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Since green fluorescent protein (GFP) has revolutionized molecular and cellular biology for about three decades, there has been a keen interest in understanding, designing, and controlling the fluorescence properties of GFP chromophore ( i.e. , HBDI) derivatives from the protein matrix to solution. Amongst these cross-disciplinary efforts, the elucidation of excited-state dynamics of HBDI derivatives holds the key to correlating the light-induced processes and fluorescence quantum yield (FQY). Herein, we implement steady-state electronic spectroscopy, femtosecond transient absorption (fs-TA), femtosecond stimulated Raman spectroscopy (FSRS), and quantum calculations to study a series of mono- and dihalogenated HBDI derivatives (X = F, Cl, Br, 2F, 2Cl, and 2Br) in basic aqueous solution, gaining new insights into the photophysical reaction coordinates. In the excited state, the halogenated “floppy” chromophores exhibit an anti-heavy atom effect, reflected by strong correlations between FQY vs. Franck–Condon energy ( E FC ) or Stokes shift, and k nr vs. E FC , as well as a swift bifurcation into the I-ring (major) and P-ring (minor) twisting motions. In the ground state, both ring-twisting motions become more susceptible to sterics and exhibit spectral signatures from the halogen-dependent hot ground-state absorption band decay in TA data. We envision this type of systematic analysis of the halogenated HBDI derivatives to provide guiding principles for the site-specific modification of GFP chromophores, and expand design space for brighter and potentially photoswitchable organic chemical probes in aqueous solution with discernible spectral signatures throughout the photocycle. 

Strategic incorporation of ameta‐dimethylamino (–NMe₂) group on the conformationally locked green fluorescent protein (GFP) model chromophore (m‐NMe₂‐LpHBDI) has drastically altered molecular electronic properties, counterintuitively enhancing fluorescence of only the neutral and cationic chromophores in aqueous solution. A ~200‐fold decrease in fluorescence quantum yield ofm‐NMe₂‐LpHBDI in alcohols (e.g., MeOH, EtOH and 2‐PrOH) supports this GFP‐derived compound as a fluorescence turn‐on water sensor, with large fluorescence intensity differences between H₂O and ROH emissions in various H₂O/ROH binary mixtures. A combination of steady‐state electronic spectroscopy, femtosecond transient absorption, ground‐state femtosecond stimulated Raman spectroscopy (FSRS) and quantum calculations elucidates an intermolecular hydrogen‐bonding chain between a solvent –OH group and the chromophore phenolic ring –NMe₂and –OH functional groups, wherein fluorescence differences arise from an extended hydrogen‐bonding network beyond the first solvation shell, as opposed to fluorescence quenching via a dark twisted intramolecular charge‐transfer state. The absence of ameta‐NMe₂group twisting coordinate upon electronic excitation was corroborated by experiments on control samples without themeta‐NMe₂group or with bothmeta‐NMe₂andpara‐OH groups locked in a six‐membered ring. These deep mechanistic insights stemming from GFP chromophore scaffold will enable rational design of organic, compact and environmentally friendly water sensors.

 

Fluorescence‐activating proteins (FAPs) that bind a chromophore and activate its fluorescence have gained popularity in bioimaging. The fluorescence‐activating and absorption‐shifting tag (FAST) is a light‐weight FAP that enables fast reversible fluorogen binding, thus advancing multiplex and super‐resolution imaging. However, the rational design of FAST‐specific fluorogens with large fluorescence enhancement (FE) remains challenging. Herein, a new fluorogen directly engineered from green fluorescent protein (GFP) chromophore by a unique double‐donor‐one‐acceptor strategy, which exhibits an over 550‐fold FE upon FAST binding and a high extinction coefficient of approximately 100,000 M⁻¹ cm⁻¹, is reported. Correlation analysis of the excited state nonradiative decay rates and environmental factors reveal that the large FE is caused by nonpolar protein−fluorogen interactions. Our deep insights into structure‐function relationships could guide the rational design of bright fluorogens for live‐cell imaging with extended spectral properties such as redder emissions.