor, side chains and secondary structure. Here, we describe a protocol to study the response of cofactor and protein in living cells via in-cell infrared difference (ICIRD) spectroscopy using the attenuated total reflection (ATR) configuration. Proteins are overexpressed in , the cells are transferred into saline solution and the copy number per cell is determined using fluorescence spectroscopy. The suspension is centrifuged and the concentrated cells transferred onto the ATR cell inside the FTIR spectrometer. The thermostatted cell is sealed and illuminated from the top with an LED. Intensity spectra are recorded before and after illumination to generate the difference spectrum of the receptor inside the living cell. With ICIRD spectroscopy, structural changes of soluble photoreceptors are resolved in a near-native environment. The approach works in HO at ambient conditions, is label free, without any limitations in protein size and does not require any purification step.