Light-Induced Conformational Heterogeneity Induces Positive Photoswitching in Photoconvertible Fluorescent Proteins of the EosFP Family DOI
Jip Wulffelé, Arijit Maity, Isabel Ayala

et al.

Journal of the American Chemical Society, Journal Year: 2025, Volume and Issue: unknown

Published: March 14, 2025

Green-to-red photoconvertible fluorescent proteins (PCFPs) of the EosFP family are commonly used in ensemble pulse-chase and single-molecule localization or tracking approaches. However, these exhibit highly complex photophysical behaviors. In green-form, recent NMR experiments revealed that mEos4b other PCFP variants exist two different conformational states at thermal equilibrium, which limits their effective photoconversion efficiency. Here, we investigate heterogeneity photoconverted red-form, employing a combination solution NMR, UV-vis spectroscopy fluorescence imaging. Only single red population is observed equilibrium. second emerges under illumination with 405 488 nm light, slowly decays dark can be swiftly reverted 561 light. This manifests itself through pH-dependent positive photoswitching mechanism adds to already characterized negative assigned cis-trans isomerization chromophore. Our data indicate photoswitching, instead, results from light-induced formation state cis configuration chromophore exhibits substantially increased pKa. Such mechanism, suggested result rewiring H-bonding network around first amino acid chromophore, panoply switching scenarios proteins. It bears consequences for spectroscopic characterization PCFPs, reduces apparent brightness generates short-lived off-times perturbing microscopy applications.

Language: Английский

A General Strategy to Control Viscosity Sensitivity of Molecular Rotor‐Based Fluorophores DOI
Songtao Ye, Han Zhang,

Jinyu Fei

et al.

Angewandte Chemie International Edition, Journal Year: 2020, Volume and Issue: 60(3), P. 1339 - 1346

Published: Sept. 29, 2020

Molecular rotor-based fluorophores (RBFs) have been widely used in many fields. However, the lack of control their viscosity sensitivity limits application. Herein, this problem is resolved by chemically installing extended π-rich alternating carbon-carbon linkages between rotational electron donors and acceptors RBFs. The data reveal that length linkage strongly influences sensitivity, likely resulting from varying height energy barriers fluorescent planar dark twisted configurations. Three RBF derivatives span a wide range sensitivities were designed. These RBFs demonstrated, through dual-color imaging strategy, they can differentiate misfolded protein oligomers insoluble aggregates, both test tubes live cells. Beyond RBFs, it envisioned chemical mechanism might be generally applicable to photoisomerizable aggregation-induced emission fluorophores.

Language: Английский

Citations

157
Quantum–classical simulations of rhodopsin reveal excited-state population splitting and its effects on quantum efficiency DOI
Xuchun Yang, Madushanka Manathunga, Samer Gozem

et al.

Nature Chemistry, Journal Year: 2022, Volume and Issue: 14(4), P. 441 - 449

Published: March 3, 2022

Language: Английский

Citations

47
Unifying structural descriptors for biological and bioinspired nanoscale complexes DOI Open Access
Minjeong Cha, Emine Sumeyra Turali Emre, Xiongye Xiao

et al.

Nature Computational Science, Journal Year: 2022, Volume and Issue: 2(4), P. 243 - 252

Published: April 28, 2022

Language: Английский

Citations

40
The β-subunit of tryptophan synthase is a latent tyrosine synthase DOI
Patrick J. Almhjell, Kadina E. Johnston, Nicholas J. Porter

et al.

Nature Chemical Biology, Journal Year: 2024, Volume and Issue: 20(8), P. 1086 - 1093

Published: May 14, 2024

Language: Английский

Citations

9
Ordered Solvents and Ionic Liquids Can Be Harnessed for Electrostatic Catalysis DOI
Longkun Xu,

Ekaterina I. Izgorodina,

Michelle L. Coote

et al.

Journal of the American Chemical Society, Journal Year: 2020, Volume and Issue: 142(29), P. 12826 - 12833

Published: July 1, 2020

Herein, we employ classical molecular dynamics simulations using the Drude oscillator-based polarizable force field, quantum chemical calculations, and ONIOM multiscale calculations to study (a) how an external field orders solvent environment in a reaction then (b) whether absence of this same applied ordered alone can electrostatically catalyze when compared with corresponding disordered solvent. Our results show that 0.2 V/Å electric which is below threshold for bond breaking molecules, leads significant ordering bulk methanol ionic liquid [EMIM][BF4]. Importantly, lowers activation energy hydrogen-transfer o-alkylphenyl ketones excess 20 kcal/mol by over 30 Even 0.1 has effects ca. 10 kcal/mol, respectively. This work suggests possible strategy scaling electrostatic catalysis applying pulsed medium maintain while allowing proceed largely field.

Language: Английский

Citations

52
Large Stokes shift fluorescence activation in an RNA aptamer by intermolecular proton transfer to guanine DOI Creative Commons
Mateusz Mieczkowski, Christian Steinmetzger, Irene Bessi

et al.

Nature Communications, Journal Year: 2021, Volume and Issue: 12(1)

Published: June 10, 2021

Abstract Fluorogenic RNA aptamers are synthetic functional RNAs that specifically bind and activate conditional fluorophores. The Chili aptamer mimics large Stokes shift fluorescent proteins exhibits high affinity for 3,5-dimethoxy-4-hydroxybenzylidene imidazolone (DMHBI) derivatives to elicit green or red fluorescence emission. Here, we elucidate the structural mechanistic basis of activation by crystallography time-resolved optical spectroscopy. Two co-crystal structures with positively charged DMHBO + DMHBI ligands revealed a G-quadruplex trans -sugar-sugar edge G:G base pair immobilize ligand π-π stacking. A Watson-Crick G:C in fluorophore binding site establishes short hydrogen bond between N7 guanine phenolic OH ligand. Ultrafast excited state proton transfer (ESPT) from neutral chromophore was found time constant 130 fs mode action fluorogenic aptamer.

Language: Английский

Citations

42
Oriented internal electrostatic fields: an emerging design element in coordination chemistry and catalysis DOI Creative Commons
Alexander B. Weberg, Ryan P. Murphy, Neil C. Tomson

et al.

Chemical Science, Journal Year: 2022, Volume and Issue: 13(19), P. 5432 - 5446

Published: Jan. 1, 2022

The power of oriented electrostatic fields (ESFs) to influence chemical bonding and reactivity is a phenomenon rapidly growing interest. presence strong ESFs has recently been implicated as one the most significant contributors activity select enzymes, wherein alignment substrate's changing dipole moment with strong, local field shown be responsible for majority enzymatic rate enhancement. Outside enzymology, researchers have studied impacts "internal"

Language: Английский

Citations

36
Directed Evolution of a Bright Variant of mCherry: Suppression of Nonradiative Decay by Fluorescence Lifetime Selections DOI
Srijit Mukherjee, Premashis Manna, Sheng‐Ting Hung

et al.

The Journal of Physical Chemistry B, Journal Year: 2022, Volume and Issue: 126(25), P. 4659 - 4668

Published: June 16, 2022

The approximately linear scaling of fluorescence quantum yield (ϕ) with lifetime (τ) in fluorescent proteins (FPs) has inspired engineering brighter fluorophores based on screening for increased lifetimes. Several recently developed FPs such as mTurquoise2, mScarlet, and FusionRed-MQV which have become useful live cell imaging are products selection strategies. However, the underlying photophysical basis improved brightness not been scrutinized. In this study, we focused understanding outcome lifetime-based directed evolution mCherry, is a popular red-FP (RFP). We identified four positions (W143, I161, Q163, I197) near FP chromophore that can be mutated to create mCherry-XL (eXtended Lifetime: ϕ = 0.70; τ 3.9 ns). 3-fold higher par brightest RFP date, mScarlet. examined selected variants within trajectory found near-linear consistent blue-shifts absorption emission spectra. find improvement primarily due decrease nonradiative decay excited state. addition, our analysis revealed rate limited blue-shift energy gap changes state reorganization energy. Our findings suggest mechanisms beyond scope energy-gap models Englman-Jortner model suppressed trajectory.

Language: Английский

Citations

35
Local Electric Fields Drives the Proton-Coupled Electron Transfer within Cytochrome P450 Reductase DOI
Ningning Li,

Shengheng Yan,

Peng Wu

et al.

ACS Catalysis, Journal Year: 2024, Volume and Issue: 14(10), P. 7893 - 7900

Published: May 6, 2024

Cytochrome P450 enzymes play a pivotal role in biosynthetic and metabolic transformations. Especially, cytochrome reductase (CPR) acts as the key electron donor for oxygen activation by monoxygenases, but transfer mechanism within CPR is largely elusive. Here, extensive molecular dynamics (MD) quantum mechanics/molecular mechanics (QM/MM) calculations were performed to elucidate CPR's mechanism. We found that from FADH– FMN occurs through proton-coupled (PCET) Glu142 transfers proton via two-water-molecule chain, concurrent with FMN. The subsequent ET FADH• FMNH• involves an Asp675-mediated PCET process, where Ser457-assisted Asp675 coupled FMNH•. Notably, local electric field doubly protonated His180 significantly enhances reactions both kinetically thermodynamically. This study highlights vital of facilitating biological enzymatic reactions.

Language: Английский

Citations

7
Molecular mechanisms and evolutionary robustness of a color switch in proteorhodopsins DOI Creative Commons
Jiafei Mao, Xinsheng Jin,

Man Shi

et al.

Science Advances, Journal Year: 2024, Volume and Issue: 10(4)

Published: Jan. 24, 2024

Proteorhodopsins are widely distributed photoreceptors from marine bacteria. Their discovery revealed a high degree of evolutionary adaptation to ambient light, resulting in blue- and green-absorbing variants that correlate with conserved glutamine/leucine at position 105. On the basis an integrated approach combining sensitivity-enhanced solid-state nuclear magnetic resonance (ssNMR) spectroscopy linear-scaling quantum mechanics/molecular mechanics (QM/MM) methods, this single residue is shown be responsible for variety synergistically coupled structural electrostatic changes along retinal polyene chain, ionone ring, within binding pocket. They collectively explain observed color shift. Furthermore, analysis differences chemical shift between nuclei same residues green blue proteorhodopsins also reveals correlation respective conservation. Our data show highly change mainly affects other residues, illustrating robustness phenotype sequence variation.

Language: Английский

Citations

6