The molecular mechanisms for the photoconversion of fluorescent proteins remain elusive owing to the challenges of monitoring chromophore structural dynamics during the light-induced processes.W ei mplemented time-resolved electronic and stimulated Raman spectroscopies to reveal two hidden species of an engineered ancestral GFP-like protein LEA, involving semi-trapped protonated and trapped deprotonated chromophores en route to photoconversion in pH 7.9 buffer.Anew dual-illumination approach was examined, using 400 and 505 nm light simultaneously to achieve faster conversion and higher color contrast. Substitution of UV irradiation with visible light benefits bioimaging,w hile the spectral benchmark of atrapped chromophore with characteristic ring twisting and bridge-H bending motions enables rational design of functional proteins.With the improved H-bonding network and structural motions,t he photoexcited chromophore could increase the photoswitching-aided photoconversion while reducing trapped species.Supportinginformation and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.
Organics emiconductor materials have recently gained momentum due to their non-toxicity,l ow cost, and sustainability.X ylindein is ar emarkably photostable pigment secreted by fungi that grow on decaying wood, and its relativelys trong electronic performance is enabled by p-p stacking and hydrogen-bonding network that promote charget ransport. Herein, femtosecondt ransienta bsorption spectroscopy with an ear-IRp robe was used to unveil ar apid excited-state intramolecular protont ransfer reaction. Conformational motions potentially lead to a conical intersection that quenches fluorescence in the monomeric state. In concentrated solutions, nascent aggregates exhibit af aster excited state lifetime due to excimer formation, confirmed by the excimer!charge-transfer excited-state absorption band of the xylindein thin film, thus limiting its optoelectronic performance. Therefore, extending the xylindein sidechains with branched alkyl groups mayh indert he excimer formation and improve optoelectronic properties of naturally derived materials.
Green-to-red photoconvertible fluorescent proteins (FPs) are vital biomimetic tools for powerful techniques such as super-resolution imaging. A unique Kaede-type FP named the least evolved ancestor (LEA) enables delineation of the evolutionary step to acquire photoconversion capability from the ancestral green fluorescent protein (GFP). A key residue, Ala69, was identified through several steady-state and time-resolved spectroscopic techniques that allows LEA to effectively photoswitch and enhance the green-to-red photoconversion.However, the inner workings of this functional protein have remained elusive due to practical challenges of capturing the photoexcited chromophore motions in real time. Here, we implemented femtosecond stimulated Raman spectroscopy and transient absorption on LEA-A69T, aided by relevant crystal structures and control FPs, revealing that Thr69 promotes a stronger π-π stacking interaction between the chromophore phenolate (P-)ring and His193 in FP mutants that cannot photoconvert or photoswitch. Characteristic time constants of $60-67 ps are attributed to P-ring twist as the onset for photoswitching in LEA (major) and LEA-A69T (minor) with photoconversion capability, different from $16/29 ps in correlation with the Gln62/His62 side-chain twist in ALL-GFP/ALL-Q62H, indicative of the light-induced conformational relaxation preferences in various local environments. A minor subpopulation of LEA-A69T capable of positive photoswitching was revealed by time-resolved electronic spectroscopies with targeted light irradiation wavelengths. The unveiled chromophore structure and dynamics inside engineered FPs in an aqueous buffer solution can be generalized to improve other green-to-red photoconvertible FPs from the bottom up for deeper biophysics with molecular biology insights and powerful bioimaging advances. K E Y W O R D Schromophore ring twist, excited state dynamics, femtosecond stimulated Raman spectroscopy, green-to-red photoconversion, photoswitchable fluorescent proteins, protein rational design, transient structural motions
Organic semiconductors have attracted increasing attention due to their low cost, solution processability, and tunable properties. Of special interest are molecules with enhanced environmental stability. We have recently reported on the (opto)electronic properties of a remarkably stable, naturally derived pigment xylindein. Here, we establish that one particular aspect of xylindein's molecular structure, namely the presence of hydroxyl (OH) groups, is critical for enabling its enhanced stability and relatively high electron mobility. In particular, we synthesized a methylated derivative of xylindein, dimethylxylindein, where the OH groups are replaced with OCH 3 groups, and compared photophysics and the (opto)electronic properties of dimethylxylindein and xylindein. We reveal the presence of a long-lived excited state in dimethylxylindein, in contrast to xylindein, which has an efficient fast nonradiative pathway to the ground state. This results in significantly reduced photostability of dimethylxylindein as compared to xylindein. The effective electron mobility, obtained from space-charge-limited currents, in amorphous xylindein films was found to be 4 orders of magnitude higher than that in amorphous and crystalline dimethylxylindein films. In contrast, the photosensitivity of dimethylxylindein is about 2 orders of magnitude higher than that of xylindein. The mechanism of charge transport in all films was thermally activated hopping, with the xylindein films characterized by considerably shallower charge traps than dimethylxylindein films, attributed to hydrogen bonding via hydroxyl groups promoting an efficient conductive network in xylindein.
Methylation occurs in a myriad of systems with protective and regulatory functions. 8-methoxypyrene-1,3,6-trisulfonate (MPTS), a methoxy derivative of a photoacid, serves as a model system to study effects of methylation on the excited state potential energy landscape. A suite of spectroscopic techniques including transient absorption, wavelength-tunable femtosecond stimulated Raman spectroscopy (FSRS), and fluorescence quantum yield measurements via steady-state electronic spectroscopy reveal the energy dissipation pathways of MPTS following photoexcitation. Various solvents enable a systematic characterization of the H-bonding interaction, viscosity, and dynamic solvation that influence the ensuing relaxation pathways. The formation of a charge-transfer state out of the Franck–Condon region occurs on the femtosecond-to-picosecond solvation timescale before encountering a rotational barrier. The rotational relaxation correlates with the H-bond donating strength of solvent, while the rotational time constant lengthens as solvent viscosity increases. Time-resolved excited-state FSRS, aided by quantum calculations, provides crucial structural dynamics knowledge and reveals the sulfonate groups playing a dominant role during solvation. Several prominent vibrational motions of the pyrene ring backbone help maneuver the population toward the more fluorescent state. These ultrafast correlated electronic and nuclear motions ultimately govern the fate of the photoexcited chromophore in solution. Overall, MPTS in water displays the highest probability to fluoresce, while the aprotic and more viscous dimethyl sulfoxide enhances the nonradiative pathways. These mechanistic insights may apply robustly to other photoexcited chromophores that do not undergo excited-state proton transfer or remain trapped in a broad electronic state and also provide design principles to control molecular optical responses with site-specific atomic substitution.
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