Fundamental mechanisms and application aspects of protein-mediated carotenoid transport

类胡萝卜素在光合生物中大量存在，是动物必需的营养物质。在光合作用中具有显色、能量收集和激发能量耗散、光保护和抗氧化活性等多种功能，是维生素、视觉色素和激素的前体。虽然类胡萝卜素是疏水的，并且会分解到脂质隔室或膜中，但只有很少的特定类胡萝卜素结合蛋白是已知的，这些蛋白使类胡萝卜素保持水溶性的形式，以便在细胞膜、胡萝卜素蛋白之间或生物体内的细胞之间穿梭。其中一个例子是35 kda的橙色类胡萝卜素蛋白（OCP），它在蓝藻光保护中起着核心作用。在蓝光照射下，OCP从基本的橙色OCPo光转化为红色信号形式OCPr，后者猝灭藻胆异构体（PBs）天线的荧光，从而防止过多的能量流到光系统进行非光化学猝灭（NPQ）。在我们对一系列OCP变异的研究中，我们发现类胡萝卜素可以在OCP的分离结构域之间转移。生化和光谱数据暗示了这一过程的几个中间产物，表明这种独特的类胡萝卜素转移机制具有多步骤的性质。后来的研究表明，OCP c端结构域（CTDH蛋白）的天然同源物能够通过将类胡萝卜素从膜转移到OCP n端结构域（HCP蛋白）的同源物中，从而调节几种缺乏全长OCP的蓝藻的光保护，这些同源物能够不断地猝灭PBs的荧光。这种对光合作用活动的新型调控还只是表面上的研究。考虑到CTDHs数量众多，序列差异巨大，以及存在与CTDHs空间结构相似的真核蛋白（核转运因子2 [NTF-2]家族），可以详细探讨蛋白质介导类胡萝卜素转运的原理。我们已经表明，各种CTDH蛋白特异性结合某些类型的类胡萝卜素，这强烈影响光谱和结构特性，以及CTDH蛋白与膜的相互作用。由于其他ntf -2样蛋白也能特异性结合类胡萝卜素（例如灵长类视网膜中与叶黄素结合的StARD3蛋白），因此鉴定类胡萝卜素运输系统的共同机制是可以实现的。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，并利用多学科系统方法（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）建立分子细节的总体功能原理。作为项目结果，将探索使用CTDH和HCP蛋白变体作为抗氧化剂靶向递送模块的潜力。

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.