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

红外差谱法在活细胞上的应用为研究受体在接近天然环境下的作用机制提供了新的机会。在之前的资助期间，我们开发了细菌细胞中可溶性光感受器的透射和衰减全反射配置的细胞内光谱方法。在这里，我们将进一步发展该方法，并将重点放在隐色素的细胞内光谱方法上，隐色素在动物和植物中作为中央蓝光受体和生物钟的关键元素。我们将在体外和细胞中仔细研究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片段的反应。光诱导的盐桥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解决c端延伸解离动力学作为信号传导的关键步骤。为此，我们将建立具有毫秒时间分辨率的不可逆系统的时间分辨胞内红外光谱。最后，我们将把红外差分光谱的应用扩展到人类细胞系。隐色素在光遗传工具的光照下驱动液-液相分离，可能在自然界也是如此。我们将在接近自然条件下的人类细胞系红外光谱区研究这些过程，以更好地了解液滴的形成条件和隐色素的光体形成。综上所述，细胞内红外差分光谱是探索活细胞中天然受体机制的现代工具。我们将把这项技术应用于人类细胞系，作为具有医学意义的表达宿主，旨在揭示细胞隐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.