GFP Variants with Alternative β-Strands and Their Application as Light-driven Protease Sensors: A Tale of Two Tails

Do, Keunbong; Boxer, Steven G.

doi:10.1021/ja4037274

Cited by 26 publications

(36 citation statements)

References 20 publications

(36 reference statements)

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“…This observation extends to a general trend that trans fluorescent proteins are less fluorescent and more photoswitchable than cis proteins (60). Moreover, we speculate based on our observations that it is this flexibility of the trans state, especially from s7-s10, that also allows trans split proteins [or constituent parts linked with a long loop (61)] to dissociate in the dark rather than the motion of cis-trans isomerization itself (i.e., cis-trans isomerization and strand dissociation are not concerted). Whether cis-trans isomerization is achieved by one-bond flip (62) or volume-conserving hula twist (36,63) is not relevant, because the trans structure does not depend on the pathway of isomerization.…”

Section: Resultssupporting

confidence: 81%

Mechanism and bottlenecks in strand photodissociation of split green fluorescent proteins (GFPs)

Lin

Both

et al. 2017

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

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Split GFPs have been widely applied for monitoring protein-protein interactions by expressing GFPs as two or more constituent parts linked to separate proteins that only fluoresce on complementing with one another. Although this complementation is typically irreversible, it has been shown previously that light accelerates dissociation of a noncovalently attached β-strand from a circularly permuted split GFP, allowing the interaction to be reversible. Reversible complementation is desirable, but photodissociation has too low of an efficiency (quantum yield <1%) to be useful as an optogenetic tool. Understanding the physical origins of this low efficiency can provide strategies to improve it. We elucidated the mechanism of strand photodissociation by measuring the dependence of its rate on light intensity and point mutations. The results show that strand photodissociation is a two-step process involving light-activated cis-trans isomerization of the chromophore followed by light-independent strand dissociation. The dependence of the rate on temperature was then used to establish a potential energy surface (PES) diagram along the photodissociation reaction coordinate. The resulting energetics-function model reveals the rate-limiting process to be the transition from the electronic excited-state to the ground-state PES accompanying cis-trans isomerization. Comparisons between split GFPs and other photosensory proteins, like photoactive yellow protein and rhodopsin, provide potential strategies for improving the photodissociation quantum yield.split GFP | potential energy surface | photodissociation | cis-trans isomerization | photosensory protein O ptical techniques for investigating biological processes in vivo can achieve subcellular spatial and millisecond temporal resolution by using genetically encoded light-responsive proteins (1). Photoactivatable proteins are used as either imaging tools, such as reversibly photoswitchable fluorescent proteins (RSFPs), with fluorescence that can be modulated by light (2) or optogenetic switches that convert light input into physiological outputs, such as channelrhodopsins, which can regulate ion flow through membranes in response to light (3). Split GFPs have been widely applied for imaging as fluorescent reporters of cellular processes, because they are small (∼25 kDa), are stable in cytosol, produce chromophores autocatalytically, and are amenable to mutation and circular permutation (4). Typically, the protein is expressed as two or more constituent parts linked to separate proteins that only fluoresce on complementing with one another, offering readouts of protein-protein colocalizaton with low background and high specificity (5). However, this technique can generate misleading results, because the GFP complexes, after being formed and fluorescing, are bound irreversibly, which can be highly perturbative to the processes being studied, especially if the protein interaction being probed is ordinarily reversible (6). Although split GFP complexes essentially do not dissocia...

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Section: Resultssupporting

confidence: 81%

Mechanism and bottlenecks in strand photodissociation of split green fluorescent proteins (GFPs)

Lin

Both

et al. 2017

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

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“…Pioneering work by a number of research groups has led to the discovery of several photoactivatable proteins, such as LOV (light, oxygen, and voltage) domains [20, 21, 22], phytochrome B (PhyB) [23, 24], cryptochrome 2 (CRY2) [25], UV resistance Locus 8 (UVR8) [26, 27], and Dronpa [28] ( Box 1 and Table 1 ). Some photoactivatable proteins, such as split GFPs [29, 30] have yet to be used in controlling live-cell signal transduction, but there has been recent success in using light-controlled protein-protein interactions to regulate intracellular signaling pathways in live cells ( Table 2 ). The mechanisms of these photoactivatable systems are well known [19].…”

Section: Optogenetic Control Of Cell Signalingmentioning

confidence: 99%

Optogenetic control of intracellular signaling pathways

Zhang

Cui

2015

Trends in Biotechnology

212

186

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Cells employ a plethora of signaling pathways to make their life-and-death decisions. Extensive genetic, biochemical, and physiological studies have led to the accumulation of knowledge about signaling components and their interactions within signaling networks. These conventional approaches, though useful, lack the ability to control the spatial and temporal aspects of signaling processes. The recently emerged optogenetic tools open up exciting opportunities by enabling signaling regulation with superior temporal and spatial resolution, easy delivery, rapid reversibility, fewer off-target side effects, and the ability to dissect complex signaling networks. Here we review recent achievements in using light to control intracellular signaling pathways, and discuss future prospects for the field, including integration of new genetic approaches into optogenetics.

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“…(1,2) It is widely used in imaging applications that rely on its structural stability [to thermal and high-pressure unfolding (3), in fusion constructs (4), and to circular permutation (5)] or its optical response to environment, such as in biosensors for force transduction (6-9), calcium concentration (10), protease activity (11), and pH (12,13). Understanding the relationship between protein structure and fluorescence is essential for these applications.…”

mentioning

confidence: 99%

Mechanically switching single-molecule fluorescence of GFP by unfolding and refolding

Ganim

Rief

2017

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

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Green fluorescent protein (GFP) variants are widely used as genetically encoded fluorescent fusion tags, and there is an increasing interest in engineering their structure to develop in vivo optical sensors, such as for optogenetics and force transduction. Ensemble experiments have shown that the fluorescence of GFP is quenched upon denaturation. Here we study the dependence of fluorescence on protein structure by driving single molecules of GFP into different conformational states with optical tweezers and simultaneously probing the chromophore with fluorescence. Our results show that fluorescence is lost during the earliest events in unfolding, 3.5 ms before secondary structure is disrupted. No fluorescence is observed from the unfolding intermediates or the ensemble of compact and extended states populated during refolding. We further demonstrate that GFP can be mechanically switched between emissive and dark states. These data definitively establish that complete structural integrity is necessary to observe single-molecule fluorescence of GFP.optical tweezers | protein folding | fluorescent protein | mechanoswitch G reen fluorescent protein (GFP) is a 27-kDa β-barrel protein with an intrinsic chromophore. (1, 2) It is widely used in imaging applications that rely on its structural stability [to thermal and high-pressure unfolding (3), in fusion constructs (4), and to circular permutation (5)] or its optical response to environment, such as in biosensors for force transduction (6-9), calcium concentration (10), protease activity (11), and pH (12, 13). Understanding the relationship between protein structure and fluorescence is essential for these applications. Photophysical properties of the chromophore are sensitive to its hydrogen bonding, solvation, isomerization state, and binding-pocket structure. (1,2,14). It is known that fluorescence is quenched upon denaturation and that several intermediates have been observed in folding experiments and simulations (3,(15)(16)(17)(18)(19)(20)(21), although it has not been possible to definitively probe the fluorescence properties of partially structured intermediates in isolation from the native state. Speculation exists regarding whether single-molecule photobleaching can be reversed by unfolding and refolding the protein (22). It is not known whether tension on the native state alters fluorescence in similarity to the compressive, pressure-induced elastic effect (3) and whether its emission can be reversibly mechanically switched. In this study, we address these questions by making an explicit connection between structure and fluorescence, using single-molecule methods.Single-molecule experiments are widely used to reveal distributions in biophysical structure and kinetics that underlie ensemble-averaged properties. Single-molecule manipulation using force experiments such as optical tweezers can be used to observe states nominally at negligible concentration in a bulk distribution (23)(24)(25). Single-molecule fluorescence experiments have been used to probe the ...

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GFP Variants with Alternative β-Strands and Their Application as Light-driven Protease Sensors: A Tale of Two Tails

Cited by 26 publications

References 20 publications

Mechanism and bottlenecks in strand photodissociation of split green fluorescent proteins (GFPs)

Mechanism and bottlenecks in strand photodissociation of split green fluorescent proteins (GFPs)

Optogenetic control of intracellular signaling pathways

Mechanically switching single-molecule fluorescence of GFP by unfolding and refolding

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