A Combination of Library Screening and Rational Mutagenesis Expands the Available Color Palette of the Smallest Fluorogen-Activating Protein Tag nanoFAST.

NanoFAST is the smallest fluorogen-activating protein, consisting of only 98 amino acids, used as a genetically encoded fluorescent tag. Previously, only a single fluorogen with an orange color was revealed for this protein. In the present paper, using rational mutagenesis and in vitro screening of fluorogens libraries, we expanded the color palette of this tag. We discovered that E46Q is one of the key substitutions enabling the range of possible fluorogens to be expanded. The introduction of this and several other substitutions has made it possible to use not only orange but also red and green fluorogens with the modified protein.

NanoLuc Luciferase as a Fluorogen-Activating Protein for GFP Chromophore Based Fluorogens.

In this work, we showed that the well-known NanoLuc luciferase can act as a fluorogen activating protein for various arylidene-imidazolones structurally similar to the Kaede protein chromophore. We showed that such compounds can be used as fluorescent sensors for this protein and can also be used in pairs with it in fluorescent microscopy as a genetically encoded tag.

Meta-CF3-Substituted Analogues of the GFP Chromophore with Remarkable Solvatochromism.

In this work, we have shown that the introduction of a trifluoromethyl group into the me-ta-position of arylidene imidazolones (GFP chromophore core) leads to a dramatic increase in their fluorescence in nonpolar and aprotic media. The presence of a pronounced solvent-dependent gradation of fluorescence intensity makes it possible to use these substances as fluorescent polarity sensors. In particular, we showed that one of the created compounds could be used for selective labeling of the endoplasmic reticulum of living cells.

Conjugated Dienoic Acid Peroxides as Substrates in Chaetopterus Bioluminescence System.

Biochemistry of bioluminescence of the marine parchment tubeworm Chaetopterus has been in research focus for over a century; however, the results obtained by various groups contradict each other. Here, we report the isolation and structural elucidation of three compounds from Chaetomorpha linum algae, which demonstrate bioluminescence activity with Chaetopterus luciferase in the presence of Fe2+ ions. These compounds are derivatives of polyunsaturated fatty acid peroxides. We have also obtained their structural analogues and demonstrated their activity in the bioluminescence reaction, thus confirming the broad substrate specificity of the luciferase.

Color Tuning of Fluorogens for FAST Fluorogen-Activating Protein.

Using benzylidene imidazolone core, we created a panel of color-shifted fluorogenic ligands for FAST protein without compromise to the binding efficiency and the utility for live-cell protein labeling. This study highlights the potential of benzylidene imidazolones derivatives for rapid expansion of a pallet of live-cell fluorogenic labeling tools.

Developing Bright Green Fluorescent Protein (GFP)-like Fluorogens for Live-Cell Imaging with Nonpolar Protein-Chromophore Interactions.

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.

Shedding light on ultrafast ring-twisting pathways of halogenated GFP chromophores from the excited to ground state.

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 (EFC) or Stokes shift, and knrvs. EFC, 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.

Designing Red-Shifted Molecular Emitters Based on the Annulated Locked GFP Chromophore Derivatives.

Bioimaging techniques require development of a wide variety of fluorescent probes that absorb and emit red light. One way to shift absorption and emission of a chromophore to longer wavelengths is to modify its chemical structure by adding polycyclic aromatic hydrocarbon (PAH) fragments, thus increasing the conjugation length of a molecule while maintaining its rigidity. Here, we consider four novel classes of conformationally locked Green Fluorescent Protein (GFP) chromophore derivatives obtained by extending their aromatic systems in different directions. Using high-level ab initio quantum chemistry calculations, we show that the alteration of their electronic structure upon annulation may unexpectedly result in a drastic change of their fluorescent properties. A flip of optically bright and dark electronic states is most prominent in the symmetric fluorene-based derivative. The presence of a completely dark lowest-lying excited state is supported by the experimentally measured extremely low fluorescence quantum yield of the newly synthesized compound. Importantly, one of the asymmetric modes of annulation provides a very promising strategy for developing red-shifted molecular emitters with an absorption wavelength of ∼600 nm, having no significant impact on the character of the bright S-S1 transition.

Ultrafast excited-state proton transfer dynamics in dihalogenated non-fluorescent and fluorescent GFP chromophores.

Green fluorescent protein (GFP) has enabled a myriad of bioimaging advances due to its photophysical and photochemical properties. To deepen the mechanistic understanding of such light-induced processes, novel derivatives of GFP chromophore p-HBDI were engineered by fluorination or bromination of the phenolic moiety into superphotoacids, which efficiently undergo excited-state proton transfer (ESPT) in aqueous solution within the short lifetime of the excited state, as opposed to p-HBDI where efficient ESPT is not observed. In addition, we tuned the excited-state lifetime from picoseconds to nanoseconds by conformational locking of the p-HBDI backbone, essentially transforming the nonfluorescent chromophores into highly fluorescent ones. The unlocked superphotoacids undergo a barrierless ESPT without much solvent activity, whereas the locked counterparts exhibit two distinct solvent-involved ESPT pathways. Comparative analysis of femtosecond transient absorption spectra of these unlocked and locked superphotoacids reveals that the ESPT rates adopt an "inverted" kinetic behavior as the thermodynamic driving force increases upon locking the backbone. Further experimental and theoretical investigations are expected to shed more light on the interplay between the modified electronic structure (mainly by dihalogenation) and nuclear motions (by conformational locking) of the functionalized GFP derivatives (e.g., fluorescence on and off).

Short Duplex Module Coupled to G-Quadruplexes Increases Fluorescence of Synthetic GFP Chromophore Analogues.

Aptasensors became popular instruments in bioanalytical chemistry and molecular biology. To increase specificity, perspective signaling elements in aptasensors can be separated into a G-quadruplex (G4) part and a free fluorescent dye that lights up upon binding to the G4 part. However, current systems are limited by relatively low enhancement of fluorescence upon dye binding. Here, we added duplex modules to G4 structures, which supposedly cause the formation of a dye-binding cavity between two modules. Screening of multiple synthetic GFP chromophore analogues and variation of the duplex module resulted in the selection of dyes that light up after complex formation with two-module structures and their RNA analogues by up to 20 times compared to parent G4s. We demonstrated that the short duplex part in TBA25 is preferable for fluorescence light up in comparison to parent TBA15 molecule as well as TBA31 and TBA63 stabilized by longer duplexes. Duplex part of TBA25 may be partially unfolded and has reduced rigidity, which might facilitate optimal dye positioning in the joint between G4 and the duplex. We demonstrated dye enhancement after binding to modified TBA, LTR-III, and Tel23a G4 structures and propose that such architecture of short duplex-G4 signaling elements will enforce the development of improved aptasensors.

Red-Shifted Substrates for FAST Fluorogen-Activating Protein Based on the GFP-Like Chromophores.

A genetically encoded fluorescent tag for live cell microscopy is presented. This tag is composed of previously published fluorogen-activating protein FAST and a novel fluorogenic derivative of green fluorescent protein (GFP)-like chromophore with red fluorescence. The reversible binding of the novel fluorogen and FAST is accompanied by three orders of magnitude increase in red fluorescence (580-650 nm). The proposed dye instantly stains target cellular proteins fused with FAST, washes out in a minute timescale, and exhibits higher photostability of the fluorescence signal in confocal and widefield microscopy, in contrast with previously published fluorogen:FAST complexes.

Fluorescence lifetime multiplexing with fluorogen activating protein FAST variants.

In this paper, we propose a fluorescence-lifetime imaging microscopy (FLIM) multiplexing system based on the fluorogen-activating protein FAST. This genetically encoded fluorescent labeling platform employs FAST mutants that activate the same fluorogen but provide different fluorescence lifetimes for each specific protein-dye pair. All the proposed probes with varying lifetimes possess nearly identical and the smallest-in-class size, along with quite similar steady-state optical properties. In live mammalian cells, we target these chemogenetic tags to two intracellular structures simultaneously, where their fluorescence signals are clearly distinguished by FLIM. Due to the unique structure of certain fluorogens under study, their complexes with FAST mutants display a monophasic fluorescence decay, which may facilitate enhanced multiplexing efficiency by reducing signal cross-talks and providing optimal prerequisites for signal separation upon co-localized and/or spatially overlapped labeling.

Fluorescence Modulation of ortho-Green Fluorescent Protein Chromophores Following Ultrafast Proton Transfer in Solution.

Photophysical and photochemical properties of the green fluorescent protein (GFP) chromophore and derivatives underlie their bioimaging applications. To date, ultrafast spectroscopic tools represent the key for unraveling fluorescence mechanisms toward rational design of this powerful biomimetic framework. To correlate the excited-state intramolecular proton transfer (ESIPT) with chromophore emission properties, we implement experimental and computational tool sets to elucidate real-time electronic and structural dynamics of two archetypal ortho-GFP chromophores (o-HBDI and o-LHBDI) possessing an intramolecular hydrogen bond to undergo efficient ESIPT, only differing in a bridge-bond constraint. Using excited-state femtosecond stimulated Raman spectroscopy (FSRS), a low-frequency phenolic (P)-ring-deformation mode (∼562 cm-1) was uncovered to accompany ESIPT. The tautomerized chromophore undergoes either rapid P-ring isomerization to reach the ground state with essentially no fluorescence for o-HBDI or enhanced (up to an impressive 180-fold in acetonitrile) and solvent-polarity-dependent fluorescence by P-ring locking in o-LHBDI. The significant dependence of the fluorescence enhancement ratio on solvent viscosity confirms P-ring isomerization as the dominant nonradiative decay pathway for o-HBDI. This work provides crucial insights into the dynamic solute-solvent electrostatic and steric interactions, enabling the application-specific improvement of ESIPT-capable molecules as versatile fluorescence-based sensors and imaging agents from large Stokes shift emission to brighter probes in physiological environments.

Excited-State Dynamics of a meta-Dimethylamino Locked GFP Chromophore as a Fluorescence Turn-on Water Sensor.

Strategic incorporation of a meta-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 of m-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 H2 O and ROH emissions in various H2 O/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 -NMe2 and -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 a meta-NMe2 group twisting coordinate upon electronic excitation was corroborated by experiments on control samples without the meta-NMe2 group or with both meta-NMe2 and para-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.

NanoFAST: structure-based design of a small fluorogen-activating protein with only 98 amino acids.

One of the essential characteristics of any tag used in bioscience and medical applications is its size. The larger the label, the more it may affect the studied object, and the more it may distort its behavior. In this paper, using NMR spectroscopy and X-ray crystallography, we have studied the structure of fluorogen-activating protein FAST both in the apo form and in complex with the fluorogen. We showed that significant change in the protein occurs upon interaction with the ligand. While the protein is completely ordered in the complex, its apo form is characterized by higher mobility and disordering of its N-terminus. We used structural information to design the shortened FAST (which we named nanoFAST) by truncating 26 N-terminal residues. Thus, we created the shortest genetically encoded tag among all known fluorescent and fluorogen-activating proteins, which is composed of only 98 amino acids.