Primary Role of the Chromophore Bond Length Alternation in Reversible Photoconversion of Red Fluorescence Proteins

Drobizhev, Mikhail; Hughes, Thomas E.; Stepanenko, Yuriy; Wnuk, Paweł; O’Donnell, Kieran J.; Scott, J. Nathan; Callis, Patrik R.; Mikhaylov, Alexander; Dokken, Leslie; Rebane, Aleksander

doi:10.1038/srep00688

Cited by 32 publications

(33 citation statements)

References 49 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…28 The photoproducts with the same fluorescence spectra, peaking between 590 and 600 nm, can also be created by one-photon irradiation of mFruits (Supporting Information Figure 8) and were also previously assigned to the trans isomers. 29−31 On the basis of these observations, we attribute the peak near 590–600 nm (Figure 2) to the anionic trans isomer of the DsRed-like chromophore in all RFPs studied here.…”

Section: Resultsmentioning

confidence: 63%

Multiphoton Photochemistry of Red Fluorescent Proteins in Solution and Live Cells

Drobizhev

Stoltzfus²,

Topol

et al. 2014

J. Phys. Chem. B

Self Cite

View full text Add to dashboard Cite

Genetically encoded fluorescent proteins (FPs), and biosensors based on them, provide new insights into how living cells and tissues function. Ultimately, the goal of the bioimaging community is to use these probes deep in tissues and even in entire organisms, and this will require two-photon laser scanning microscopy (TPLSM), with its greater tissue penetration, lower autofluorescence background, and minimum photodamage in the out-of-focus volume. However, the extremely high instantaneous light intensities of femtosecond pulses in the focal volume dramatically increase the probability of further stepwise resonant photon absorption, leading to highly excited, ionizable and reactive states, often resulting in fast bleaching of fluorescent proteins in TPLSM. Here, we show that the femtosecond multiphoton excitation of red FPs (DsRed2 and mFruits), both in solution and live cells, results in a chain of consecutive, partially reversible reactions, with individual rates driven by a high-order (3–5 photon) absorption. The first step of this process corresponds to a three- (DsRed2) or four-photon (mFruits) induced fast isomerization of the chromophore, yielding intermediate fluorescent forms, which then subsequently transform into nonfluorescent products. Our experimental data and model calculations are consistent with a mechanism in which ultrafast electron transfer from the chromophore to a neighboring positively charged amino acid residue triggers the first step of multiphoton chromophore transformations in DsRed2 and mFruits, consisting of decarboxylation of a nearby deprotonated glutamic acid residue.

show abstract

Section: Resultsmentioning

confidence: 63%

Multiphoton Photochemistry of Red Fluorescent Proteins in Solution and Live Cells

Drobizhev

Stoltzfus²,

Topol

et al. 2014

J. Phys. Chem. B

Self Cite

View full text Add to dashboard Cite

show abstract

“…This is a fast relaxation typically associated with the p,p* excitation of p-conjugated systems. [32,33] A similar excited state relaxation was reported for lumiflavin using computations of comparable quality (i.e. with the symmetry adapted clusterconfiguration interaction method).…”

supporting

confidence: 53%

A Conical Intersection Controls the Deactivation of the Bacterial Luciferase Fluorophore

et al. 2014

View full text Add to dashboard Cite

The photophysics of flavins is highly dependent on their environment. For example, 4a-hydroxy flavins display weak fluorescence in solution, but exhibit strong fluorescence when bound to a protein. To understand this behavior, we performed temperature-dependent fluorescent studies on an N(5)-alkylated 4a-hydroxy flavin: the putative bacterial luciferase fluorophore. We find an increase in fluorescence quantum yield upon reaching the glass transition temperature of the solvent. We then employ multiconfigurational quantum chemical methods to map the excited-state deactivation path of the system. The result reveals a shallow but barrierless excited state deactivation path that leads to a conical intersection displaying an orthogonal out-of-plane distortion of the terminal pyrimidine ring. The intersection structure readily explains the observed spectroscopic behavior in terms of an excitedstate barrier imposed by the rigid glass cavity.Oxidized flavin cofactors like flavin mononucleotide (FMN) contain a planar, stiff flavin moiety that displays intense fluorescence in solutions (with a lifetime of ca. 5 ns).[1] Such a fluorescence is quenched when the cofactor is proteinbound due to fast excited-state electron transfer from the flavin to neighboring aromatic amino acid residues (such as tyrosine or tryptophan). This behavior of oxidized flavins has been exploited to follow the conformational changes of flavoproteins over the course of enzymatic reactions. [2][3][4][5] In contrast, the 1,5 and 4a,5 flavin adducts (see Scheme 1 a) such as those found in reduced (FMNH 2 ) [6] and 4a-hydroxylated (FMNHOH) cofactors, respectively, [7] display a weak fluorescence in solution. [8,9] However, when the same cofactors are protein-bound, the confinement [6,7] leads to an enhanced fluorescence emission. [10][11][12][13][14][15] The exact geometry and electronic structure of the emitting conformer and the mechanism preventing the efficient internal conversion occurring in solution are presently unknown.A spectacular manifestation of the effects of protein confinement on flavin cofactors is seen in bacterial luciferases where a 4a,5 flavin adduct is held responsible for the bioluminescence of a vast group of marine eubacteria. In organisms such as Vibrio harveyi, the emission of the fluorophore may be strong enough to cause a blue luminescence of the microbial population that is so intense as to be observable from satellites.[16] Moreover, such flavin adducts are frequent intermediates in enzymatic catalysis by flavinbased monooxygenases, which activate molecular oxygen and catalyze the insertion of atomic oxygen into a number of substrates (e.g. in hydroxylations, sulfoxidations, epoxidations and Baeyer-Villiger oxidations).[17] A specific type of such monooxygenase activity is responsible for the bacterial luciferase fluorescence, [18][19][20][21][22] where the oxidation of aldehydes to acids via molecular oxygen would generate the 4a-hydroxy flavin byproduct as the putative light-emitting intermediate (FMNHOH in Sche...

show abstract

“…These modes are known to play an important role in the photophysics of conjugated dyes generally 65,66,[87][88][89][90] and in fluorescent protein chromophores particularly. [91][92][93][94] The relevance of bridge stretching modes to the development of charge-transfer behavior is implicit in Scheme 2. The omission of these modes is in keeping with our current narrow goal of achieving a simple model of the coupling between twisting and charge-transfer behavior; strategies for the incorporation of other important modes are deferred for later work.…”

Section: B Computational Quantum Chemistrymentioning

confidence: 99%

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

Olsen

McKenzie

2012

The Journal of Chemical Physics

View full text Add to dashboard Cite

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.

show abstract

Primary Role of the Chromophore Bond Length Alternation in Reversible Photoconversion of Red Fluorescence Proteins

Cited by 32 publications

References 49 publications

Multiphoton Photochemistry of Red Fluorescent Proteins in Solution and Live Cells

Multiphoton Photochemistry of Red Fluorescent Proteins in Solution and Live Cells

A Conical Intersection Controls the Deactivation of the Bacterial Luciferase Fluorophore

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

Contact Info

Product

Resources

About