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

海洋隐花色素的太阳光与月光反应以及分子相互作用

• 批准号: 538823817
• 负责人: Professorin Dr. Eva Wolf
• 金额: --
• 依托单位: Arbeitsgruppe Strukturelle Chronobiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/538823817?language=en
• 关键词: Sun; versus; moonlight; responses; molecular

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.

许多海洋生物使用内部月历（也称为周月时钟或周月振荡器）根据月球周期来确定其成熟和繁殖的时间。海洋刚毛虫 Platynereis dumerilii (Pdu) 具有一种光敏隐花色素 (Cry) 蛋白，称为 L-Cry，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其周月时钟将其繁殖与满月相同步。我们之前表明，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，主要是核暗状态，其光敏辅因子黄素腺嘌呤二核苷酸 (FAD) 处于完全氧化形式，2. 单体，主要是细胞质，光激活阳光状态，具有完全还原的阴离子 FAD°- 自由基和 3. a 半还原，主要是核，月光状态。在这里，我们将结合蛋白质生化、光谱、3D 结构和细胞方法，进一步阐明 L-Cry 对阳光和月光差异反应的分子机制。我们的具体目标是了解 L-Cry 极高的光敏感性的分子基础，使其能够感知非常暗淡的月光，揭示神秘的 L-Cry 月光态的分子结构，并剖析 L-Cry 的黑暗状态、阳光状态和月光状态之间转变过程中发生的构象重排和中间状态。此外，我们将确定光强度范围的上限和下限，从而导致 L-Cry 功能性月光或日光状态的形成。这将使我们能够预测人为夜间光污染对 L-Cry 月光信号和蠕虫绕月行为的影响。为了阐明周月振荡器迄今为止未知的分子基础，我们还将分析 L-Cry 向周月钟发出的下游信号。为此，我们将使用下拉/质谱法寻找新的 L-Cry 相互作用因子，并在体外和体内从机制和功能上表征它们与 L-Cry 的相互作用。