Thermodynamics, Kinetics, and Photochemistry of β-Strand Association and Dissociation in a Split-GFP System

Do, Keunbong; Boxer, Steven G.

doi:10.1021/ja207985w

Cited by 41 publications

(80 citation statements)

References 20 publications

(34 reference statements)

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“…60,61 In this paper, we describe a simple two-state model Hamiltonian that can describe the potential energy and the charge-transfer dependence of monomethine dyes at different detuning from the cyanine limit. The model is a generalized Mulliken-Hush electron transfer model, 62,63 wherein the diabatic state energies and electron transfer matrix element are coupled to twisting displacements about the methine bridge bonds.…”

Section: Introductionmentioning

confidence: 99%

A two-state model of twisted intramolecular charge-transfer in monomethine dyes

Olsen

McKenzie

2012

The Journal of Chemical Physics

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Articles you may be interested inModeling opto-electronic properties of a dye molecule in proximity of a semiconductor nanoparticle J. Chem. Phys. 139, 024105 (2013) A two-state model Hamiltonian is proposed, which can describe the coupling of twisting displacements to charge-transfer behavior in the ground and excited states of a general monomethine dye molecule. This coupling may be relevant to the molecular mechanism of environment-dependent fluorescence yield enhancement. The model is parameterized against quantum chemical calculations on different protonation states of the green fluorescent protein chromophore, which are chosen to sample different regimes of detuning from the cyanine (resonant) limit. The model provides a simple yet realistic description of the charge transfer character along two possible excited state twisting channels associated with the methine bridge. It describes qualitatively different behavior in three regions that can be classified by their relationship to the resonant (cyanine) limit. The regimes differ by the presence or absence of twist-dependent polarization reversal and the occurrence of conical intersections. We find that selective biasing of one twisting channel over another by an applied diabatic biasing potential can only be achieved in a finite range of parameters near the cyanine limit.

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Section: Introductionmentioning

confidence: 99%

A two-state model of twisted intramolecular charge-transfer in monomethine dyes

Olsen

McKenzie

2012

The Journal of Chemical Physics

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“…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 dissociate spontaneously after they are formed, we have discovered that dissociation into a peptide and a truncated protein can be induced by light irradiation in certain cases (7). Photodissociation introduces the possibility of active control of protein-protein interactions with light.…”

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confidence: 99%

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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“…Boxer and co-workers have demonstrated that GFP can undergo photo-induced displacement and exchange of bstrand 10 [49,50] (Figure 1c). This exchange can be used to shift GFP's emission from green to yellow fluorescence due to the presence of Tyr203 (associated with yellow emission) on one copy of b-strand 10 (s10 T203Y ), but not the one that is exchanged with (s10 WT ).…”

Section: Light-driven Fp-based Biosensormentioning

confidence: 97%