Chapter 2 - Light-induced difference Fourier-transform infrared spectroscopy of photoreceptive proteins

Abstract

Light-induced difference Fourier-transform infrared (FTIR) spectroscopy is a powerful, sensitive, and informative method for studying protein structural changes in photoreceptive proteins. Strong absorption of water in the IR region is always an issue in this method. However, if water content in the sample is controlled during measurements, this method can provide detailed structural information on a single protein-bound water molecule. We have established three sample preparation methods, including hydrated film, redissolved sample, and concentrated solution. The hydrated film-based method is preferably used for the FTIR measurements of many photoreceptive proteins such as microbial and animal rhodopsins, LOV and BLUF domains. In this method, accurate difference FTIR spectra are obtained in the whole mid-IR region (4000–800cm−1). On the other hand, redissolved sample- and concentrated solution-based methods are used for the measurements with enzymatic turnover in photolyase, and for samples less tolerant to drying. In this chapter, we describe how light-induced difference FTIR spectroscopy was applied to study photoreceptive proteins, along with a summary of the outcome on our understanding of microbial rhodopsins, animal rhodopsins, and flavoproteins.