Fundamental mechanisms and application aspects of protein-mediated carotenoid transport

类胡萝卜素在光合作用的生物体中含量丰富，是动物的必需营养物质。这700多种植物具有多方面的功能，包括光合作用中的着色、能量收集和激发能量耗散、光保护和抗氧化活性，它们是维生素、视觉色素和激素的前体。尽管类胡萝卜素是疏水性的，并被分配到脂室或膜中，但人们只知道很少有特定的类胡萝卜素结合蛋白，这种结合蛋白保持类胡萝卜素的水溶性，使它们在有机体的膜之间、胡萝卜素蛋白之间或细胞之间穿梭。其中一个例子是35 kDa的橙色类胡萝卜素蛋白(OCP)，它在蓝藻的光保护中发挥着核心作用。在蓝光照射下，OCP从基本的橙色OCPo光转化为红色信号OCPr，后者猝灭藻胆体(PBS)触角的荧光，从而防止过多的能量流向光系统产生非光化学猝灭(NPQ)。在对一系列OCP变体的研究中，我们发现类胡萝卜素可以在OCP的孤立结构域之间转移。生化和光谱数据暗示了这一过程的几个中间体，暗示了这一独特的类胡萝卜素转移机制的多步骤性质。后来的研究表明，天然存在的OCP C-末端结构域的同系物(CTDH蛋白)能够通过将类胡萝卜素从膜转移到OCP N-末端结构域的同系物(HCP蛋白)来调节几种缺乏全长OCP的蓝藻的光保护作用，这些同系物能够持续地猝灭PBS荧光。对这种新型的光合作用活动调节方式的研究还很肤浅。鉴于CTDH的数量巨大、序列差异很大，以及在空间结构上与CTDH相似的真核蛋白的存在(核运输因子2[NTF-2]家族)，蛋白质介导的类胡萝卜素运输的原理可以得到详细的探索。我们已经证明，不同的CTDH蛋白可以特异性地结合某些类型的类胡萝卜素，这强烈地影响了CTDH蛋白的光谱和结构性质，以及CTDH蛋白与膜的相互作用。由于其他NTF-2样蛋白也能与类胡萝卜素特异结合(例如灵长类视网膜中与叶黄素结合的StARD3蛋白)，识别类胡萝卜素运输系统的常见机制是遥不可及的。该项目旨在探索蛋白质介导的类胡萝卜素运输的这些基本特征，并从分子细节上建立最主要的功能原理，受益于多学科的系统方法(生物化学、生物物理学、结构和分子生物学、稳态和时间分辨光谱学)。作为一个项目的结果，将探索使用CTDH和HCP蛋白质变体作为靶向输送抗氧化剂的模块的潜力。

Carotenoids abound in photosynthetic organisms and are essential nutrients for animals. The more than 700 species perform multifaceted functions ranging from coloration, energy harvesting and excitation energy dissipation in photosynthesis, light protection and antioxidant activity, and they are precursors for vitamins, visual pigments and hormones. Though carotenoids are hydrophobic and partition into lipid compartments or membranes, only very few specific carotenoid-binding proteins are known, which keep carotenoids in water-soluble form to shuttle them between membranes, between carotenoproteins or between cells in the organism. One of these examples is the 35-kDa Orange Carotenoid Protein (OCP), which plays a central role in cyanobacterial photoprotection. Upon illumination with blue light, OCP is photoconverted from the basic, orange form OCPo into the red signaling form OCPr, the latter quenching the fluorescence of phycobilisome (PBs) antennae, thus preventing excessive energy flow to the photosystems to enact non-photochemical quenching (NPQ).During our studies on a series of OCP variants, we discovered that carotenoids can be transferred between the isolated domains of OCP. Biochemical and spectroscopic data hinted at several intermediates of this process suggesting a multi-step nature of this unique carotenoid transfer mechanism. It was later shown that naturally occurring homologs of the OCP C-terminal domain (CTDH proteins) are able to regulate photoprotection in several cyanobacteria lacking full-length OCP by transferring the carotenoid from membranes to homologs of the OCP N-terminal domain (HCP proteins), which are constantly able to quench PBs fluorescence. This novel type of regulation of photosynthetic activity is only superficially studied yet. Given the huge number of CTDHs with vastly different sequences, as well as the existence of eukaryotic proteins, which are similar to CTDHs in spatial structure (nuclear transport factor 2 [NTF-2] family), the principles of protein-mediated carotenoid transport can be explored in detail. We have shown that various CTDH proteins specifically bind certain types of carotenoids, which strongly influences the spectral and structural properties, and the interaction of CTDH proteins with membranes. Since other NTF-2-like proteins specifically bind carotenoids as well (e.g. the lutein-binding StARD3 protein in primate retinae), the identification of common mechanisms of carotenoid transport systems is within reach. This project aims to explore these fundamental features of protein-mediated carotenoid transport and establish the overarching functional principles in molecular detail, taking benefit from a multidisciplinary, systemic approach (biochemistry, biophysics, structural and molecular biology, steady-state and time-resolved spectroscopy). As a project result, the potentials for using CTDH and HCP protein variants as modules for the targeted delivery of antioxidants will be explored.