In-Cell Infrared Difference Spectroscopy on Cryptochromes with Millisecond Time Resolution and in Human Cell Lines

活细胞的红外差异光谱为研究近自然环境中受体的机制开辟了新的机会。在之前的资助期间，我们开发了在细胞内传输光谱的方法，并衰减了细菌细胞中可溶性光感受器上的全反射构型。在这里，我们将进一步发展这一方法，并将重点放在细胞内光谱方法上，隐花色素是动物和植物的中央蓝光受体和生物钟的关键元件。我们将在体外和细胞中仔细观察靠近黄素辅助因子的保守盐桥是否将光化学反应的信号传递给蛋白质部分的反应。光诱导盐桥的断裂可能代表了隐色素的统一机制。此外，我们将用细胞内光谱学解决C-末端延伸的解离动力学，作为信号传递的关键步骤。为此，我们将为不可逆系统建立时间分辨的细胞内红外光谱，时间分辨率为毫秒。最后，我们将把红外差示光谱的应用扩展到人类细胞系中。在光遗传工具中以及在自然界中，隐色素在光照下驱动液-液相分离。我们将在近自然条件下研究红外光谱区人类细胞系中的这些过程，以更好地了解液滴中的条件和隐色素形成光体的情况。综上所述，细胞内红外差异光谱是探索活细胞天然受体机制的一种现代工具。我们将把这项技术应用到人类细胞系中，作为具有医学意义的表达宿主，并旨在揭示细胞内隐花色素信号通路的机制和动力学的关键步骤。

Infrared difference spectroscopy on living cells opens up new opportunities to study the mechanisms of receptors in a near-native environment. In the previous funding period we developed the approach for in-cell spectroscopy in transmission and attenuated total reflection configuration on soluble photoreceptors in bacterial cells. Here, we will develop the method further and focus with in-cell spectroscopic methods on cryptochromes, which act as central blue light receptors and key elements of the circadian clock in animals and plants. We will scrutinize in vitro and in cells whether a conserved salt bridge close to the flavin cofactor relays the signal from the photochemical reaction to the response of the protein moiety. The light-induced breaking of the salt bridge might represent a unifying mechanism for cryptochromes. Moreover, we will resolve with in-cell spectroscopy the kinetics of dissociation of the C-terminal extension as a key step in signaling. To this end we will establish time-resolved in-cell infrared spectroscopy with millisecond time resolution for irreversible systems. Finally, we will extend application of infrared difference spectroscopy to human cell lines. Cryptochromes drive liquid-liquid phase separation upon illumination in optogenetic tools and presumably also in nature. We will study these processes in human cell lines in the infrared spectral region under near-native conditions to better understand the conditions in droplets and the photobody formation by cryptochromes. In summary, in-cell infrared difference spectroscopy is a modern tool to explore native receptor mechanisms in living cells. We will extend application of this technique to human cell lines as an expression host with medical relevance and aim to reveal key steps in mechanism and kinetics of signaling pathways within cryptochromes in cells.