Goett-Zink Lukas, Karsten Lennard, Mann Charlotte, Horstmeier Hendrik, Spang Jonas, Müller Kristian M, Kottke Tilman
Biophysical Chemistry and Diagnostics, Faculty of Chemistry, Bielefeld University, Bielefeld 33615, Germany.
Biophysical Chemistry and Diagnostics, Medical School OWL, Bielefeld University, Bielefeld 33615, Germany.
J Am Chem Soc. 2025 Mar 19;147(11):9676-9685. doi: 10.1021/jacs.4c17815. Epub 2025 Mar 7.
In-cell experiments on proteins have revealed that the cellular environment can exert a considerable influence on protein mechanism and structure. Here, we introduce in-cell infrared difference spectroscopy (ICIRD) as a method to study soluble receptors in living human embryonic kidney cells by applying the attenuated total reflection approach. We demonstrate on the sensory domains of plant cryptochrome and aureochrome1a, a light, oxygen, or voltage (LOV) protein, that experiments can be performed using stable and transient transfection. Cells were cultivated and transfected on an internal reflection element directly inside the spectrometer, while their viability and growth were monitored by infrared spectroscopy. Using ICIRD, we then resolved the photoreactions of oxidized flavin to the flavin neutral radical in cryptochrome and to the flavin-cysteine adduct in LOV inside eukaryotic cells, to our knowledge for the first time, and thus confirmed their photochemical mechanisms in living human cells. However, we observed for LOV a significant upshift in signals of the carbonyl stretching modes of oxidized flavin and cysteine adduct compared to measurements, which could not be rationalized by effects of molecular crowding, dehydration, or temperature. Accordingly, we identified a strong impact of the eukaryotic cellular environment on the hydrogen bonding network and structure of flavin in LOV, which needs to be considered in physiology and optogenetic applications. In conclusion, we introduce ICIRD as a noninvasive, label-free approach to study soluble photoactivatable receptors in mammalian cells and provide insight into the in-cell mechanisms of two photoreceptors.
对蛋白质进行的细胞内实验表明，细胞环境会对蛋白质的机制和结构产生重大影响。在此，我们引入细胞内红外差示光谱法（ICIRD），作为一种通过应用衰减全反射方法来研究活的人胚肾细胞中可溶性受体的方法。我们以植物隐花色素和 aureochrome1a（一种光、氧或电压（LOV）蛋白）的传感结构域为例证明，使用稳定转染和瞬时转染均可进行实验。细胞在光谱仪内部的内反射元件上进行培养和转染，同时通过红外光谱监测其活力和生长情况。然后，我们利用 ICIRD 首次解析了真核细胞内隐花色素中氧化黄素向黄素中性自由基以及 LOV 中黄素 - 半胱氨酸加合物的光反应，从而证实了它们在活的人细胞中的光化学机制。然而，我们观察到，与测量结果相比，LOV 中氧化黄素和半胱氨酸加合物的羰基伸缩模式信号出现了显著的上移，分子拥挤、脱水或温度的影响无法解释这一现象。因此，我们确定了真核细胞环境对 LOV 中黄素的氢键网络和结构有强烈影响，在生理学和光遗传学应用中需要考虑这一点。总之，我们引入 ICIRD 作为一种无创、无标记的方法来研究哺乳动物细胞中的可溶性光激活受体，并深入了解了两种光感受器的细胞内机制。
