Their general task is to sense the metabolic state of the cells by binding in an interactive manner the metabolite status reporter molecules ATP and ADP as well as 2-oxoglutarate 2. PII signalling proteins are ubiquitous in nature, in particular in Prokaryotes and plastids of Archaeplastida 1. The novel NanoBiT sensor showed unprecedented sensitivity, which made it possible to detect even weak and transient interactions between PII variants and their interacting partners, thereby shedding new light in PII signalling processes. Therefore, we set out to develop a new NanoBiT sensor based on the interaction of PII protein from Synechocystis sp. PCC6803 with PII-interacting protein X (PipX) and N-acetyl-L-glutamate kinase (NAGK). When fused to proteins of interest, they reconstitute an active luciferase when the proteins of interest interact. The luciferase NanoLuc is divided in two subunits: a 18 kDa polypeptide termed “Large BiT” and a 1.3 kDa peptide termed “Small BiT”, which only weakly associate. To this purpose, split luciferase proteins have been used to develop a novel complementation reporter called NanoLuc Binary Technology (NanoBiT). To investigate the complexity of interactions of PII with target proteins, analytical methods that do not disrupt the native cellular context are required. Depending on bound effector molecule, PII proteins interact with and modulate the activity of multiple target proteins. The canonical PII signal proteins sense metabolic state of the cells by binding the metabolite molecules ATP, ADP and 2-oxoglutarate. PII proteins constitute a widespread signal transduction superfamily in the prokaryotic world.
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