Split Green Fluorescent Proteins: Scope, Limitations, and Outlook

Romei, Matthew G.; Boxer, Steven G.

doi:10.1146/annurev-biophys-051013-022846

Cited by 168 publications

(179 citation statements)

References 164 publications

(190 reference statements)

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“…and this approach would provide a means to capture such short-lived complexes in vivo, with the caveat that protein associations may not be detected in their endogenous time frame but can be locked together over a longer time period [reviewed in 50,51].…”

Section: Plos Geneticsmentioning

confidence: 99%

Dbp5 associates with RNA-bound Mex67 and Nab2 and its localization at the nuclear pore complex is sufficient for mRNP export and cell viability

Adams

Wente

2020

PLoS Genet

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In Saccharomyces cerevisiae, the mRNA export receptor Mex67 is recruited to mature nuclear transcripts to mediate mRNA export through the nuclear pore complex (NPC) to the cytoplasm. Mex67 binds transcripts through adaptor proteins such as the poly(A) binding protein Nab2. When a transcript reaches the cytoplasmic face of the NPC, the DEADbox protein Dbp5 acts to induce a local structural change to release Nab2 and Mex67 in an essential process termed mRNP remodeling. It is unknown how certain proteins (Nab2, Mex67) are released during Dbp5-mediated mRNP remodeling, whereas others remain associated. Here, we demonstrate that Dbp5 associates in close proximity with Mex67 and Nab2 in a cellular complex. Further, fusion of Dbp5 to Nup159 anchors Dbp5 at the cytoplasmic face of the NPC and is sufficient for cell viability. Thus, we speculate that the essential role of Dbp5 in remodeling exporting mRNPs requires its localization to the NPC and is separable from other subcellular functions of Dbp5. This work supports a model where the diverse nuclear, cytoplasmic and NPC functions of Dbp5 in the mRNA lifecycle are not interdependent and that Dbp5 is locally recruited through complex protein-protein interactions to select regions of transcripts for specific removal of transport proteins at the NPC.

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Section: Plos Geneticsmentioning

confidence: 99%

Dbp5 associates with RNA-bound Mex67 and Nab2 and its localization at the nuclear pore complex is sufficient for mRNP export and cell viability

Adams

Wente

2020

PLoS Genet

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“…Split proteins and conditional reconstitution systems are powerful tools for interrogating biology and controlling cell behavior. 1–4 These systems work by splitting a protein into two fragments to disrupt the protein’s function. Each fragment is then fused to a partner domain such that the split protein is reconstituted, and its function is restored only when the partner domains interact.…”

Section: Introductionmentioning

confidence: 99%

Computation-guided optimization of split protein systems

Dolberg¹,

Meger²,

Boucher³

et al. 2019

Preprint

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25Splitting bioactive proteins, such as enzymes or fluorescent reporters, into conditionally reconstituting 26 fragments is a powerful strategy for building tools to study and control biochemical systems. However, split 27 proteins often exhibit a high propensity to reconstitute even in the absence of the conditional trigger, which 28 limits their utility. Current approaches for tuning reconstitution propensity are laborious, context-specific, or 29 often ineffective. Here, we report a computational design-driven strategy that is grounded in fundamental 30 protein biophysics and which guides the experimental evaluation of a focused, sparse set of mutants-31 which vary in the degree of interfacial destabilization while preserving features such as stability and catalytic 32 activity-to identify an optimal functional window. We validate our method by solving two distinct split 33 protein design challenges, generating both broad insights and new technology platforms. This method will 34 streamline the generation and use of split protein systems for diverse applications. 35 36 KEYWORDS: synthetic biology, split proteins, computational protein design, protein engineering 37 38

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“…These limitations have brought biophosphors into focus, leading to Bio-HLEDs featuring performances close to those with Ir-based OPs 15,[27][28][29][30][31][32][33][34] . Here, FP-phosphors stand out, since (i) the photoluminescence features of FPs fulfills all of the above requirements 27 , (ii) the protein backbone is an effective shield against ambient oxygen 40,41 , and (iii) FPs production can be carried out in a cheap and sustainable way using bacteria (please notice that lighting and laser applications do not require high purification levels).…”

mentioning

confidence: 99%

Long-living and highly efficient bio-hybrid light-emitting diodes with zero-thermal-quenching biophosphors

et al. 2020

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Bio-hybrid light-emitting diodes (Bio-HLEDs) based on color down-converting filters with fluorescent proteins (FPs) have achieved moderate efficiencies (50 lm/W) and stabilities (300 h) due to both thermal-and photo-degradation. Here, we present a significant enhancement in efficiency (~130 lm/W) and stability (>150 days) using a zero-thermalquenching bio-phosphor design. This is achieved shielding the FP surface with a hydrophilic polymer allowing their homogenous integration into the network of a light-guiding and hydrophobic host polymer. We rationalize how the control of the mechanical and optical features of this bio-phosphor is paramount towards highly stable and efficient Bio-HLEDs, regardless of the operation conditions. This is validated by the relationships between the stiffness of the FP-polymer phosphor and the maximum temperature reached under device operation as well as the transmittance of the filters and device efficiency.

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Split Green Fluorescent Proteins: Scope, Limitations, and Outlook

Cited by 168 publications

References 164 publications

Dbp5 associates with RNA-bound Mex67 and Nab2 and its localization at the nuclear pore complex is sufficient for mRNP export and cell viability

Dbp5 associates with RNA-bound Mex67 and Nab2 and its localization at the nuclear pore complex is sufficient for mRNP export and cell viability

Computation-guided optimization of split protein systems

Long-living and highly efficient bio-hybrid light-emitting diodes with zero-thermal-quenching biophosphors

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