Engineering allosteric communication

Herde, Zachary D.; Short, Andrew E.; Kay, Valerie E; Huang, Brian D; Park, Jongwoo; Wilson, Clive

doi:10.1016/j.sbi.2020.05.004

Cited by 10 publications

(11 citation statements)

References 35 publications

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“…Given that protein–DNA interaction design rules could not be established based on the broader study from Milk et al , a quantitative explanation for the observed increases in the dynamic ranges were difficult to reconcile completely. We posited that improved pairing of allosteric networks with alternate DNA binding domains could account for much of the improved performance, consistent with the arguments we posed previously. , To reconcile an improvement in dynamic range would require a complete assessment of the permissive allosteric network, which is beyond the scope of this study.…”

Section: Resultssupporting

confidence: 77%

Engineering Alternate Ligand Recognition in the PurR Topology: A System of Novel Caffeine Biosensing Transcriptional Antirepressors

Rondon

Wilson

2021

ACS Synth. Biol.

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Recent advances in synthetic biology and protein engineering have increased the number of allosteric transcription factors used to regulate independent promoters. These developments represent an important increase in our biological computing capacity, which will enable us to construct more sophisticated genetic programs for a broad range of biological technologies. However, the majority of these transcription factors are represented by the repressor phenotype (BUFFER), and require layered inversion to confer the antithetical logical function (NOT), requiring additional biological resources. Moreover, these engineered transcription factors typically utilize native ligand binding functions paired with alternate DNA binding functions. In this study, we have advanced the state-of-the-art by engineering and redesigning the PurR topology (a native antirepressor) to be responsive to caffeine, while mitigating responsiveness to the native ligand hypoxanthinei.e., a deamination product of the input molecule adenine. Importantly, the resulting caffeine responsive transcription factors are not antagonized by the native ligand hypoxanthine. In addition, we conferred alternate DNA binding to the caffeine antirepressors, and to the PurR scaffold, creating 38 new transcription factors that are congruent with our current transcriptional programming structure. Finally, we leveraged this system of transcription factors to create integrated NOR logic and related feedback operations. This study represents the first example of a system of transcription factors (antirepressors) in which both the ligand binding site and the DNA binding functions were successfully engineered in tandem.

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Section: Resultssupporting

confidence: 77%

Engineering Alternate Ligand Recognition in the PurR Topology: A System of Novel Caffeine Biosensing Transcriptional Antirepressors

Rondon

Wilson

2021

ACS Synth. Biol.

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“…The mechanisms by which this is achieved vary vastly, ranging from rigid body intramolecular rearrangements to global (re-) structuring events 1 . Major efforts have been directed into understanding the underlying molecular mechanisms and devising approaches for engineering of artificial allosteric protein receptors 2 . Such receptors have numerous applications as diagnostic protein biosensors, signaling surface receptors, and ligand-regulated transcription factors controlling cell metabolism 3 .…”

Section: Introductionmentioning

confidence: 99%

“…Not surprisingly, most progress in understanding and engineering allostery has been made with systems that can be tested using high throughput assays (transcription factors, antibiotic resistance, and fluorescent proteins). Efforts to engineer allosteric systems broadly fall into two categories: (a) protein designs where new or re-designed allosteric sites are coupled to the active sites through long range intradomain interactions 2 , and (b) utilization of ligand-binding domains that pass their conformational changes onto reporter domains thereby controlling their activity 4 . The latter approach is more straightforward but requires ligand-binding domains that undergo large conformational changes.…”

Section: Introductionmentioning

confidence: 99%

Design of a methotrexate-controlled chemical dimerization system and its use in bio-electronic devices

et al. 2021

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Natural evolution produced polypeptides that selectively recognize chemical entities and their polymers, ranging from ions to proteins and nucleic acids. Such selective interactions serve as entry points to biological signaling and metabolic pathways. The ability to engineer artificial versions of such entry points is a key goal of synthetic biology, bioengineering and bioelectronics. We set out to map the optimal strategy for developing artificial small molecule:protein complexes that function as chemically induced dimerization (CID) systems. Using several starting points, we evolved CID systems controlled by a therapeutic drug methotrexate. Biophysical and structural analysis of methotrexate-controlled CID system reveals the critical role played by drug-induced conformational change in ligand-controlled protein complex assembly. We demonstrate utility of the developed CID by constructing electrochemical biosensors of methotrexate that enable quantification of methotrexate in human serum. Furthermore, using the methotrexate and functionally related biosensor of rapamycin we developed a multiplexed bioelectronic system that can perform repeated measurements of multiple analytes. The presented results open the door for construction of genetically encoded signaling systems for use in bioelectronics and diagnostics, as well as metabolic and signaling network engineering.

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“…Allosteric TFs serve as simple genetic switches, whereby gene expression is modulated in response to environmental, cellular, and temporal signals ( 20 , 21 ). Here, environmental signals (or INPUTS) interact with a TF’s regulatory core domain (RCD), causing an allosteric shift in the protein’s conformation to either increase or decrease the affinity of the TF’s DNA-binding domain (DBD) for specific operator DNA sequences ( 21 – 23 ). Operators, DNA sequences specific to the DBD, are in proximity to gene promoters, thus allowing TFs to either inhibit transcription by compromising RNA polymerase (RNAP) binding (repressor) or facilitate transcription by recruiting RNAP (activator).…”

mentioning

confidence: 99%

Biological signal processing filters via engineering allosteric transcription factors

Groseclose

Hersey

Huang

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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Signal processing is critical to a myriad of biological phenomena (natural and engineered) that involve gene regulation. Biological signal processing can be achieved by way of allosteric transcription factors. In canonical regulatory systems (e.g., the lactose repressor), an INPUT signal results in the induction of a given transcription factor and objectively switches gene expression from an OFF state to an ON state. In such biological systems, to revert the gene expression back to the OFF state requires the aggressive dilution of the input signal, which can take 1 or more d to achieve in a typical biotic system. In this study, we present a class of engineered allosteric transcription factors capable of processing two-signal INPUTS, such that a sequence of INPUTS can rapidly transition gene expression between alternating OFF and ON states. Here, we present two fundamental biological signal processing filters, BANDPASS and BANDSTOP, that are regulated by D-fucose and isopropyl-β-D-1-thiogalactopyranoside. BANDPASS signal processing filters facilitate OFF–ON–OFF gene regulation. Whereas, BANDSTOP filters facilitate the antithetical gene regulation, ON–OFF–ON. Engineered signal processing filters can be directed to seven orthogonal promoters via adaptive modular DNA binding design. This collection of signal processing filters can be used in collaboration with our established transcriptional programming structure. Kinetic studies show that our collection of signal processing filters can switch between states of gene expression within a few minutes with minimal metabolic burden—representing a paradigm shift in general gene regulation.

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Engineering allosteric communication

Cited by 10 publications

References 35 publications

Engineering Alternate Ligand Recognition in the PurR Topology: A System of Novel Caffeine Biosensing Transcriptional Antirepressors

Engineering Alternate Ligand Recognition in the PurR Topology: A System of Novel Caffeine Biosensing Transcriptional Antirepressors

Design of a methotrexate-controlled chemical dimerization system and its use in bio-electronic devices

Biological signal processing filters via engineering allosteric transcription factors

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