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Fundamental mechanisms and application aspects of protein-mediated carotenoid transport

Subject Area Biochemistry
Biophysics
Physical Chemistry of Molecules, Liquids and Interfaces, Biophysical Chemistry
Term from 2020 to 2023
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 429542475
 
Final Report Year 2024

Final Report Abstract

The 1204 natural carotenoids currently listed in the carotenoids database perform multiple functions from coloration, energy harvesting and dissipation, light protection and antioxidant activity, as well as being precursors for vitamins, visual pigments and hormones. Though carotenoids are hydrophobic and partition into lipid membranes, only very few specific carotenoid-binding proteins are known, which keep carotenoids in water-soluble form to mobilize them between membranes, carotenoproteins or cells. One of the few well-characterized carotenoproteins is the 35-kDa Orange Carotenoid Protein (OCP) involved in cyanobacterial photoprotection, a photoreceptor, which is photoconverted by light from the orange form OCPO into the red signaling form OCPR, the latter quenching the fluorescence of phycobilisomes to prevent excessive energy flow to the photosystems. The main goals of the CaroTrans project were (i) to characterize previously unexplored carotenoproteins, (ii) to investigate the determinants of carotenoid-binding specificity, (iii) to analyze carotenoid-transfer between carotenoproteins and/or lipid or cell membranes, and (iv) to establish experimental procedures for carotenoid loading into and delivery from a diverse set of carotenoproteins. Regarding (i) and (ii), we succeeded to produce two new carotenoid-binding holoproteins in E. coli, on which we carried out structural and functional studies to reveal the determinants of carotenoid binding. These include AstaP from Coelastrella astaxantina Ki-4 and BmCBP from the silkworm Bombyx mori, while all efforts to produce carotenoid-containing holoproteins of human STARD3 protein (a putative lutein-binding protein from human macula) or SLDCP from Sphaerobacter thermophilus (homologous to the C-terminal domain of OCP) failed, which we attribute to the lack of critical structural elements. Regarding (iii) and (iv), we tested the C-terminal domain homolog from Anabaena (AnaCTDH) in serving as a delivery module for ketocarotenoid transfer from or to liposome or mammalian cell membranes. Furthermore, we studied how the physico-chemical properties of liposomes prepared from various lipids impact carotenoid storage and delivery into AnaCTDH. By mutagenesis, we analyzed the role of individual amino acids within the C- terminal tail of AnaCTDH on the interaction with liposome membranes and carotenoid uptake kinetics and efficiency. By “atomic mutagenesis” upon insertion of a non-canonical amino acid into OCP, we altered the carotenoid-binding preference and determined the impact of one specific H-bond to the ketocarotenoid on photoactivation. Furthermore, the differential carotenoid-binding preference of two variants of the OCP protein lacking the N-terminal extension (OCP-ΔNTE) allowed us to characterize various OCP-ΔNTE complexes with the fluorescence recovery protein (FRP) by small-angle scattering techniques in solution and to correlate these complexes with OCP/FRP photocycle intermediates.

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