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The function of carotenoids in carotenoproteins is optimized by the electrostatic and steric interactions between the carotenoid and its surrounding binding site, which generally imposes distorted conformations and induces charge-transfer character. This chapter shows how the line shape of the fluorescence spectrum, the fluorescence quantum yield, and the fluorescence anisotropy of the second excited singlet state of a carotenoid, S2, can be used as probes of the structure and dynamics of carotenoids in carotenoproteins. The experimental approach and a brief introduction to the theory we used to detect hydrogen bonding interactions by ketocarotenoids in the orange carotenoid protein are introduced as an example. The fluorescence anisotropy is then introduced as a probe of a carotenoid’s excited-state conformational motion using results from a study of β-carotene in solution over a range of temperatures. 

The increased interest in sequencing cyanobacterial genomes has allowed the identification of new homologs to both the N-terminal domain (NTD) and C-terminal domain (CTD) of the Orange Carotenoid Protein (OCP). The N-terminal domain homologs are known as Helical Carotenoid Proteins (HCPs). Although some of these paralogs have been reported to act as singlet oxygen quenchers, their distinct functional roles remain unclear. One of these paralogs (HCP2) exclusively binds canthaxanthin (CAN) and its crystal structure has been recently characterized. Its absorption spectrum is significantly red-shifted, in comparison to the protein in solution, due to a dimerization where the two carotenoids are closely placed, favoring an electronic coupling interaction. Both the crystal and solution spectra are red-shifted by more than 50 nm when compared to canthaxanthin in solution. Using molecular dynamics (MD) and quantum mechanical/molecular mechanical (QM/MM) studies of HCP2, we aim to simulate these shifts as well as obtain insight into the environmental and coupling effects of carotenoid–protein interactions.