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Surveying the landscape of optogenetic methods for detection of protein–protein interactions

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Mapping the protein–protein interaction (PPi) landscape is of critical importance to furthering our understanding how cells and organisms function. Optogenetic methods, that is, approaches that utilize genetically encoded fluorophores or fluorogenic enzyme reactions, uniquely enable the visualization of biochemical phenomena in live cells with high spatial and temporal accuracy. Applying optogenetic methods to the detection of PPis requires the engineering of protein‐based systems in which an optical signal undergoes a substantial change when the two proteins of interest interact. In recent years, researchers have developed a number of creative and effective optogenetic methods that achieve this goal, and used them to further elaborate our map of the PPi landscape. In this review, we provide an introduction to the general principles of optogenetic PPi detection, and then provide a number of representative examples of how these principles have been applied. We have organized this review by categorizing methods based on whether the signal generated is reversible or irreversible in nature, and whether the signal is localized or nonlocalized at the subcellular site of the PPi. We discuss these techniques giving both their benefits and drawbacks to enable rational choices about their potential use. This article is categorized under: Laboratory Methods and Technologies > Imaging Laboratory Methods and Technologies > Macromolecular Interactions, Methods Analytical and Computational Methods > Analytical Methods
Gene‐level control protein–protein interaction (PPi) detection systems. (a) Yeast 2‐hybrid based on split Gal4 (Fields & Song, ). The Gal4 transcription factor is split into two parts: the DNA‐binding domain (DBD) and the activation domain (AD). When fused proteins of interest (X and Y) interact, transcription is initiated and the reporter gene is expressed. (b) Split TEV protease system (Wehr et al., ). A transcription factor is tethered to the membrane with a linker containing a TEV protease cut site. The interaction of proteins X and Y leads to reconstitution of TEV protease that cleaves the linker releasing the transcription factor, which then activates expression of the reporter gene. (c) Split RNA polymerase (Pu, Zinkus‐Boltz, & Dickinson, ). The PPi between X and Y brings the two parts of T7 RNA polymerase together allowing transcription of the reporter gene to occur. (d) Split Cre recombinase. Initially, the reporter gene is inactive, due to an inverted orientation or an insertion that disables the gene. The interaction of X and Y reconstitutes Cre recombinase, which flips the gene, or excises part of the DNA, to enable proper expression of the reporter gene
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Jablonski diagrams to represent various optical imaging modes: (a) Absorbance, (b) fluorescence, (c) bioluminescence, (d) Förster resonance energy transfer (FRET), and (e) bioluminescence resonance energy transfer (BRET). Undulating lines represent transitions involving photons with up and down arrows representing absorbance and emission, respectively. Hollow arrows represent nonabsorbing or nonradiative transitions. Purple arrows represent relaxation by internal conversion. All excited states have a probability to relax nonradiatively, as shown in (a)
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Fluoppi protein–protein interaction (PPi) detection system (Watanabe et al., ). Top: A schematic representation of a cell before and after the PPi. Bottom: The protein aggregate formed upon interaction of proteins X and Y, leading to the formation of large fluorescent puncta throughout the cell. These aggregates are stabilized by the tetramerization of the Azami green FP (AG) and linear oligomerization of PB1
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FlimPIA protein–protein interaction (PPi) detection system (Kurihara et al., ). In the firefly luminescent intermediate‐based protein–protein interaction assay (FlimPIA) system, the two enzymatic functions of firefly luciferase have been split into separate polypeptides. One polypeptide is the donor capable of the adenylation step, and the second polypeptide is the acceptor, which is only capable of the oxidation step. This system is potentially useful for detecting the formation of large protein complexes with dimensions that exceed the working range of Förster resonance energy transfer (FRET)
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General strategy for protein–protein interaction (PPi) detection with a split reporter enzyme. The interaction of X and Y reconstitutes the function of the split reporter enzyme which, in the presence of an appropriate substrate, can produce a product with a detectable phenotype
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The four types of research‐ready split luciferases. (a) Split D‐luciferin luciferases include Firefly (shown) (Luker et al., ), click beetle red (Hida et al., ), and Emerald luciferases (Misawa et al., ). (b) Renilla luciferase using the substrate coelenterazine (Loening, Fenn, & Gambhir, ). (c) Gaussia luciferase using the substrate coelenterazine (Remy & Michnick, ). (d) Oplophorus luciferases (NanoLuc shown) using the substrate furimazine (Hall et al., ). In all four types shown the interaction of X and Y initiates complementation and the subsequent enzymatic creation of photons. In the absence of the protein–protein interaction (PPi), no light is produced
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Protein–protein interaction (PPi) detection systems based on split fluorescent proteins (FPs) that use exogenous chromophores. (a) iSplit (Filonov & Verkhusha, ) is a split version of the iRFP protein (Filonov et al., ). Reconstitution of iRFP due to the interaction of X and Y activates the fluorescence of the bound biliverdin molecule. (b) IFP PCA (Tchekanda et al., ) is a split version of IFP1.4 that is reported to be reversible (Shu et al., ). (c) uPPI (To et al., ) is based on split UnaG and depends on the X and Y PPi to activate fluorescence of a bound bilirubin molecule
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Fluorescent protein (FP)‐based protein–protein interaction (PPi) detection systems. (a) Split FP. When the interaction of X and Y occurs, the two halves of the split FP associate to form the intact and functional FP. This process is essentially irreversible, so proteins X and Y remain associated. (b) Tripartite split FP. An FP is split into three parts that are only capable of forming their chromophore when all three are brought together by a three way PPi, schematically represented here with proteins X, Y, and Z. (c) Dimerization‐dependent FPs (ddFPs). The red fluorescence of red A‐copy (RA) is increased when the X and Y PPi brings the RA and B copies together to allow its reversible dimerization to occur. (d) FP exchange (FPX). The B copy from (c) is capable of activating the fluorescence of both RA and green A‐copy (GA), a green version of RA, but only one at a time. The interaction of X and Y serves to shift the equilibrium between the GA‐B complex and the RA‐B complex, resulting in a red‐green ratiometric change in fluorescence hue. (e) FLINC. The interaction of X and Y induces an interaction between Dronpa and TagRFP‐T, resulting in increased fluorescence fluctuations from TagRFP‐T. (f) Split intein‐based PPi detection. When X and Y interact, the Int‐n and Int‐c protein fragments associate to form a functional intein protein, which catalyzes a splicing reaction to form an intact reporter protein. The spliced reporter protein is not attached to the now‐truncated X‐Int‐n and Y‐Int‐c proteins and will diffuse away
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Analytical and Computational Methods > Analytical Methods
Laboratory Methods and Technologies > Macromolecular Interactions, Methods
Laboratory Methods and Technologies > Imaging

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