Structural characterization of carotenoid binding proteins and their role in carotenoid transport and uptake from membranes

Excess light radiation can lead to significant damage to the photosynthetic apparatus and eventually completely stop the photosynthesis process, leading to cell death. Cyanobacteria have developed a mechanism of absorbed light energy dissipation induced by the orange carotenoid protein (OCP). The OCP induces non-photochemical quenching (NPQ), a process of photoprotective thermal dissipation in which absorbed light energy is safely converted into heat. The OCP is a two-domain protein comprising an active N-terminal domain (NTD) and a regulatory C-terminal domain (CTD), both encapsulate a keto-carotenoid molecule. Homologs of the CTD (CTDH) are suggested to uptake carotenoids from the thylakoid membrane and transfer the carotenoid to helical carotenoid proteins (HCPs), which are paralogs of the NTD. Holo-HCPs have various functions as carotenoid carriers (HCP1), singlet oxygen quenchers (HCP2, HCP3) and energy quenching proteins (HCP4). HCP1-HCP3 structures have been previously reported; however, the structure and the mode of action of HCP4, which is capable of functioning as reactive oxygen species (ROS) quencher as well as an energy quencher, is not yet understood. Our study is comprised of two complementary parts: a structural part and a mechanistic part. Our first goal is to determine the high-resolution crystal structures of the holo-HCP4/holo-CTDH and to obtain the structure of OCP-like species. Our second goal is to investigate the mechanism of carotenoid binding by HCPs and carotenoid transfer from membranes to HCPs. Recently, we have obtained high-resolution diffraction data of the apo-CTDH, which will be presented here. Also, we have managed to crystalize the holo-CTDH form.