Single-fluorescent protein biosensors (SFPBs) are an important class of probes that enable the single-cell quantification of analytes in vivo. Despite advantages over other detection technologies, their use has been limited by the inherent challenges of their construction. Specifically, the rational design of green fluorescent protein (GFP) insertion into a ligand-binding domain, generating the requisite allosteric coupling, remains a rate-limiting step. Here, we describe an unbiased approach, termed domain-insertion profiling with DNA sequencing (DIP-seq), that combines the rapid creation of diverse libraries of potential SFPBs and high-throughput activity assays to identify functional biosensors. As a proof of concept, we construct an SFPB for the important regulatory sugar trehalose. DIP-seq analysis of a trehalose-binding-protein reveals allosteric hotspots for GFP insertion and results in high-dynamic range biosensors that function robustly in vivo. Taken together, DIP-seq simultaneously accelerates metabolite biosensor construction and provides a novel tool for interrogating protein allostery.

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Photo-controlled transcription factors could be powerful tools for probing the roles of transcriptional processes in a variety of settings. Previously, we designed a photo-controlled DNA binding protein based on a fusion between the bZIP region of GCN4 and photoactive yellow protein from H. halophila (Morgan et al., J. Mol. Biol. 2010, 399:94-112). Here we report a structure-based attempt to improve the degree of photoswitching observed with this chimeric protein. Using computational design tools PoPMuSiC 2.0, Rosetta, Eris and bCIPA we identified a series of single and multiple point mutations that were expected to stabilize the folded dark state of the protein and thereby enhance the degree of photoswitching. While a number of these mutations, particularly those that introduced a hydrophobic residue at position 143, did significantly enhance dark-state protein stability as judged by urea denaturation studies, dark-state stability did not correlate directly with the degree of photoswitching. Instead, the influence of mutations on the degree of photoswitching was found to be related to their effects on the degree to which DNA binding slowed the pB to pG transition in the PYP photocycle. One mutant, K143F, caused a ~10-fold slowing of the photocycle and also showed the largest difference in apparent K d for DNA binding − 3.5-fold lower upon irradiation. This change in apparent K d causes a 12-fold enhancement in fraction bound DNA upon irradiation due to the cooperativity of DNA binding by this family of proteins. The results highlight the strengths and weaknesses of current approaches to a practical problem in protein design as well as suggesting strategies for further improvement of designed photo-controlled transcription factors. Keywordscoiled-coil; bZIP; photo-control; photoactive yellow protein; PYP; optogenetics; photoisomerization; genetically encoded; LOV domain Photo-control of transcriptional processes in living cells may help to elucidate the roles of location and timing of gene expression in spatiotemporally complex settings such as occur during development and during normal functioning of the nervous system (1). Naturally occurring photo-controlled transcription factors are known (2,3), however, they are often multi-component systems so that engineering them for use as tools for the photo-control of transcription is not straightforward. Alternatively, naturally occurring light-dependent protein-protein interactions may be co-opted to photo-control transcription. Quail and Correspondence to: G. Andrew Woolley, awoolley@chem.utoronto.ca. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2012 February 22. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript colleagues used a phytochrome-GAL4-DNA-binding-domain fusion and a PIF3-GAL4-activation-domain fusion to photo-control the expression of genes that contain a promoter with a GAL4 binding site (4). A similar approach using the transactivator VP16 fused to the LOV domain of FKF1 and the ...

Photo-controlled DNA-binding proteins promise to be useful tools for probing complex spatiotemporal patterns of gene expression in living organisms. Here we report a novel photoswitchable DNA-binding protein, GCN4(S)Δ25PYP, based on a truncated GCN4-photoactive yellow protein chimera. In contrast to previously reported designed photoswitchable proteins where DNA binding affinity is enhanced upon irradiation, GCN4(S)Δ25PYP dissociates from DNA when irradiated with blue light. In addition, the rate of thermal relaxation to the ground state, part of the PYP photocycle, is enhanced by DNA binding whereas in previous reported constructs it is slowed. The origins of this reversed photoactivity are analyzed in structural terms.

Metabolic engineering offers the potential to renewably produce important classes of chemicals, particularly biofuels, at an industrial scale. DNA synthesis and editing techniques can generate large pathway libraries, yet identifying the best variants is slow and cumbersome. Traditionally, analytical methods like chromatography and mass spectrometry have been used to evaluate pathway variants, but such techniques cannot be performed with high throughput. Biosensors - genetically encoded components that actuate a cellular output in response to a change in metabolite concentration - are therefore a promising tool for rapid and high-throughput evaluation of candidate pathway variants. Applying biosensors can also dynamically tune pathways in response to metabolic changes, improving balance and productivity. Here, we describe the major classes of biosensors and briefly highlight recent progress in applying them to biofuel-related metabolic pathway engineering.