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Dissecting Optical Response and Molecular Structure of Fluorescent Proteins With Non-canonical Chromophores.

ABSTRACT: Tracking the structural dynamics of fluorescent protein chromophores holds the key to unlocking the fluorescence mechanisms in real time and enabling rational design principles of these powerful and versatile bioimaging probes. By combining recent chemical biology and ultrafast spectroscopy advances, we prepared the superfolder green fluorescent protein (sfGFP) and its non-canonical amino acid (ncAA) derivatives with a single chlorine, bromine, and nitro substituent at the ortho site to the phenolate oxygen of the embedded chromophore, and characterized them using an integrated toolset of femtosecond transient absorption and tunable femtosecond stimulated Raman spectroscopy (FSRS), aided by quantum calculations of the vibrational normal modes. A dominant vibrational cooling time constant of ~4 and 11 ps is revealed in Cl-GFP and Br-GFP, respectively, facilitating a ~30 and 12% increase of the fluorescent quantum yield vs. the parent sfGFP. Similar time constants were also retrieved from the transient absorption spectra, substantiating the correlated electronic and vibrational motions on the intrinsic molecular timescales. Key carbon-halogen stretching motions coupled with phenolate ring motions of the deprotonated chromophores at ca. 908 and 890 cm-1 in Cl-GFP and Br-GFP exhibit enhanced activities in the electronic excited state and blue-shift during a distinct vibrational cooling process on the ps timescale. The retrieved structural dynamics change due to targeted site-specific halogenation of the chromophore thus provides an effective means to design new GFP derivatives and enrich the bioimaging probe toolset for life and medical sciences.

SUBMITTER: Oscar BG

PROVIDER: S-EPMC7358599 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Dissecting Optical Response and Molecular Structure of Fluorescent Proteins With Non-canonical Chromophores.

Oscar Breland G BG Zhu Liangdong L Wolfendeen Hayati H Rozanov Nikita D ND Chang Alvin A Stout Kenneth T KT Sandwisch Jason W JW Porter Joseph J JJ Mehl Ryan A RA Fang Chong C

Frontiers in molecular biosciences 20200707

Tracking the structural dynamics of fluorescent protein chromophores holds the key to unlocking the fluorescence mechanisms in real time and enabling rational design principles of these powerful and versatile bioimaging probes. By combining recent chemical biology and ultrafast spectroscopy advances, we prepared the superfolder green fluorescent protein (sfGFP) and its non-canonical amino acid (ncAA) derivatives with a single chlorine, bromine, and nitro substituent at the <i>ortho</i> site to t ...[more]

PMID: 32733917

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Project description:A series of mono-, bis-, tris-, and tetrakis(porphinato)zinc(II) (PZn)-elaborated ruthenium(II) bis(terpyridine) (Ru) complexes have been synthesized in which an ethyne unit connects the macrocycle meso carbon atom to terpyridyl (tpy) 4-, 4'-, and 4''-positions. These supermolecular chromophores, based on the ruthenium(II) [5-(4'-ethynyl-(2,2';6',2''-terpyridinyl))-10,20-bis(2',6'-bis(3,3-dimethyl-1-butyloxy)phenyl)porphinato]zinc(II)-(2,2';6',2''-terpyridine)(2+) bis-hexafluorophosphate (RuPZn) archetype, evince strong mixing of the PZn-based oscillator strength with ruthenium terpyridyl charge resonance bands. Potentiometric and linear absorption spectroscopic data indicate that for structures in which multiple PZn moieties are linked via ethynes to a [Ru(tpy)(2)](2+) core, little electronic coupling is manifest between PZn units, regardless of whether they are located on the same or opposite tpy ligand. Congruent with these experiments, pump-probe transient absorption studies suggest that the individual RuPZn fragments of these structures exhibit, at best, only modest excited-state electronic interactions that derive from factors other than the dipole-dipole interactions of these strong oscillators; this approximate independent character of the component RuPZn oscillators enables fabrication of nonlinear optical (NLO) multipoles with extraordinary hyperpolarizabilities. Dynamic hyperpolarizability (?(?)) values and depolarization ratios (?) were determined from hyper-Rayleigh light scattering (HRS) measurements carried out at an incident irradiation wavelength (?(inc)) of 1300 nm. The depolarization ratio data provide an experimental measure of chromophore optical symmetry; appropriate coupling of multiple charge-transfer oscillators produces structures having enormous averaged hyperpolarizabilities (?(HRS) values), while evolving the effective chromophore symmetry from purely dipolar (e.g., Ru(tpy)[4-(Zn-porphyrin)ethynyl-tpy](PF(6))(2), ?(HRS) = 1280 × 10(-30) esu, ? = 3.8; Ru(tpy)[4'-(Zn-porphyrin)ethynyl-tpy](PF(6))(2), ?(HRS) = 2100 × 10(-30) esu, ? = 3.8) to octopolar (e.g., Ru[4,4''-bis(Zn-porphyrin)ethynyl-tpy](2)(PF(6))(2), ?(HRS) = 1040 × 10(-30) esu, ? = 1.46) via structural motifs that possess intermediate values of the depolarization ratio. The chromophore design roadmap provided herein gives rise to octopolar supermolecules that feature by far the largest off-diagonal octopolar first hyperpolarizability tensor components ever reported, with the effectively octopolar Ru[4,4''-bis(Zn-porphyrin)ethynyl-tpy](2)(PF(6))(2) possessing a ?(HRS) value at 1300 nm more than a factor of 3 larger than that determined for any chromophore having octopolar symmetry examined to date. Because NLO octopoles possess omnidirectional NLO responses while circumventing the electrostatic interactions that drive bulk-phase centrosymmetry for NLO dipoles at high chromophore concentrations, the advent of octopolar NLO chromophores having vastly superior ?(HRS) values at technologically important wavelengths will motivate new experimental approaches to achieve acentric order in both bulk-phase and thin film structures.

Dataset Information

Dissecting Optical Response and Molecular Structure of Fluorescent Proteins With Non-canonical Chromophores.

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Dissecting Optical Response and Molecular Structure of Fluorescent Proteins With Non-canonical Chromophores.

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