Structural insight into protein-mediated carotenoid transfer by cyanobacterial carotenoproteins

Carotenoids are a class of naturally occurring pigments with a wide range of biological functions, including light harvesting and light protection in photosynthesis, antioxidant activity and antitumour effects. Due to their hydrophobic nature, carotenoids are incorporated into membranes, membrane proteins or water-soluble carotenoid-binding proteins. However, to date, only a few classes of carotenoid-binding proteins have been characterised, which facilitate transportation of carotenoids within the aqueous medium between membranes, carotenoproteins and cells in the organism to fulfil their function at the target destination. A notable example is the orange carotenoid protein (OCP), which contributes to photoprotection during photosynthesis in cyanobacteria. In conditions of intense light, OCP undergoes a transition from the orange state to the light-adapted red state, facilitating its binding to water-soluble light-harvesting complexes, the phycobilisomes, thereby quenching excess energy. In addition, both the N-terminal and C-terminal domains of OCP, along with their homologues, possess the capacity to bind carotenoids. In particular, the C-terminal domain homologues, such as those from Anabaena sp. PCC 7120 (AnaCTDH), play a pivotal role in the processes of carotenoid uptake, storage and release within the cells. Specifically, the C-terminal tail (CTT), a structural element of AnaCTDH, is critically relevant to the uptake and delivery of carotenoids. However, the mechanism of carotenoid mobilisation and the factors that influence the lipid-carotenoid-protein interaction and result in effective protein-mediated carotenoid transfer remain to be fully researched. Therefore, the present dissertation investigates the structural impact of the interaction partners on carotenoid extraction from liposomes, serving as a biomembrane model, by OCP and OCP-related proteins. The variation in the chemical composition and, consequently, the physicochemical properties of the carotenoid-loaded liposomes revealed a direct impact on both the incorporation yield and the long-term storage rate of the carotenoids canthaxanthin (CAN) and echinenone (ECN) in the liposomes. Moreover, the composition exerts a substantial influence on the rate of carotenoid uptake by the AnaCTDH apoprotein, which served as a model protein. In the context of this dissertation, liposomes composed of the lipids DMPG or DOPC are identified as the most suitable for the formation of AnaCTDH holoproteins. Thus, this finding established the foundation for further investigations in this thesis. The application of the carotenoid-loaded DMPG liposomes demonstrated their aptitude for the efficient formation of photoactive OCP holoproteins with CAN and ECN. Moreover, in addition to the compact, orange OCP state, a further species was identified upon CAN extraction depending on the OCP concentration. Chromatographic and spectroscopic analyses revealed that this species represents an extended OCP dimer, with both C-terminal domains bridged via a CAN molecule. This phenomenon manifests exclusively in the presence of CAN and high protein concentrations, resulting in the loss of photoswitchability of the CAN-bridged dimer. Furthermore, the present thesis confirms the previously hypothesised preference of the isolated N-terminal domain of OCP, termed as red carotenoid protein (RCP), for CAN over ECN. Moreover, the significantly decelerated and reduced formation of RCP holoproteins precludes their additional function as an effective carotenoid extractor, alongside the quenching of excitation energy. The present dissertation additionally provides valuable insights into the functions of structural elements and specific amino acids of the CTT of the AnaCTDH protein in carotenoid uptake. The interaction of the generated AnaCTDH variants, containing point mutations in the CTT region, with the structurally different DMPG/DOPC liposomes was analysed spectroscopically for this purpose. The results reveal the importance of the amino acid residue Glu-132 as a membrane anchor and highlight the significance of the amino acid residues Arg-137 and Arg-138 for the process of carotenoid uptake. Furthermore, the reduction in the hydrophobicity of the CTT results in a substantial impairment of carotenoid extraction. By changing the flexibility of the CTT, the postulated perpendicular arrangement of the CTT to both the main AnaCTDH protein and the membrane during extraction is confirmed, and the potential for optimising carotenoid transfer with regard to specific applications is demonstrated.