Sun- versus moonlight responses and molecular interactions of a marine Cryptochrome

许多海洋生物使用内月历，也被称为圆月时钟或圆周振荡器，根据月球周期对它们的成熟和繁殖进行计时。海洋猪毛蠕虫Platynereis umerilii(PDU)有一种名为L-Cry的光敏隐色素蛋白质，可以区分月光和阳光以及不同的月相。这使得蠕虫能够通过它的圆周时钟将它的繁殖与满月同步。我们之前已经证明L-Cry可以形成三种不同的分子状态，具有独特的生化和功能特性：1.二聚体，以核为主，暗状态，其光敏辅因子黄嘌呤二核苷酸(FAD)以完全氧化的形式存在；2.单体，主要是胞质，光激活的日光状态，具有完全还原的阴离子FAD°-自由基；以及3.半还原，以核为主的月光状态。在这里，我们将结合蛋白质生化、光谱、3D结构和基于细胞的方法，进一步阐明L哭对阳光和月光的差异反应的分子机制。我们的具体目标是了解L-Cry异常高的光敏性使其能够感知非常暗的月光的分子基础，揭示神秘的L-Cry月光态的分子结构，并剖析在L-Cry暗状态、日光状态和月光状态之间发生的构象重排和中间态。此外，我们将确定导致L哭功能月光或日照状态形成的光强度范围的上限和下限。这将使我们能够预测人为的夜间光污染对L哭月光信号的影响以及对蠕虫圆周行为的影响。为了阐明迄今未知的圆月振荡器的分子基础，我们还将分析L-Cry对圆月钟的下游信号。为此，我们将利用下拉/质谱仪寻找新的L-Cry相互作用分子，并从力学和功能上表征它们在体外和体内与L-Cry的相互作用。

Many marine organisms time their maturation and reproduction according to the lunar cycle using an inner monthly calendar, also referred to as circalunar clock or circalunar oscillator. The marine bristle worm Platynereis dumerilii (Pdu) has a light-sensitive cryptochrome (Cry) protein, called L-Cry, that can discriminate between moonlight and sunlight and between different moon phases. This enables the worm to synchronize its reproduction to the full moon phase via its circalunar clock. We previously showed that L-Cry can form three distinct molecular states with unique biochemical and functional properties: 1. a dimeric, predominantly nuclear, dark state with its light-sensing cofactor flavine adenine dinucleotide (FAD) in the fully oxidized form, 2. a monomeric, predominantly cytosolic, photoactivated sunlight state with a fully reduced anionic FAD°- radical and 3. a half-reduced, predominantly nuclear, moonlight state. Here, we will further elucidate the molecular mechanisms underlying the differential responses of L-Cry to sunlight and moonlight, using a combination of protein biochemical, spectroscopic, 3D-structural and cell-based approaches. Our specific aims are to understand the molecular basis of the exceptionally high light sensitivity of L-Cry that enables it to sense very dim moonlight, to uncover the molecular structure of the enigmatic L-Cry moonlight state, and to dissect conformational rearrangements and intermediate states occurring during the transitions between the dark-, sunlight- and moonlight states of L-Cry. Furthermore, we will determine the upper and lower limits of the light intensity ranges, that lead to the formation of functional moonlight- or sunlight states of L-Cry. This will enable us to predict the impact of anthropogenic artificial nocturnal light pollution on L-Cry moonlight signalling and on the circalunar behaviour of the worms. To elucidate the hitherto unknown molecular basis of circalunar oscillators, we will also analyse L-Cry’s downstream signalling to the circalunar clock. To this end, we will search for new L-Cry interactors using pulldown/mass spectrometry, and mechanistically and functionally characterize their interactions with L-Cry in vitro and in vivo.