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Multiphoton photochemistry of red fluorescent proteins in solution and live cells.

Drobizhev M, Stoltzfus C, Topol I, Collins J, Wicks G, Mikhaylov A, Barnett L, Hughes TE, Rebane A - J Phys Chem B (2014)

Bottom Line: Genetically encoded fluorescent proteins (FPs), and biosensors based on them, provide new insights into how living cells and tissues function.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics and ‡Department of Cell Biology and Neuroscience, Montana State University , Bozeman, Montana 59717, United States.

ABSTRACT
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.

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Schematic diagram ofthe mCherry (left) and DsRed (right) chromophoreenvironments.41,44 Hydrogen bonds are presentedby dashed lines, charged groups are highlighted with color (blue,positive; red, negative), and water molecules are designated as W.
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fig8: Schematic diagram ofthe mCherry (left) and DsRed (right) chromophoreenvironments.41,44 Hydrogen bonds are presentedby dashed lines, charged groups are highlighted with color (blue,positive; red, negative), and water molecules are designated as W.

Mentions: Finally, the −CH2• radical,produced in this reaction, should recombine with either a hydrogenatom or accept an electron and proton. Because the hydrogen atom orproton can be transferred only on short distances, the hydrogen-donatinggroup should be in close contact with the decarboxylated residue.In the DsRed2 protein, the E215 residue makes a close hydrogen-bondingcontact with the protonated ε-amino group of K70 (Figure 8, right). As mentioned above, the −NH3+ group of K70 turns into the ammonium radial −NH3• after electron transfer from the chromophore.It is known that −NH3• in theground state is metastable with respect to the −NH3• → NH2 + H• reaction.43,45,46 Because of the close distance between K70 and E215, the releasedhydrogen may eventually recombine with the remaining −CH2• radical of E215.


Multiphoton photochemistry of red fluorescent proteins in solution and live cells.

Drobizhev M, Stoltzfus C, Topol I, Collins J, Wicks G, Mikhaylov A, Barnett L, Hughes TE, Rebane A - J Phys Chem B (2014)

Schematic diagram ofthe mCherry (left) and DsRed (right) chromophoreenvironments.41,44 Hydrogen bonds are presentedby dashed lines, charged groups are highlighted with color (blue,positive; red, negative), and water molecules are designated as W.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4126731&req=5

fig8: Schematic diagram ofthe mCherry (left) and DsRed (right) chromophoreenvironments.41,44 Hydrogen bonds are presentedby dashed lines, charged groups are highlighted with color (blue,positive; red, negative), and water molecules are designated as W.
Mentions: Finally, the −CH2• radical,produced in this reaction, should recombine with either a hydrogenatom or accept an electron and proton. Because the hydrogen atom orproton can be transferred only on short distances, the hydrogen-donatinggroup should be in close contact with the decarboxylated residue.In the DsRed2 protein, the E215 residue makes a close hydrogen-bondingcontact with the protonated ε-amino group of K70 (Figure 8, right). As mentioned above, the −NH3+ group of K70 turns into the ammonium radial −NH3• after electron transfer from the chromophore.It is known that −NH3• in theground state is metastable with respect to the −NH3• → NH2 + H• reaction.43,45,46 Because of the close distance between K70 and E215, the releasedhydrogen may eventually recombine with the remaining −CH2• radical of E215.

Bottom Line: Genetically encoded fluorescent proteins (FPs), and biosensors based on them, provide new insights into how living cells and tissues function.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics and ‡Department of Cell Biology and Neuroscience, Montana State University , Bozeman, Montana 59717, United States.

ABSTRACT
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 MeSH
Related in: MedlinePlus