Goett-Zink Lukas, Klocke Jessica L, Kottke Tilman
Department of Chemistry, Bielefeld University, Bielefeld, Germany.
Medical School OWL, Bielefeld University, Bielefeld, Germany.
Bio Protoc. 2021 Feb 5;11(3):e3909. doi: 10.21769/BioProtoc.3909.
Several in-cell spectroscopic techniques have been developed recently to investigate the structure and mechanism of proteins in their native environment. Conditions differ dramatically from those selected for experiments. Accordingly, the cellular environment can affect the protein mechanism for example by molecular crowding or binding of small molecules. Fourier transform infrared (FTIR) difference spectroscopy is a well-suited method to study the light-induced structural responses of photoreceptors including changes in cofactor, side chains and secondary structure. Here, we describe a protocol to study the response of cofactor and protein in living cells via in-cell infrared difference (ICIRD) spectroscopy using the attenuated total reflection (ATR) configuration. Proteins are overexpressed in , the cells are transferred into saline solution and the copy number per cell is determined using fluorescence spectroscopy. The suspension is centrifuged and the concentrated cells transferred onto the ATR cell inside the FTIR spectrometer. The thermostatted cell is sealed and illuminated from the top with an LED. Intensity spectra are recorded before and after illumination to generate the difference spectrum of the receptor inside the living cell. With ICIRD spectroscopy, structural changes of soluble photoreceptors are resolved in a near-native environment. The approach works in HO at ambient conditions, is label free, without any limitations in protein size and does not require any purification step.
最近已经开发了几种细胞内光谱技术来研究蛋白质在其天然环境中的结构和机制。这些条件与为实验选择的条件有很大不同。因此，细胞环境可以例如通过分子拥挤或小分子结合来影响蛋白质机制。傅里叶变换红外（FTIR）差示光谱法是一种非常适合研究光感受器的光诱导结构响应的方法，包括辅因子、侧链和二级结构的变化。在这里，我们描述了一种通过使用衰减全反射（ATR）配置的细胞内红外差示（ICIRD）光谱法来研究活细胞中辅因子和蛋白质响应的方案。蛋白质在细胞中过表达，将细胞转移到盐溶液中，并使用荧光光谱法确定每个细胞的拷贝数。将悬浮液离心，然后将浓缩的细胞转移到FTIR光谱仪内的ATR池上。将恒温池密封并用LED从顶部照射。在光照前后记录强度光谱，以生成活细胞内受体的差示光谱。通过ICIRD光谱法，可以在近乎天然的环境中解析可溶性光感受器的结构变化。该方法在环境条件下的水中有效，无需标记，对蛋白质大小没有任何限制，也不需要任何纯化步骤。
