Enhanced UnaG With Minimal Labeling Artifact for Single-Molecule Localization Microscopy

Ko, Sangyoon; Kwon, Jiwoong; Shim, Sang Hee

doi:10.3389/fmolb.2021.647590

Cited by 4 publications

(5 citation statements)

References 44 publications

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“…Green fluorescent protein (GFP) has revolutionized many scientific fields. − Its utility has spurred the creation of a broad spectrum of related molecules with properties tuned for a variety of uses − such that 25,900 papers were published in 2022 containing the phrase fluorescent protein . An increasing number of studies examine structural dynamics of the fluorescent protein (FP) chromophore and its surroundings such that molecular dynamics (MD) simulations are often employed (7440 of those 25,900 also contain the words molecular , dynamics , and simulation ).…”

Section: Introductionmentioning

confidence: 99%

“…An entire field of discovery employs mutations in the chromophore and to nearby regions of the surrounding protein to control the emission properties of the resulting proteins. Properties such as the emission wavelength, pH dependence, and photostability can all be controlled and optimized. ,,,− ,− …”

Section: Introductionmentioning

confidence: 99%

“…Properties such as the emission wavelength, pH dependence, and photostability can all be controlled and optimized. 6 , 7 , 11 , 14 − 17 , 37 − 42 …”

Section: Introductionmentioning

confidence: 99%

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Molecular Dynamics Force Field Parameters for the EGFP Chromophore and Some of Its Analogues

Breyfogle,

Blood,

Rosnik

et al. 2023

J. Phys. Chem. B

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Fluorescent proteins (FPs) have had an enormous impact on molecular and cellular biology and are employed in a wide range of studies of molecular structure and dynamics. Yet, only a modest number of papers have published molecular dynamics (MD) parameters describing FPs. And despite the development of a wide range of FPs, there has been no careful development of MD parameters across a series of FPs. In this work, we present MD parameters describing six fluorescent protein chromophores (EGFP, EBFP, EYFP, ECFP, mCherry, and DsRed) for use with the Cornell et al. (J. Am. Chem. Soc.199511751795197) family of AMBER force fields, including ff14SB and ff19SB. We explore a wide range of solvent dielectric constants for determining the chromophore equilibrium geometry and evaluate the impact of the modeled solvent on the final atomic charges. We also present our methodological approach in which we considered all six chromophores together with a focus on modularity, transferability, and balance with existing force fields. The parameters given here make it easy to employ MD simulations to study any of the six systems, whereas the methodology makes it easy for anyone to extend this work to develop consistent parameters for additional fluorescent proteins. The results of our own MD simulations are presented, showing that the classical MD parameters yield chromophore structural distributions that compare well with QM/MM simulations.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Molecular Dynamics Force Field Parameters for the EGFP Chromophore and Some of Its Analogues

Breyfogle,

Blood,

Rosnik

et al. 2023

J. Phys. Chem. B

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“…A prominent example is Point Accumulation for Imaging in Nanoscale Topography (PAINT) and its variants, which rely on stochastic fluorophore binding to generate localizable single molecules [7][8][9] . Recently, FP-based PAINT was demonstrated using the bilirubin-activating protein UnaG 10 and its brightness-enhanced mutant eUnaG 11 . UnaG is derived from Japanese eel muscle and fluoresces when non-covalently bound to the bilirubin chromophore 12 .…”

mentioning

confidence: 99%

Near-infrared PAINT localization microscopy via chromophore replenishment of phytochrome-derived fluorescent tag

Lu,

Wazawa,

Matsuda

et al. 2024

Commun Biol

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Bacterial phytochromes are attractive molecular templates for engineering fluorescent proteins (FPs) because their near-infrared (NIR) emission significantly extends the spectral coverage of GFP-like FPs. Existing phytochrome-based FPs covalently bind heme-derived tetrapyrrole chromophores and exhibit constitutive fluorescence. Here we introduce Rep-miRFP, an NIR imaging probe derived from bacterial phytochrome, which interacts non-covalently and reversibly with biliverdin chromophore. In Rep-miRFP, the photobleached non-covalent adduct can be replenished with fresh biliverdin, restoring fluorescence. By exploiting this chromophore renewal capability, we demonstrate NIR PAINT nanoscopy in mammalian cells using Rep-miRFP.

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“…Such applications necessitate FPs with superior properties such as high FQY, fraction of time remaining fluorescent, outstanding photostability, and structural stability ( Bourgeois and Adam, 2012 ; Nienhaus and Nienhaus, 2016 ; Woodhouse et al, 2020 ). In this issue, a bilirubin-activated photoswitching protein called eUnaG was developed with the highest bulk fluorescence to date, comparable to organic dyes ( Ko et al, 2021 ). The superior performance of eUnaG is primarily due to its increased stability, leading to reduced aggregation and fewer labeling artifacts.…”

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confidence: 99%

Editorial: Mechanisms of Fluorescent Proteins

Fang

Drobizhev

et al. 2021

Front. Mol. Biosci.

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Editorial on the Research Topic Mechanisms of Fluorescent ProteinsThis collection of papers for the Research Topic "Mechanisms of Fluorescent Proteins" (FPs) samples a broad range of research on physical mechanisms, applications, and molecular engineering strategies. The papers demonstrate a combination of experimental and computational approaches and are of broad interest to researchers working on FPs, microscopy, and spectroscopy.In bioimaging with FPs, increasing the penetration depth and decreasing unwanted scattering are desirable, which have motivated efforts for engineered FPs with redder emission and higher brightness (Subach and Verkhusha, 2012;Dedecker et al., 2013). In a combined experimental and computational work (Gorbachev et al., 2020), the green/red photoconversion of EGFP with reducing agents was investigated and a novel green-emitting state only present under low-oxygen conditions was identified. Following photoconversion, the distinct orange and red-emitting forms (565 and 600 nm emission maxima) differ from the reported red-emitting form (607 nm emission) via oxidative reaction. This work showcases a complex interplay between the chromophore and protein environment, generating a neutral quinoid-like green-emitting chromophore (525 nm emission) as an intermediate. This step leads to a zwitterionic form of the photoexcited chromophore via charge transfer that bifurcates into the orange and red-emitting forms. Such a general oxidative mechanism enriches the FP application toolset (Bourgeois and Adam, 2012;Jung, 2012;Krueger et al., 2020;Nasu et al., 2021) with additional tunable "knobs" of oxygen levels and redox-active compounds to control photoconversion and achieve redder emission.To brighten the generally dim red and far-red FPs, a systematic study of nonradiative relaxation in red FPs (RFPs) (Drobizhev et al., 2021) reveals a dominant role of the twisted intramolecular charge transfer mechanism over the energy gap law. This work substantiates local electrical field control of fluorescence quantum yield (FQY) of RFPs. Aided by one-and two-photon absorption spectroscopy and quantum calculations of seven different RFPs with the same chromophore structure, a spectroscopic method of evaluating local electric fields (amplitude and direction in E x and E y ) at the protein chromophore enables separation of contributing factors to the nonradiative relaxation rate. A small range of positive or negative values for E x and E y (-10 to +10 MV/cm) was revealed to facilitate both a red-shifted absorption and a high FQY, providing rational design principles for sitespecific mutagenesis using RFP scaffold like DsRed.On a fundamental level, the fluorescence mechanism via excited-state proton transfer (Chattoraj et al., 1996;Fang et al., 2009;Tonge and Meech, 2009;Fang and Tang, 2020) is elucidated further by a computational study (Coppola et al., 2020) on the complex hydrogen (H)-bond equilibrium dynamics for neutral, intermediate, and anionic chromophore forms inside GFP. An accurate

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Enhanced UnaG With Minimal Labeling Artifact for Single-Molecule Localization Microscopy

Cited by 4 publications

References 44 publications

Molecular Dynamics Force Field Parameters for the EGFP Chromophore and Some of Its Analogues

Molecular Dynamics Force Field Parameters for the EGFP Chromophore and Some of Its Analogues

Near-infrared PAINT localization microscopy via chromophore replenishment of phytochrome-derived fluorescent tag

Editorial: Mechanisms of Fluorescent Proteins

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