Fluorescence imaging using synthetic GFP chromophores

Walker, Christopher L; Lukyanov, Konstantin A.; Yampolsky, Ilia V.; Mishin, Alexander S.; Bommarius, Andreas S.; Duraj-Thatte, Anna; Azizi, Bahareh; Tolbert, Laren M.; Solntsev, Kyril M.

doi:10.1016/j.cbpa.2015.06.002

Cited by 121 publications

(93 citation statements)

References 66 publications

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“…These energy gaps correlate well with electrostatic interactions experienced by the chromophores (74)(75)(76). Fluorescence quantum yields can be increased by raising the excited-state barrier height either sterically (as with T203Y) (35) or electrostatically (77) to suppress undesired internal conversion or isomerization. Because charge transfer is dominant during the isomerization process (36,55,70,78,79), barrier heights can, in principle, be adjusted systematically via electrostatics (80,81).…”

Section: Resultsmentioning

confidence: 84%

“…The resulting E a,fwd values are 14 ± 2 and 22 ± 2 kcal/mol for s10 and s7 circular permutants, respectively. These energy barriers experienced by the chromophore during isomerization are caused by the protein environment as shown by model GFP chromophores, which isomerize efficiently instead of fluorescing in fluid solutions unless frozen (32)(33)(34)(35). This viewpoint is also supported by various computational studies (24,(36)(37)(38)(39).…”

Section: Resultsmentioning

confidence: 84%

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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: Resultsmentioning

confidence: 84%

Section: Resultsmentioning

confidence: 84%

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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“…2 Variations of the GFP chromophore, 4-hydroxybenzylidene-imidazolinone (HBI, Figure 1), have expanded FPs with diverse photophysical properties, including spectral range, quantum yield, photostability, and photoswitchability. 3 These chromophores, however, become mostly nonfluorescent when synthesized outside their protein cavity, largely due to rapid nonradiative decay via twisted-intramolecular charge transfer (TICT) (Figure 1). 4 Although such behavior undermines the application of FP analogues, both chemical and biological restriction of TICT restores fluorescence of synthetic FP chromophores.…”

mentioning

confidence: 99%

“…4 For instance, fluorophores have been developed to inhibit TICT of FP chromophores by structural modifications. 3 In addition, FP analogues locked in supra-molecular hosts, 4b metal−organic frameworks, 5 aggregated solids 6 and host proteins 7 have been reported to fluoresce strongly. In biological imaging, HBI analogues have been used to visualize RNA aptamers and DNA quadruplex.…”

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confidence: 99%

Modulation of Fluorescent Protein Chromophores To Detect Protein Aggregation with Turn-On Fluorescence

Liu¹,

et al. 2018

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We present a fluorogenic method to visualize misfolding and aggregation of a specific protein-of-interest in live cells using structurally modulated fluorescent protein chromophores. Combining photo-physical analysis, X-ray crystallography, and theoretical calculation, we show that fluorescence is triggered by inhibition of twisted-intramolecular charge transfer of these fluorophores in the rigid microenvironment of viscous solvent or protein aggregates. Bioorthogonal conjugation of the fluorophore to Halo-tag fused protein-of-interests allows for fluorogenic detection of both misfolded and aggregated species in live cells. Unlike other methods, our method is capable of detecting previously invisible misfolded soluble proteins. This work provides the first application of fluorescent protein chromophores to detect protein conformational collapse in live cells.

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“…6 There are certain advantages of genetic tagging over chemical labeling of expressed proteins. 7 For example, because the fluorescent protein is fused to the target protein, they are colocalized with absolute specificity, which obviates the problem of nonspecific postsynthetic labeling.…”

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confidence: 99%

Synthesis and Evaluation of a Library of Fluorescent Dipeptidomimetic Analogues as Substrates for Modified Bacterial Ribosomes

et al. 2016

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Described herein are the synthesis and photophysical characterization of a library of aryl-substituted oxazole- and thiazole-based dipeptidomimetic analogues, and their incorporation into position 66 of green fluorescent protein (GFP) in lieu of the natural fluorophore. These fluorescent analogues resemble the fluorophore formed naturally by GFP. As anticipated, the photophysical properties of the analogues varied as a function of the substituents at the para position of the phenyl ring. The fluorescence emission wavelength maxima of compounds in the library varied from ~365 nm (near-UV region) to ~490 nm (visible region). The compounds also exhibited a large range of quantum yields (0.01–0.92). The analogues were used to activate a suppressor tRNACUA and were incorporated into position 66 of GFP using an in vitro protein biosynthesizing system that employed engineered ribosomes selected for their ability to incorporate dipeptides. Four analogues with interesting photophysical properties and reasonable suppression yields were chosen, and the fluorescent proteins (FPs) containing these fluorophores were prepared on a larger scale for more detailed study. When the FPs were compared with the respective aminoacyl-tRNAs and the actual dipeptide analogues, the FPs exhibited significantly enhanced fluorescence intensities at the same concentrations. Part of this was shown to be due to the presence of the fluorophores as an intrinsic element of the protein backbone. There were also characteristic shifts in the emission maxima, indicating the environmental sensitivity of these probes. Acridon-2-ylalanine and oxazole 1a were incorporated into positions 39 and 66 of GFP, respectively, and were shown to form an efficient Förster resonance energy transfer (FRET) pair, demonstrating that the analogues can be used as FRET probes.

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Fluorescence imaging using synthetic GFP chromophores

Cited by 121 publications

References 66 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)

Modulation of Fluorescent Protein Chromophores To Detect Protein Aggregation with Turn-On Fluorescence

Synthesis and Evaluation of a Library of Fluorescent Dipeptidomimetic Analogues as Substrates for Modified Bacterial Ribosomes

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