Two independent routes of post-translational chemistry in fluorescent protein FusionRed

Muslinkina, Liya; Плетнев, В. З.; Pletneva, N.V.; Ruchkin, Dmitry A.; Kolesov, Danila V.; Bogdanov, Alexey M.; Kost, Liubov A.; Rakitina, Tatiana V.; Agapova, Yu. K.; Shemyakina, Irina I.; Chudakov, Dmitriy M.; Pletnev, Sergei

doi:10.1016/j.ijbiomac.2020.03.244

Cited by 18 publications

(54 citation statements)

References 40 publications

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“…Crystal structure data suggests conformational freedom of the sidechain at position 42 in FR. 51 The position 42 lies in close proximity to the acylimine end of the chromophore and based on our previous work, is critical to the hydrogen-bond network on that end of the chromophore in closely related FPs mKate and TagRFP-675. 38 A smaller site-saturated library of 20 possible mutants at position 42 allowed us to carry out a plate based screen on FR-M, where Gln stood out as the best possible sidechain residue in terms of increases in  and .…”

Section: Discussionmentioning

confidence: 87%

“…Crystal structures indicate that the phenol group of FP chromophores can occupy either the trans (non-fluorescing) or the cis (fluorescing) configuration 52 , , 53 and in FR, the latter can potentially form a hydrogen bond with the hydroxy group of the S144 residue (Figure 2). 51 The location of C159 suggests that it may play a similar role for the trans form of the chromophore. The position 159 was recently discussed to be critical in terms of molecular brightness for FR.…”

Section: Discussionmentioning

confidence: 99%

“…The position 159 was recently discussed to be critical in terms of molecular brightness for FR. 51 The propensity to switch to the trans form may be reduced when the residue at this position is substituted for an aliphatic group. The cis to trans isomerization is a reversible process and is manifested as the reversible component of the photobleaching trace under continuous and pulsed (ms to s) illumination.…”

Section: Discussionmentioning

confidence: 99%

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Structure-guided point mutations on FusionRed produce a brighter red fluorescent protein

Mukherjee

Hung

Douglas

et al. 2020

Preprint

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The development of fluorescent proteins (FPs) has revolutionized biological imaging. FusionRed, a monomeric red FP (RFP), is known for its low cytotoxicity and appropriate localization of target fusion proteins in mammalian cells but is limited in application by low fluorescence brightness. We report a brighter variant of FusionRed, FusionRed-MQV, which exhibits an extended fluorescence lifetime (2.8 ns), enhanced quantum yield (0.53), higher extinction coefficient (~140,000 M -1 cm -1 ), increased radiative rate constant and reduced non-radiative rate constant with respect to its precursor. The properties of FusionRed-MQV derive from three mutations -M42Q, C159V and the previously identified L175M. A structure-guided approach was used to identify and mutate candidate residues around the phenol and the acylimine ends of the chromophore. The C159V mutation was identified via lifetime-based flow cytometry screening of a library in which multiple residues adjacent to the phenol end of the chromophore were mutated. The M42Q mutation is located near the acylimine end of the chromophore and was discovered using sitedirected mutagenesis guided by x-ray crystal structures. FusionRed-MQV exhibits 3.4-fold higher molecular brightness and a 5-fold increase in the cellular brightness in HeLa cells (based on FACS) compared to FusionRed. It also retains the low cytotoxicity and high-fidelity localization of FusionRed, as demonstrated through assays in mammalian cells.

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Section: Discussionmentioning

confidence: 87%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Structure-guided point mutations on FusionRed produce a brighter red fluorescent protein

Mukherjee

Hung

Douglas

et al. 2020

Preprint

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“…In the early days on GFP structural research, a vast number of works explored similar changes arising from single point mutations of highly conserved residues in the nearest chromophore environment and the chromophore itself. Their effect ranged from complete cessation of the chromophore maturation to highly precise change of the fluorescence corresponding to different steps of the chromophore maturation [36] , [50] , [51] , [52] . A distinctive feature of the EYFP-F165G structure is the presence of two species at the space typically occupied by the chromophore.…”

Section: Discussionmentioning

confidence: 99%

Amino acid residue at the 165th position tunes EYFP chromophore maturation. A structure-based design

Pletneva

Maksimov

Protasova

et al. 2021

Computational and Structural Biotechnology Journal

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“…53 Together with findings and from other studies, including crystal structure data, indicate a possible interconversion of the FusionRed chromophore from a fluorescent cis to a dark trans isomer. 56,57 Moreover, in the low irradiance regime of 1-10 W/cm 2 , the temperature increase in the vicinity of an FP molecule can be considered negligible. 58 Based on these observations, we hypothesized that the rate of recovery to the ground state (kGSR) is independent of the excitation rate (kEx) under low irradiances for FusionRed and FusionRed-MQ.…”

Section: Introductionmentioning

confidence: 99%

Characterizing Dark State Kinetics and Single Molecule Fluorescence of FusionRed and FusionRed-MQ at Low Irradiances

Mukherjee

Thomas

Wilson

et al. 2022

Preprint

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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 that make several super-resolved single-molecule localization microscopy (SMLM) methods possible. However, studies quantifying the timescales and nature of dark state behavior for commonly used FPs under conditions typical of widefield and total internal reflection fluorescence (TIRF) microscopy remain scarce and pre-date many new SMLM techniques. FusionRed is a relatively bright red FP exhibiting fluorescence intermittency and has thus been identified as a potential candidate for SMLM. We herein characterize the rates for dark-state conversion and the subsequent ground-state recovery of FusionRed and its 2.5-fold brighter descendent FusionRed L175M M42Q (FusionRed-MQ) at low irradiances (1-10 W/cm2), which were previously unexplored experimental conditions. We characterized the kinetics of dark state transitions in these two FPs by using single molecule blinking and ensemble photobleaching experiments bridged with a dark state kinetic model. We find that at low irradiances, the recovery process to the ground state is minimally light-driven and FusionRed-MQ has a 1.3-fold higher ground state recovery time indicating a conformationally restricted dark-state chromophore in comparison to FusionRed. Our studies indicate that the brighter FusionRed-MQ exhibits higher tendency in dark state conversion, thus it is potentially a better candidate for SMLM applications than its progenitor FusionRed.

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Two independent routes of post-translational chemistry in fluorescent protein FusionRed

Cited by 18 publications

References 40 publications

Structure-guided point mutations on FusionRed produce a brighter red fluorescent protein

Structure-guided point mutations on FusionRed produce a brighter red fluorescent protein

Amino acid residue at the 165th position tunes EYFP chromophore maturation. A structure-based design

Characterizing Dark State Kinetics and Single Molecule Fluorescence of FusionRed and FusionRed-MQ at Low Irradiances

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