Site-Specific Fragmentation of Green Fluorescent Protein Induced by Blue Light.

Green fluorescent protein (GFP) and related fluorescent proteins have multiple applications in cell biology, and elucidating their functions has been at the focus of biophysical research for about three decades. Fluorescent proteins can be bleached by intense irradiation, and a number of them undergo photoconversion. Rare cases have been reported where distant functional relatives of GFP exhibit UV-light-induced protein fragmentation. Here, we show that irreversible bleaching of two different variants of GFP (sfGFP, EGFP) with visible light is paralleled by successive backbone fragmentation of the protein. Mass spectrometry revealed that the site of fragmentation resides at the fluorophore, between residue positions 65 and 66.

Mechanism of Color and Photoacidity Tuning for the Protonated Green Fluorescent Protein Chromophore.

The neutral or A state of the green fluorescent protein (GFP) chromophore is a remarkable example of a photoacid naturally embedded in the protein environment and accounts for the large Stokes shift of GFP in response to near UV excitation. Its color tuning mechanism has been largely overlooked, as it is less preferred for imaging applications than the redder anionic or B state. Past studies, based on site-directed mutagenesis or solvatochromism of the isolated chromophore, have concluded that its color tuning range is much narrower than its anionic counterpart. However, as we performed extensive investigation on more GFP mutants, we found that the color of the neutral chromophore can be more sensitive to protein electrostatics than can the anionic counterpart. Electronic Stark spectroscopy reveals a fundamentally different electrostatic color tuning mechanism for the neutral state of the chromophore that demands a three-form model as compared to that of the anionic state, which requires only two forms ( J. Am. Chem. Soc. 2019, 141, 15250-15265). Specifically, an underlying zwitterionic charge-transfer state is required to explain its sensitivity to electrostatics. As the Stokes shift is tightly linked to excited-state proton transfer (ESPT) of the protonated chromophore, we infer design principles of the GFP chromophore as a photoacid through the color tuning mechanisms of both protonation states. The three-form model could also be applied to similar biological and nonbiological dyes and complements the failure of the two-form model for donor-acceptor systems with localized ground-state electronic distributions.

Dramatic changes in the excited-state behaviour of the green fluorescent protein chromophore by a strong π-donating group through significantly lowering the excited-state potential energy surface with photoinduced intramolecular charge transfer.

A strong π-donating group like p-NMe2 dramatically changes the excited-state behavior of the green fluorescent protein (GFP) chromophore, such as realizing charge-transfer absorption and executing significant photoinduced intramolecular charge transfer (ICT), which makes a planar first singlet (S1) excited-state minimum disappear and significantly lowers the S1 excited-state potential energy surface (PES), leading to barrierless τ-torsion and φ-torsion excited-state relaxation and eliciting the φ-torsion S1 excited-state minimum. This finding is critical since a strong π-donating group like p-NMe2 may do the same things to other fluorophores.

Influence of the First Chromophore-Forming Residue on Photobleaching and Oxidative Photoconversion of EGFP and EYFP.

Enhanced green fluorescent protein (EGFP)-one of the most widely applied genetically encoded fluorescent probes-carries the threonine-tyrosine-glycine (TYG) chromophore. EGFP efficiently undergoes green-to-red oxidative photoconversion ("redding") with electron acceptors. Enhanced yellow fluorescent protein (EYFP), a close EGFP homologue (five amino acid substitutions), has a glycine-tyrosine-glycine (GYG) chromophore and is much less susceptible to redding, requiring halide ions in addition to the oxidants. In this contribution we aim to clarify the role of the first chromophore-forming amino acid in photoinduced behavior of these fluorescent proteins. To that end, we compared photobleaching and redding kinetics of EGFP, EYFP, and their mutants with reciprocally substituted chromophore residues, EGFP-T65G and EYFP-G65T. Measurements showed that T65G mutation significantly increases EGFP photostability and inhibits its excited-state oxidation efficiency. Remarkably, while EYFP-G65T demonstrated highly increased spectral sensitivity to chloride, it is also able to undergo redding chloride-independently. Atomistic calculations reveal that the GYG chromophore has an increased flexibility, which facilitates radiationless relaxation leading to the reduced fluorescence quantum yield in the T65G mutant. The GYG chromophore also has larger oscillator strength as compared to TYG, which leads to a shorter radiative lifetime (i.e., a faster rate of fluorescence). The faster fluorescence rate partially compensates for the loss of quantum efficiency due to radiationless relaxation. The shorter excited-state lifetime of the GYG chromophore is responsible for its increased photostability and resistance to redding. In EYFP and EYFP-G65T, the chromophore is stabilized by π-stacking with Tyr203, which suppresses its twisting motions relative to EGFP.

Why does a para-amino group make the green fluorescent protein chromophore non-fluorescent: coherent intramolecular charge transfer reduces the Z/E-photoisomerization barrier.

A para-N-phenyl-amino group significantly increases the fluorescence quantum yield of N-phenyl-4-aminostilbene by the "amino conjugation effect", but, in contrast, a para-amino group in the para-amino analogue (p-ABDI) of the green fluorescent protein (GFP) chromophore makes p-ABDI non-fluorescent because the coherent photo-induced intramolecular charge transfer reduces the barrier of the Z/E-photoisomerization.

A critical comparison of lanthanide based upconversion nanoparticles to fluorescent proteins, semiconductor quantum dots, and carbon dots for use in optical sensing and imaging.

The right choice of a fluorescent probe is essential for successful luminescence imaging and sensing and especially concerning in vivo and in vitro applications, the development of new classes have gained more and more attention in the last years. One of the most promising class are upconversion nanoparticles (UCNPs)-inorganic nanocrystals capable to convert near-infrared light in high energy radiation. In this review we will compare UCNPs with other fluorescent probes in terms of (a) the optical properties of the probes, such as their brightness, photostability and excitation wavelength; (b) their chemical properties such as the dispersibility, stability under experimental or physiological conditions, availability of chemical modification strategies for labelling; and (c) the potential toxicity and biocompatibility of the probe. Thereby we want to provide a better understanding of the advantages and drawbacks of UCNPs and address future challenges in the design of the nanocrystals.

Ultrafast internal conversion dynamics of bilirubin bound to UnaG and its N57A mutant.

Fluorescent proteins (FPs) have become fundamental tools for live cell imaging. Most FPs currently used are members of the green fluorescent protein super-family, but new fluorophores such as bilin-FPs are being developed and optimized. In particular, the UnaG FP incorporates bilirubin (BR) as a chromophore, enhancing its fluorescence quantum yield by three orders of magnitude relative to that in solution. To investigate the mechanism of this dramatic enhancement and provide a basis for further engineering of UnaG and other tetrapyrrole-based fluorophores, we performed picosecond fluorescence and femtosecond transient absorption measurements of BR bound to UnaG and its N57A site-directed mutant. The dynamics of wt-UnaG, which has a fluorescence QY of 0.51, are largely homogeneous, showing an excited state relaxation of ∼200 ps, and a 2.2 ns excited-state lifetime decay with a kinetic isotope effect (KIE) of 1.1 for D2O vs. H2O buffer. In contrast, for UnaG N57A (fluorescence QY 0.01) the results show a large spectral inhomogeneity with excited state decay timescales of 47 and 200 ps and a KIE of 1.4. The non-radiative deactivation of the excited state is limited by proton transfer. The loss of direct hydrogen bonds to the endo-vinyl dipyrrinone moiety of BR leads to high flexibility and structural heterogeneity of UnaG N57A, as seen in the X-ray crystal structure.

Lighting Up Live Cells with Smart Genetically Encoded Fluorescence Probes from GMars Family.

As a special kind of delicate light-controllable genetically encoded optical device, reversibly photoswitchable fluorescent proteins (RSFPs) have been widely applied in many fields, especially various kinds of advanced nanoscopy approaches in recent years. However, there are still necessities for exploring novel RSFPs with specific biochemical or photophysical properties not only for bioimaging or biosensing applications but also for fluorescent protein (FP) mechanisms study and further knowledge-based molecular sensors or optical actuators' rational design and evolution. Besides previously reported GMars-Q and GMars-T variants, herein, we reported the development and applications of other RSFPs from GMars family, especially some featured RSFPs with desired optical properties. In the current work, in vitro FP purification, spectra measurements, and live-cell RESOLFT nanoscopy approaches were applied to characterize the basic properties and test the imaging performances of the selected RSFPs. As demonstrated, GMars variants such as GMars-A, GMars-G, or remarkable photofatigue-resistant GMars-L were found with beneficial properties to be capable of parallelized RESOLFT nanoscopy in living cells, while other featured GMars variants such as dark GMars-P may be a good candidate for further biosensor or actuator design and applications.

Optogenetic precision toolkit to reveal form, function and connectivity of single neurons.

All-optical methods enable the control and monitoring of neuronal activity with minimal perturbation of the system. Although imaging and optogenetic manipulations can be performed at cellular resolution, the morphology of single cells in a dense neuronal population has often remained unresolvable. Here we describe in detail two recently established optogenetic protocols for systematic description of function and morphology of single neurons in zebrafish. First, the Optobow toolbox allows unbiased mapping of excitatory functional connectivity. Second, the FuGIMA technique enables selective labeling and anatomical tracing of neurons that are responsive to a given sensory stimulus or correlated with a specific behavior. Both strategies can be genetically targeted to a neuronal population of choice using the Gal4/UAS system. As these in vivo approaches are non-invasive, we envision useful applications for the study of neuronal structure, function and connectivity during development and behavior.

Ultrafast trans-cis photoisomerization of the neutral chromophore in green fluorescent proteins: Surface-hopping dynamics simulation.

Reversible photoswitching fluorescent protein can reversibly switch between on-state (fluorescent) and off-state (dark). Anionic cis and neutral trans chromophores are the on- and off-states in green fluorescent proteins (GFPs), respectively. We investigated the ultrafast trans-cis photoisomerization mechanisms of the neutral GFP chromophore upon excitation to the S1 state by means of surface-hopping dynamics simulations based on the Zhu-Nakamura theory. Two trans isomers, located in the S0 state, were taken into consideration in dynamics simulation. After these two trans isomers are excited to the S1 state, the molecule moves to a excited-state minimum by increasing the imidazolinone-bridge bond length and decreasing the phenol-bridge bond length. The twist of imidazolinone-bridge bond drives the molecule toward a conical intersection, and internal conversion occurs. Then, a cis or trans conformer will be obtained in the S0 state. The torsion around the imidazolinone-bridge bond plays a key role in the ultrafast photoisomerization of a neutral chromophore. The torsional motion around the phenol-bridge bond is restricted in the S1 state, while it may occur in the S0 state. The isomerization reaction of this molecule is predicted to be not sensitive to solvent viscosity, and time-dependent density functional theory (TDDFT) calculations indicate that the fast excited-state decay from the Franck-Condon region of the trans isomer to the excited-state minimum was almost independent of solvent polarity.

Monitoring Microtubule Dynamics in the Mouse Egg Using Photoactivatable-GFP-Tubulin.

Fluorescence photoactivation provides a strategy for monitoring protein kinetics within living cells. In particular, fluorescence photoactivation of a subpopulation of microtubule subunits within the spindle using photoactivatable fluorescent tubulin constructs has proven useful for assessing a variety of features of spindle microtubule dynamics, including poleward microtubule movement, microtubule depolymerization, and microtubule turnover, in various cellular settings. The current chapter describes a method for monitoring microtubule dynamics within the mouse egg spindle by photoactivation of photoactivatable-GFP-tubulin, followed by time-lapse confocal imaging.

Photoactivation of Actin in Mouse Oocyte.

Development of fluorescence distribution assays like FRAP (fluorescence recovery after photobleaching) or photoactivation has had a great impact in studying intracellular protein dynamics. In particular, the cytoskeleton field largely benefited from these techniques, with lots of new information provided about the dynamics and organization of actin networks whithin cells.In mouse oocyte, actin photoactivation has been very useful to determine the dynamics of different actin structures involved in meiotic divisions, including a cytoplasmic meshwork and a subcortical actin layer.Here, we describe a method, actin photoactivation, to determine the dynamics of the actin cytoplasmic meshwork and the subcortical actin layer during the first meiotic division in the mouse oocyte, that could be adapted to other actin structures or other stages of meiotic divisions.

Photoinduced proton transfer inside an engineered green fluorescent protein: a stepwise-concerted-hybrid reaction.

Photoactivated proton transfer (PT) wire is responsible for the glow of green fluorescent protein (GFP), which is crucial for bioimaging and biomedicine. In this work, a new GFP-S65T/S205V double mutant is developed from wild-type GFP in which the PT wire is significantly modified. We implement femtosecond transient absorption (fs-TA) and femtosecond stimulated Raman spectroscopy (FSRS) to delineate the PT process in action. The excited state proton transfer proceeds on the ∼110 ps timescale, which infers that the distance of one key link (water to T203) in the PT wire of GFP-S205V is shortened by the extra S65T mutation. The rise of an imidazolinone ring deformation mode at ∼871 cm-1 in FSRS further suggests that this PT reaction is in a concerted manner. A ∼4 ps component prior to large-scale proton dissociation through the PT wire is also retrieved, indicative of some small-scale proton motions and heavy-atom rearrangement in the vicinity of the chromophore. Our work provides deep insights into the novel hybrid PT mechanism in engineered GFP and demonstrates the power of tunable FSRS methodology in tracking ultrafast photoreactions with the desirable structural specificity in physiological environments.

Time-resolved stimulated emission depletion and energy transfer dynamics in two-photon excited EGFP.

Time and polarization-resolved stimulated emission depletion (STED) measurements are used to investigate excited state evolution following the two-photon excitation of enhanced green fluorescent protein (EGFP). We employ a new approach for the accurate STED measurement of the hitherto unmeasured degree of hexadecapolar transition dipole moment alignment α40 present at a given excitation-depletion (pump-dump) pulse separation. Time-resolved polarized fluorescence measurements as a function of pump-dump delay reveal the time evolution of α40 to be considerably more rapid than predicted for isotropic rotational diffusion in EGFP. Additional depolarization by homo-Förster resonance energy transfer is investigated for both α20 (quadrupolar) and α40 transition dipole alignments. These results point to the utility of higher order dipole correlation measurements in the investigation of resonance energy transfer processes.

Polarized two-photon photoselection in EGFP: Theory and experiment.

In this work, we present a complete theoretical description of the excited state order created by two-photon photoselection from an isotropic ground state; this encompasses both the conventionally measured quadrupolar (K = 2) and the "hidden" degree of hexadecapolar (K = 4) transition dipole alignment, their dependence on the two-photon transition tensor and emission transition dipole moment orientation. Linearly and circularly polarized two-photon absorption (TPA) and time-resolved single- and two-photon fluorescence anisotropy measurements are used to determine the structure of the transition tensor in the deprotonated form of enhanced green fluorescent protein. For excitation wavelengths between 800 nm and 900 nm, TPA is best described by a single element, almost completely diagonal, two-dimensional (planar) transition tensor whose principal axis is collinear to that of the single-photon S0 → S1 transition moment. These observations are in accordance with assignments of the near-infrared two-photon absorption band in fluorescent proteins to a vibronically enhanced S0 → S1 transition.

Ultrafast action chemistry in slow motion: atomistic description of the excitation and fluorescence processes in an archetypal fluorescent protein.

We report quantum mechanical/molecular mechanical non-adiabatic molecular dynamics simulations on the electronically excited state of green fluorescent protein mutant S65T/H148D. We examine the driving force of the ultrafast (τ < 50 fs) excited-state proton transfer unleashed by absorption in the A band at 415 nm and propose an atomistic description of the two dynamical regimes experimentally observed [Stoner Ma et al., J. Am. Chem. Soc., 2008, 130, 1227]. These regimes are explained in terms of two sets of successive dynamical events: first the proton transfers quickly from the chromophore to the acceptor Asp148. Thereafter, on a slower time scale, there are geometrical changes in the cavity of the chromophore that involve the distance between the chromophore and Asp148, the planarity of the excited-state chromophore, and the distance between the chromophore and Tyr145. We find two different non-radiative relaxation channels that are operative for structures in the reactant region and that can explain the mismatch between the decay of the emission of A* and the rise of the emission of I*, as well as the temperature dependence of the non-radiative decay rate.

A Single-Chain Photoswitchable CRISPR-Cas9 Architecture for Light-Inducible Gene Editing and Transcription.

Optical control of CRISPR-Cas9-derived proteins would be useful for restricting gene editing or transcriptional regulation to desired times and places. Optical control of Cas9 functions has been achieved with photouncageable unnatural amino acids or by using light-induced protein interactions to reconstitute Cas9-mediated functions from two polypeptides. However, these methods have only been applied to one Cas9 species and have not been used for optical control of different perturbations at two genes. Here, we use photodissociable dimeric fluorescent protein domains to engineer single-chain photoswitchable Cas9 (ps-Cas9) proteins in which the DNA-binding cleft is occluded at baseline and opened upon illumination. This design successfully controlled different species and functional variants of Cas9, mediated transcriptional activation more robustly than previous optogenetic methods, and enabled light-induced transcription of one gene and editing of another in the same cells. Thus, a single-chain photoswitchable architecture provides a general method to control a variety of Cas9-mediated functions.

Structural Insight into the Photochemistry of Split Green Fluorescent Proteins: A Unique Role for a His-Tag.

Oligohistidine affinity tags (His-tags) are commonly fused to proteins to aid in their purification via metal affinity chromatography. These His-tags are generally assumed to have minimal impact on the properties of the fusion protein, as they have no propensity to form ordered elements, and are small enough not to significantly affect the solubility or size. Here we report structures of two variants of truncated green fluorescent protein (GFP), i.e., split GFP with a ß-strand removed, that were found to behave differently in the presence of light. In these structures, the N-terminal His-tag and several neighboring residues play a highly unusual structural and functional role in stabilizing the truncated GFP by substituting as a surrogate ß-strand in the groove vacated by the native strand. This finding provides an explanation for the seemingly very different peptide binding and photodissociation properties of split proteins involving ß-strands 10 and 11. We show that these truncated GFPs can bind other non-native sequences, and this promiscuity invites the possibility for rational design of sequences optimized for strand binding and photodissociation, both useful for optogenetic applications.

Fluorescent cell-selective ablation using an adaptive photodynamic method.

Intravital ablation of particular cell populations is necessary to decipher their roles under spatiotemporal conditions. Energy transfer-based photodynamic therapy presented a conditional range for specifically inducing the death of GFP expressing cells, with little effect on normal cells. This novel system enables easy access to the functional study of cells.

Chimeric Autofluorescent Proteins as Photophysical Model System for Multicolor Bimolecular Fluorescence Complementation.

The yellow fluorescent protein (YFP) is frequently used in a protein complementation assay called bimolecular fluorescence complementation (BiFC), and is employed to visualize protein-protein interactions. In this analysis, two different, nonfluorescent fragments of YFP are genetically attached to proteins of interest. Upon interaction of these proteins, the YFP fragments are brought into proximity close enough to reconstitute their original structure, enabling fluorescence. BiFC allows for a straightforward readout of protein-protein interactions and furthermore facilitates their functional investigation by in vivo imaging. Furthermore, it has been observed that the available color range in BiFC can be extended upon complementing fragments of different proteins that are, like YFP, derived from the Aequorea victoria green fluorescent protein, thereby allowing for a multiplexed investigation of protein-protein interactions. Some spectral characteristics of "multicolor" BiFC (mcBiFC) complexes have been reported before; however, no in-depth analysis has been performed yet. Therefore, little is known about the photophysical characteristics of these mcBiFC complexes because a proper characterization essentially relies on in vitro data. This is particularly difficult for fragments of autofluorescent proteins (AFPs) because they show a very strong tendency to form supramolecular aggregates which precipitate ex vivo. In this study, this intrinsic difficulty is overcome by directly fusing the coding DNA of different AFP fragments. Translation of the genetic sequence in Escherichia coli leads to fully functional, highly soluble fluorescent proteins with distinct properties. On the basis of their construction, they are designated chimeric AFPs, or BiFC chimeras, here. Comparison of their spectral characteristics with experimental in vivo BiFC data confirmed the utility of the chimeric proteins as a BiFC model system. In this study, nine different chimeras were thoroughly analyzed at both the ensemble and the single-molecular level. The data indicates that mutations believed to be photophysically silent significantly alter the properties of AFPs.