Steady-state and time-resolved spectroscopic studies of green-to-red photoconversion of fluorescent protein Dendra2

Makarov, Nikolay S.; Cirloganu, Claudiu M.; Perry, Joseph W.; Lukyanov, Konstantin A.; Solntsev, Kyril M.

doi:10.1016/j.jphotochem.2014.02.001

Cited by 13 publications

(28 citation statements)

References 44 publications

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“…While only the neutral and not the anionic chromophore in Kaede-like proteins possesses the capacity to photoconvert to the red state, Dendra (and its monomeric variant Dendra2) can be photoconverted from both neutral and anionic forms. The PC quantum yields of Dendra2 suggest a 25-fold more efficient green-to-red conversion when initiated in the neutral form (Makarov et al, 2014). In numerous instances it has been noted that there is no simple one-to-one correlation between the pK a of the chromophore hydroxyphenyl group and the PC efficiency (Moeyaert et al, 2014).…”

Section: Discussionmentioning

confidence: 99%

“…It has been demonstrated that in Kaede-like proteins only the neutral and not the anionic chromophore is able to photoconvert into the red state. A remarkable exception from this rule is DendFP and its monomeric variant Dendra2, which can be photoconverted in a microscope by continuous-wave blue light at about 490 nm, albeit with a much lower efficiency compared with UV/violet light (Gurskaya et al, 2006;Chudakov et al, 2007;Makarov et al, 2014). A recent extensive wavelength-and pH-dependent study of Dendra2 PC suggested that the green-to-red conversion occurs following the absorption of one photon directly from the excited state of each (neutral and anionic) form of the green protein without intermediate excited states or species requiring the absorption of additional photons (Makarov et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

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Crystal structure of the fluorescent protein fromDendronephthyasp. in both green and photoconverted red forms

Pletneva

Pletnev

Пахомов

et al. 2016

Acta Cryst Sect D Struct Biol

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The fluorescent protein from Dendronephthya sp. (DendFP) is a member of the Kaede-like group of photoconvertible fluorescent proteins with a His62-Tyr63-Gly64 chromophore-forming sequence. Upon irradiation with UV and blue light, the fluorescence of DendFP irreversibly changes from green (506 nm) to red (578 nm). The photoconversion is accompanied by cleavage of the peptide backbone at the C(α)-N bond of His62 and the formation of a terminal carboxamide group at the preceding Leu61. The resulting double C(α)=C(β) bond in His62 extends the conjugation of the chromophore π system to include imidazole, providing the red fluorescence. Here, the three-dimensional structures of native green and photoconverted red forms of DendFP determined at 1.81 and 2.14 Å resolution, respectively, are reported. This is the first structure of photoconverted red DendFP to be reported to date. The structure-based mutagenesis of DendFP revealed an important role of positions 142 and 193: replacement of the original Ser142 and His193 caused a moderate red shift in the fluorescence and a considerable increase in the photoconversion rate. It was demonstrated that hydrogen bonding of the chromophore to the Gln116 and Ser105 cluster is crucial for variation of the photoconversion rate. The single replacement Gln116Asn disrupts the hydrogen bonding of Gln116 to the chromophore, resulting in a 30-fold decrease in the photoconversion rate, which was partially restored by a further Ser105Asn replacement.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Crystal structure of the fluorescent protein fromDendronephthyasp. in both green and photoconverted red forms

Pletneva

Pletnev

Пахомов

et al. 2016

Acta Cryst Sect D Struct Biol

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“…The violet conversionl ight is absorbed by the neutraln on-fluorescent green chromophore, causing a bond cleavage between the Na and Ca atoms andt he formation of ad ouble bond between the Ca and the Cb atoms of the chromophore histidine. Apart from ar eport by Makarov et al [43] statingt hat also the anionic form of Dendra2c ould minimally contribute to violetlight-induced conversion,f ormation of the red speciesb y means of this "traditional" photoconversion process is believed to rely on absorption by the neutrals pecies. The detailed mechanism underlying this process has been subject of debate for many years (reviewed in reference [42]).…”

Section: Mechanism Of Near-uv Photoconversionmentioning

confidence: 99%

“…The detailed mechanism underlying this process has been subject of debate for many years (reviewed in reference [42]). Apart from ar eport by Makarov et al [43] statingt hat also the anionic form of Dendra2c ould minimally contribute to violetlight-induced conversion,f ormation of the red speciesb y means of this "traditional" photoconversion process is believed to rely on absorption by the neutrals pecies. Recently,o ur group reportedafundamentally different mechanism,c alled primedc onversion, which acts mainly on the anionic chromophore instead.…”

Section: Mechanism Of Near-uv Photoconversionmentioning

confidence: 99%

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Primed Conversion: The New Kid on the Block for Photoconversion

Mohr

Pantazis

2018

Chemistry A European J

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In 2015, a novel way to convert photoconvertible fluorescent proteins was reported that uses the intercept of blue and far-red light instead of traditional violet or near-UV light illumination. This Minireview describes and contrasts this distinct two-step mechanism termed primed conversion with traditional photoconversion. We provide a comprehensive overview of what is known to date about primed conversion and focus on the molecular requirements for it to take place. We provide examples of its application to axially confined photoconversion in complex tissues as well as super-resolution microscopy. Further, we describe why and when it is useful, including its advantages and disadvantages, and give an insight into potential future development in the field.

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Targeting Ultrafast Spectroscopic Insights into Red Fluorescent Proteins

Krueger,

Chen,

Fang

2023

Chemistry An Asian Journal

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Red fluorescent proteins (RFPs) represent an increasingly popular class of genetically encodable bioprobes and biomarkers that can advance next‐generation breakthroughs across the imaging and life sciences. Since the rational design of RFPs with improved functions or enhanced versatility requires a mechanistic understanding of their working mechanisms, while fluorescence is intrinsically an ultrafast event, a suitable toolset involving steady‐state and time‐resolved spectroscopic techniques has become powerful in delineating key structural features and dynamic steps which govern irreversible photoconverting or reversible photoswitching RFPs, and large Stokes shift (LSS)RFPs. The pertinent cis‐trans isomerization and protonation state change of RFP chromophores in their local environments, involving key residues in protein matrices, lead to rich and complicated spectral features across multiple timescales. In particular, ultrafast excited‐state proton transfer in various LSSRFPs showcases the resolving power of wavelength‐tunable femtosecond stimulated Raman spectroscopy (FSRS) in mapping a photocycle with crucial knowledge about the red‐emitting species. Moreover, recent progress in noncanonical RFPs with a site‐specifically modified chromophore provides an appealing route for efficient engineering of redder and brighter RFPs, highly desirable for bioimaging. Such an effective feedback loop involving physical chemists, protein engineers, and biomedical microscopists will enable future successes to expand fundamental knowledge and improve human health.

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Steady-state and time-resolved spectroscopic studies of green-to-red photoconversion of fluorescent protein Dendra2

Cited by 13 publications

References 44 publications

Crystal structure of the fluorescent protein fromDendronephthyasp. in both green and photoconverted red forms

Crystal structure of the fluorescent protein fromDendronephthyasp. in both green and photoconverted red forms

Primed Conversion: The New Kid on the Block for Photoconversion

Targeting Ultrafast Spectroscopic Insights into Red Fluorescent Proteins

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