PubMed · 11689845
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I Willner. 2001. Protein hinges for bioelectronics.. https://doi.org/10.1038/nbt1101-1023
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Post-translational control enables rapid and precise regulation of cell behavior. Despite these advantages, general strategies to build phosphorylation-based synthetic circuits are limited. Here we reasoned that engineered allostery, a technique that has been applied to design light- and chemically gated protein switches, could also be used to engineer phosphorylation-controlled protein switches (phospho-switches). Using an allosterically controllable Gal4 transcription factor as a scaffold, we show that a classic kinase Förster resonance energy transfer biosensor architecture can be used as a starting point for phospho-switch design. We optimize all features of the phospho-switch to develop an ERK-controlled transcription factor with a 20-fold phosphorylation-dependent change in transcriptional output. The resulting synthetic ERK-responsive transcription factor responds with comparable sensitivity to the c-fos promoter and reveals spatial ERK signaling patterns in mammalian developmental organoids. We further show that our switch architecture can be generalized to other input kinases and allosterically controlled targets. This work provides a general platform for a new generation of kinase-responsive tools for biosensing and synthetic biology applications.
The structural transition induced by ligand binding in human hemoglobin encompasses quaternary structure changes at the interfaces between the two alphabeta dimers. In contrast, the interfaces between alpha and beta subunits within the same dimer (i.e., alpha1beta1 and alpha2beta2 interfaces) are structurally invariant. Previous work from this laboratory using NMR spectroscopy has identified four sites at the intradimeric alpha1beta1 and alpha2beta2 interfaces that, although structurally invariant, experience significant changes in the rates of proton exchange upon ligand binding. These sites are Hisalpha103(G10) and Hisalpha122(H5) in each alpha subunit of the hemoglobin tetramer. In the present work, we show that the proton exchange at the Hisalpha103(G10) sites is affected by the interactions of hemoglobin with chloride ions. Increasing concentrations of chloride ions at pH 6.45 and at 37 degrees C enhance the exchange rate of the Hisalpha103(G10) N(epsilon 2) proton. The enhancement is greater in deoxygenated than in ligated hemoglobin. In the framework of the local unfolding model for proton exchange, these results suggest that the structural free energy and/or the proton transfer reactions at the Hisalpha103(G10) sites depend on the concentration of chloride ions. Therefore, the ligand-induced changes at the Hisalpha103(G10) sites are modulated by the allosteric effect of chloride ions on hemoglobin.
Two distinct allosteric inhibitors of glycogen phosphorylase, 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) and CP-91149 (an indole-2-carboxamide), were investigated for their effects on the phosphorylation state of the enzyme in hepatocytes in vitro. CP-91149 induced inactivation (dephosphorylation) of phosphorylase in the absence of hormones and partially counteracted the phosphorylation caused by glucagon. Inhibition of glycogenolysis by CP-91149 can be explained by dephosphorylation of phosphorylase a. This was associated with activation of glycogen synthase and stimulation of glycogen synthesis. DAB, in contrast, induced a small degree of phosphorylation of phosphorylase. This was associated with inactivation of glycogen synthase and inhibition of glycogen synthesis. Despite causing phosphorylation (activation) of phosphorylase, DAB is a very potent inhibitor of glycogenolysis in both the absence and presence of glucagon. This is explained by allosteric inhibition of phosphorylase a, which overrides the increase in activation state. In conclusion, two potent phosphorylase inhibitors exert different effects on glycogen metabolism in intact hepatocytes as a result of opposite effects on the phosphorylation state of both phosphorylase and glycogen synthase.