PubMed · 8561049
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K E van Holde, K I Miller. 1995. Hemocyanins.. https://doi.org/10.1016/s0065-3233(08)60545-8
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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.
Neurosteroid modulation of GABAA receptors has been observed with all subunit combinations investigated; however, hetero-oligomeric GABAA receptors containing delta subunits were not studied previously. We describe the effect of delta subunit expression on 3alpha,21-dihydroxy-5alpha-pregnan-20-1 (THDOC)-induced potentiation of GABA-gated currents in transfected HEK 293 cells and in cerebellar granule cells in vitro. THDOC (100 nM) significantly potentiated GABA-gated currents in cells transfected with combinations of alpha1, alpha6, beta3, and gamma2 subunit cDNAs, whereas cotransfection of delta subunit cDNA inhibited this potentiation. In contrast, the direct Cl- channel activation by THDOC at higher concentrations (1-10 microM) was not significantly dependent on delta subunit cotransfection. These results suggest that the presence of the delta subunit inhibits GABAA receptor modulation but not the direct activation by neurosteroids. Cotransfection with delta subunit also affected the negative allosteric modulation by pregnenolone sulfate. THDOC potentiation of GABA-gated currents was greater in cerebellar granule cell cultures at 4 d in vitro (DIV) compared with those at 14 DIV. Single-cell reverse transcription-PCR analysis of the mRNAs expressed in cultured cerebellar granule cells shows that an increased number of granule cells at 14 DIV express delta subunit mRNAs as compared with 4 DIV granule cells. The presence of delta subunit mRNAs detected in individual cells correlated well with the lack of sensitivity to THDOC. These results suggest that developmental expression of GABAA receptor delta subunits may play an important role in determining the region-specific neurosteroid-induced modification of fast inhibitory synaptic function.
Subunit-hybrid enzymes of mutant tetrameric L-lactate dehydrogenases from Bifidobacterium longum were studied in an examination of the mechanism of allosteric activation by fructose 1,6-bisphosphate. We earlier developed an in vivo method for subunit hybridization in Escherichia coli and the hybrids formed were a mixture with different subunit compositions. The B. longum hybrids were separated by anion-exchange chromatography with a mutational tag. Hybrids formed between fructose 1,6-bisphosphate-desensitized subunits and wild-type subunits and also between fructose 1, 6-bisphosphate-desensitized subunits and catalytically inactive subunits. Kinetic analyses of the hybrid enzymes showed that (i) those residues from two symmetrically related subunits that constituted the fructose 1,6-bisphosphate-binding site could bind fructose 1,6-bisphosphate and activate the enzyme only if intact, (ii) hybrids with only one functional fructose 1, 6-bisphosphate-binding site were fully sensitive to fructose 1, 6-bisphosphate, but the allosteric equilibrium had shifted partially, and (iii) activation by fructose 1,6-bisphosphate at the fructose 1, 6-bisphosphate-binding site was transmitted to the active sites through a quaternary structural change, not through direct conformational change within a subunit. These results are evidence of the validity of the concerted allosteric model of this enzyme based on T- and R-state structures in the same crystal lattice proposed earlier.