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WIREs Nanomed Nanobiotechnol
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Deconstruction of complex protein signaling switches: a roadmap toward engineering higher‐order gene regulators

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The control of gene expression is an important tool for metabolic engineering, the design of synthetic gene networks, gene‐function analysis, and protein manufacturing. The most successful approaches to date are based on modulating messenger RNA (mRNA) synthesis via their inducible coupling to transcriptional effectors, which requires biosensing functionality. A hallmark of biological sensing is the conversion of an exogenous signal, usually a small molecule or environmental cue such as a protein–ligand interaction, into a useful output or response. One of the most utilized regulatory proteins is the lactose repressor (LacI). In this review we will (1) explore the mechanochemical structure–function relationship of LacI; (2) discuss how the physical attributes of LacI can be leveraged to identify and understand other regulatory proteins; (3) investigate the designability (tunability) of LacI; (4) discuss the potential of the modular design of novel regulatory proteins, fashioned after the topology and mechanochemical properties of LacI. WIREs Nanomed Nanobiotechnol 2017, 9:e1461. doi: 10.1002/wnan.1461 This article is categorized under: Diagnostic Tools > Biosensing Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Biology-Inspired Nanomaterials > Protein and Virus-Based Structures
Molecular function of repressor phenotypes. (a) The wildtype repressor phenotype (I+, blue) negatively regulates its associated gene by binding the operator sequence in the absence of inducer (hexagon). Upon binding inducer, the repressor undergoes a conformational change from the repressor active (R) to inactive states (R*), reducing its affinity for the operator. Dissociation of the repressor from the operator enables gene transcription. Inability to complete any of these steps results in expression of an inducer insensitive repressor phenotype (IS, red). IS repressors result from mutations that: (b) increase affinity between the operator and the repressors R* conformation, or prohibit the conformational change between the R to R* configuration of the repressor, or (c) inhibit inducer binding the repressor. The third repressor phenotype (I, purple) interferes with the repressor binding the operator preventing regulation of gene expression (d–f). Mutations that: (d) disrupt formation of the dimer repressor complex, (e) prevent the adoption of the R conformation, or (f) reduce affinity of the R conformation for its operator, results in a non‐functional repressor variant. (g) A template of transcription factor topology and functional units.
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Engineering anti‐lacs. Engineering alternate routes for cooperative communication in the LacI scaffold (a) is initiated by first introducing point mutations (i.e., Is mutations) that cut off allosteric communication between the effector‐binding site and the DNA binding (b). To ‘re‐route’ communication in each of the Is variants, compensatory mutations to the insensitive LacI parental mutant are introduced by way of a library of random (unbiased) mutations throughout the entire core domain (i.e., N‐ and C‐subdomains), introduced via EP‐PCR.
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Modular design of two orthogonal and logic gates. Chimeric repressors are constructed baring either the wildtype LacI DNA binding domain (blue) recognizing the PLAC operator or the LacI/Y17T/Q18A/R22N DNA binding domain variant (orange) recognizing the PTAN operator. Each DNA binding domain is fused to one of four regulatory domains: GalS (blue) recognizing fructose (A), RbsR (red) recognizing ribose (B), TreR (green) recognizing trehalose (C), and LacI (purple) recognizing IPTG (D). The first two chimeras repress the expression of GFP while the second two repressors inhibit the expression of RFP. The expression signals observed are tabulated as a function of inducer combinations that are interrogated by the bi‐AND logic gate expression system.
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Variation in functional inducer binding sites. LacI heterodimeric chimeras were constructed containing (a) two functional inducer binding sites, (b) a single functional site, or (c) no functional inducer binding sites.
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Recognition of chimeric operators by control over repressor association specificity. (a) A mixture of chimeric repressors baring DNA binding domains recognizing PLAC (blue) and PTAN (orange) operators are fused to LacI regulatory domains. This mixture of repressors can associate into two homodimeric species (containing either 2 PLAC binding domains or 2 PTAN binding domains) or a heterodimer (containing one of each domain). (b) To construct an orthogonal repressor incapable of associating as a homodimer to repress genes under the control of PLAC or PTAN, a non‐functional (I) LacI repressor variant fused to the PLAC DNA binding domain is screened against PTAN containing chimeras incapable of associating and repressing the chimeric operator. (c) Mutagenesis is employed to identify a complementary repressor baring the PTAN binding domain enforcing exclusive formation of the heterodimer incapable of repressing either individual PLAC or PTAN operator. Four of the TAN LacI variants were selected for detailed characterization: HET1 (4A20: Y17T/Q18A/R22N + L251M/D278N/Y282F, forming a heterodimer with LacI Y282A), HET2 (4S29: Y17T/Q18A/R22N + L251K/R255L/D278T/C281W/Y282T, forming a heterodimer with LacI Y282S), HET3 (4S31: Y17T/Q18A/R22N + L251K/R255W/D278T/C281Q/Y282W, forming a heterodimer with LacI Y282S), and HET4 (4S36: Y17T/Q18A/R22N + L251K/R255K/D278S/C281Y/Y282W, forming a heterodimer with LacI Y282S).
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Characterization of mutations to LacI linker positions. Mutation to positions on the LacI linker segment result in one of three induction profiles. (a) Positions tolerant to mutation resulting in minor changes to the induction profile are referred to as neutral positions (green). (b) Mutations to positions that either preserve or eliminate function are referred to as toggle switch positions (red). And, (c) amino acid substitutions conferring a range of induction profiles while conserving repressor function are referred to as rheostat positions (blue).
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First generation LacI/GalR family chimeras. Modular construction and function of LacI, PurR, and GalR chimeras. Parent proteins LacI (a, blue), PurR (b, orange), and GalR (c, purple) bind the small molecule effectors: IPTG, hypoxanthine, and fructose, respectively. LacI and GalR negatively regulate their respective genes inhibiting gene expression by binding operators lacO1 and gal in the absence of inducer. PurR functions as a positive regulator of gene expression that binds its operator pur in the presence of its co‐repressor. (d) Construction of the LLhP chimera by fusion of the PurR regulatory domain (orange) to the LacI DNA binding domain (blue) results in a co‐repressor that associates with the lacO1 operator in the presence of hypoxanthine. (E) Construction of the LLhG chimera by fusion of the GalR regulatory domain (purple) to the LacI DNA binding domain (blue) results in a repressor that dissociates from the lacO1 operator in the presence of fructose.
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Biology-Inspired Nanomaterials > Protein and Virus-Based Structures
Nanotechnology Approaches to Biology > Nanoscale Systems in Biology
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