Antonenko Anastasiia, Pomorski Adam, Singh Avinash Kumar, Kapczyńska Katarzyna, Krężel Artur
Department of Chemical Biology, Faculty of Biotechnology, University of Wroclaw, Joliot-Curie 14a, 50-383 Wrocław, Poland.
Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester 55901, Minnesota, United States.
ACS Omega. 2024 Oct 30;9(45):45127-45137. doi: 10.1021/acsomega.4c05828. eCollection 2024 Nov 12.
To understand how particular post-translational modifications (PTMs) affect the function of a target protein, it is essential to first prepare and investigate the target with the modification at the desired position. This drives the continuous development of site-specific protein modification technologies. Here, we present the chemical synthesis and application of the biarsenical linker SrtCrAsH-EDT, which has a dual labeling functionality. This linker, containing a sortase A recognition motif, can be conjugated with any protein containing the LPXTG motif at the C terminus, such as ubiquitin and the SUMO tag, and then attached to a protein of interest (POI) containing a terminal or bipartite (intramolecularly placed) tetracysteine motif. This modification of the POI facilitates the straightforward and rapid incorporation of PTMs, which are further highlighted by the fluorescent biarsenical probe. Consequently, this directly correlates proteins' physical properties and cellular roles under various physiological conditions or in disease states. The proposed one-pot labeling methodology can be utilized to explore the effects of PTMs on proteins, affecting their structure, function, localization, and interactions within the cellular environment. Understanding these effects is crucial for uncovering the complex mechanisms that regulate cellular function and dysfunction.
为了解特定的翻译后修饰（PTM）如何影响目标蛋白的功能，首先在所需位置制备并研究带有该修饰的目标蛋白至关重要。这推动了位点特异性蛋白修饰技术的不断发展。在此，我们展示了双功能生物砷酸连接子SrtCrAsH-EDT的化学合成及应用。该连接子含有分选酶A识别基序，可与任何在C端含有LPXTG基序的蛋白（如泛素和SUMO标签）缀合，然后连接到含有末端或二分体（分子内放置）四半胱氨酸基序的目标蛋白（POI）上。POI的这种修饰有助于直接且快速地引入PTM，荧光生物砷酸探针进一步突出了这一点。因此，这直接关联了蛋白质在各种生理条件或疾病状态下的物理性质和细胞作用。所提出的一锅法标记方法可用于探索PTM对蛋白质的影响，这些影响涉及蛋白质在细胞环境中的结构、功能、定位和相互作用。了解这些影响对于揭示调节细胞功能和功能障碍的复杂机制至关重要。
