Characterizing dark state kinetics and single molecule fluorescence of FusionRed and FusionRed-MQ at low irradiances

Mukherjee, Srijit; Thomas, Connor; Wilson, Ryan N.; Simmerman, Emma M.; Hung, Sheng-Ting; Jimenez, Ralph

doi:10.1039/d2cp00889k

Cited by 8 publications

(9 citation statements)

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“…The structure and dynamics of the chromophore environment are obviously critical for controlling ultrafast dynamics which lead to nonradiative decay of the excited state, thus limiting the fluorescence quantum yield. − In most bright red FPs (RFPs), the chromophore occupies a cis -phenolate conformation in the ground state with the phenolate exhibiting a single-bond character and the negative charge localized on the O atom of the phenol moiety. , Many recent studies have identified the rotation of the chromophore’s P-bond as a primary pathway of ultrafast nonradiative decay. ,− For example, in mCherry-like FPs, Drobhizev and co-workers examined the roles of several side chains near the MYG chromophore in restricting access to twisted intramolecular charge transfer (TICT) states through phenolate-bond (P-Bond) rotation . The presence of these TICT states permits ultrafast nonradiative decay through a conical intersection seam on the excited state surface.…”

Section: Introductionmentioning

confidence: 99%

Conformational Dynamics of mCherry Variants: A Link between Side-Chain Motions and Fluorescence Brightness

et al. 2022

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The 3-fold higher brightness of the recently developed mCherry-XL red fluorescent protein (FP) compared to its progenitor, mCherry, is due to a significant decrease in the nonradiative decay rate underlying its increased fluorescence quantum yield. To examine the structural and dynamic role of the four mutations that distinguish the two FPs and closely related variants, we employed microsecond time scale, all-atom molecular dynamics simulations. The simulations revealed that the I197R mutation leads to the formation of multiple hydrogen-bonded contacts and increased rigidity of the β-barrel. In particular, mCherryXL showed reduced nanosecond time scale breathing of the gap between the β7 and β10-strands, which was previously shown to be the most flexible region of mCherry. Together with experimental results, the simulations also reveal steric interactions of residue 161 and a network of hydrogen-bonding interactions of the chromophore with residues at positions 59, 143, and 163 that are critical in perturbing the chromophore electronic structure. Finally, we shed light on the conformational dynamics of the conserved residues R95 and S146, which are hydrogen-bonded to the chromophore, and provide physical insights into the observed photophysics. To the best of our knowledge, this is the first study that evaluates the conformational space for a set of closely related FPs generated by directed evolution.

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Section: Introductionmentioning

confidence: 99%

Conformational Dynamics of mCherry Variants: A Link between Side-Chain Motions and Fluorescence Brightness

et al. 2022

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“…The “dark state” in fluorescence, which is the unsupported concept that there is a time lag between the moment of excitation and the beginning of emission is believed to be very short, even shorter than the emission, and has been called “nonexistent”,with evidence that “remain scarce” [ 65 , 68 ]. All of these times are orders of magnitude shorter that the dwell time of the odorant ligands on the OR which are on a millisecond timescale [ 69 ].The combination of a brief, if even existent “dark state”, along with ns and ps fluorescence lifetimes indicates the release of the multiple photons of an odorant set resulting from the decay of high energy states of odorant molecules in ORs following IET would be on a time-scale so brief that they would be experienced as a set by the brain, providing for the time basis for the interpretation of sets of ONs of individual odorant molecules.…”

Section: Luminescence Timingmentioning

confidence: 99%

The Luminescence Hypothesis of Olfaction

Willeford

2023

Sensors

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A new hypothesis for the mechanism of olfaction is presented. It begins with an odorant molecule binding to an olfactory receptor. This is followed by the quantum biology event of inelastic electron tunneling as has been suggested with both the vibration and swipe card theories. It is novel in that it is not concerned with the possible effects of the tunneled electrons as has been discussed with the previous theories. Instead, the high energy state of the odorant molecule in the receptor following inelastic electron tunneling is considered. The hypothesis is that, as the high energy state decays, there is fluorescence luminescence with radiative emission of multiple photons. These photons pass through the supporting sustentacular cells and activate a set of olfactory neurons in near-simultaneous timing, which provides the temporal basis for the brain to interpret the required complex combinatorial coding as an odor. The Luminescence Hypothesis of Olfaction is the first to present the necessity of or mechanism for a 1:3 correspondence of odorant molecule to olfactory nerve activations. The mechanism provides for a consistent and reproducible time-based activation of sets of olfactory nerves correlated to an odor.The hypothesis has a biological precedent: an energy feasibility assessment is included, explaining the anosmia seen with COVID-19, and can be confirmed with existing laboratory techniques.

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“…24,25 Many recent studies have identified the rotation of the chromophore's P-bond as a primary pathway of ultrafast non-radiative decay. 9,[26][27][28][29][30][31][32] For example, Drobhizev and co-workers illustrated the roles of several sidechains in proximity of the MYGchromophore in mCherry-like proteins in restricting the ability of the chromophore to access twisted intramolecular charge transfer (TICT) states through phenolate-bond (P-Bond) rotation. 29 The presence of these TICT states permits ultrafast non-radiative decay through a conical intersection seam on the excitedstate surface.…”

Section: Introductionmentioning

confidence: 99%

Conformational dynamics of mCherry variants: a link between sidechain motions and fluorescence brightness

Mukherjee

Manna

Douglas

et al. 2022

Preprint

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We recently developed the red fluorescent protein (RFP) mCherry-XL, which is threefold brighter than its predecessor, mCherry, with a directed evolution approach using fluorescence-lifetime flow cytometry selections. The enhanced brightness is due to a significant decrease in the non-radiative decay rate underlying its increase in fluorescence quantum yield. To examine the dynamic role of the four mutations that distinguish the two RFPs and closely-related variants, we employed microsecond-timescale, all-atom molecular dynamics simulations to sample their ground state conformational landscapes. The simulations revealed the significance of the I197R mutation, which leads to the formation of multiple hydrogen-bonded contacts. The triad of interactions observed between residues K70, E148 and 197R is also seen in mScarlet, another RFP of unrelated origin, but of comparably high brightness. These substitutions in mCherry-XL increased the rigidity of the β-barrel, for example as shown by increased hydrogen-bonding in the chromophore region and decreased root-mean-square backbone deviations. Furthermore, mCherry-XL showed significantly less ns-timescale breathing of the gap between β-7 and β-10 strands. This gap was previously shown to be the most flexible region of mCherry, permitting entry of O2 into the barrel. We also characterize the role played by the sidechain of residue 161 using a combination of MD simulations and in-vitro experimental characterization. We find this position is critical to the steric interactions that perturb the chromophore electronic structure. MD simulations also help us to recognize a network of hydrogen-bonded interactions between the chromophore, the residue 143, 163 and 59, which can potentially impact the electron distribution of the chromophore. Finally, we shed light on the conformational dynamics of the conserved residues R95 and S146, which are hydrogen bonded to the chromophore, and provide physical insights into the observed photophysics. To the best of our knowledge, this is the first study that evaluates the conformational space for a set of closely related FPs generated by directed evolution.

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Characterizing dark state kinetics and single molecule fluorescence of FusionRed and FusionRed-MQ at low irradiances

Abstract: The presence of dark states causes fluorescence intermittency of single molecules due to transitions between “on” and “off” states. Genetically encodable markers such as fluorescent proteins (FPs) exhibit dark states...

Cited by 8 publications

References 93 publications

Conformational Dynamics of mCherry Variants: A Link between Side-Chain Motions and Fluorescence Brightness

Conformational Dynamics of mCherry Variants: A Link between Side-Chain Motions and Fluorescence Brightness

The Luminescence Hypothesis of Olfaction

Conformational dynamics of mCherry variants: a link between sidechain motions and fluorescence brightness

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