Orange Carotenoid Protein; Characterization with Resonance Raman Spectroscopy

Abstract

In photosynthetic organisms, high light intensities can result in cellular damage from the production of dangerous oxygen species. Cyanobacteria, also known as blue-green bacteria, blue-green algae, and Cyanophyta, is a phylum of bacteria that obtain their energy through photosynthesis. Reactive oxygen species (ROS) are created from the over-reduction of photosynthetic components, like the plastoquinone pool. Orange carotenoid protein (OCP) acts as a protective mechanism in certain cyanobacteria as it shuttles energy away from the light harvesting apparatuses (in cyanobacteria they are usually phycobilisomes). For my PhD, I am biophysically characterizing this protein with Raman spectroscopy and other techniques. OCP is a photoactive protein that changes confirmation when exposed to blue light; it contains a carotenoid ligand, 3'-hydroxyechinenone. I have purified the his-tagged OCP (from the bacteria Synechocystis PCC 6803) with a nickel column, and am currently using Raman spectroscopy to study the protein's inner carotenoid to deduce information about the vibrational sublevels, and thus the protein environment surrounding the carotenoid. My experiments consist of comparison between the orange (inactive) and red (active- after light exposure) structural forms of the protein. I will soon be using Raman spectroscopy on mutants of the protein as well. Other techniques that I will use for characterization include FTIR (fourier transform infrared), two photon absorption spectroscopy and analytical ultracentrifugation (AUC).