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Genetically encoded biosensors based on Förster resonance energy transfer (FRET) are indispensable tools for monitoring biochemical changes in cells. Green and red fluorescent protein-based FRET pairs offer advantages over the classically employed cyan and yellow fluorescent protein pairs, such as better spectral separation, lower phototoxicity, and less autofluorescence. Here, we describe the development of an mScarlet-derived green fluorescent protein (designated as mWatermelon) and its use as a FRET donor to the red fluorescent protein mScarlet-I as a FRET acceptor. We tested the functionality of this FRET pair by engineering biosensors for the detection of protease activity, Ca 2+ , and K + . Furthermore, we described a strategy to enhance the FRET efficiency of these biosensors by modulating the intramolecular association between mWatermelon and mScarlet-I.

Fluorescent proteins (FPs) are indispensable tools for noninvasive bioimaging and sensing. Measuring the free cellular calcium (Ca 2+ ) concentrations in vivo with genetically encodable FPs can be a relatively direct measure of neuronal activity due to the complex signaling role of these ions. REX-GECO1 is a recently developed red-green emission and excitation ratiometric FP-based biosensor that achieves a high dynamic range due to differences in the chromophore response to light excitation with and without calcium ions. Using steady-state electronic measurements (UV/Visible absorption and emission), along with time-resolved spectroscopic techniques including femtosecond transient absorption (fs-TA) and femtosecond stimulated Raman spectroscopy (FSRS), the potential energy surfaces of these unique biosensors are unveiled with vivid details. The ground-state structural characterization of the Ca 2+ -free biosensor via FSRS reveals a more spacious protein pocket that allows the chromophore to efficiently twist and reach a dark state. In contrast, the more compressed cavity within the Ca 2+ -bound biosensor results in a more heterogeneous distribution of chromophore populations that results in multi-step excited state proton transfer (ESPT) pathways on the sub-140 fs, 600 fs, and 3 ps timescales. These results enable rational design strategies to enlarge the spectral separation between the protonated/deprotonated forms and the Stokes shift leading to a larger dynamic range and potentially higher fluorescence quantum yield, which should be broadly applicable to the calcium imaging and biosensor communities.

Large Stokes shift (LSS) red fluorescent proteins (RFPs) are highly desirable for bioimaging advances. The RFP mKeima, with coexisting cis- and trans-isomers, holds significance as an archetypal system for LSS emission due to excited-state proton transfer (ESPT), yet the mechanisms remain elusive. We implemented femtosecond stimulated Raman spectroscopy (FSRS) and various time-resolved electronic spectroscopies, aided by quantum calculations, to dissect the cis- and trans-mKeima photocycle from ESPT, isomerization, to ground-state proton transfer in solution. This work manifests the power of FSRS with global analysis to resolve Raman fingerprints of intermediate states. Importantly, the deprotonated trans-isomer governs LSS emission at 620 nm, while the deprotonated cis-isomer's 520 nm emission is weak due to an ultrafast cis-to-trans isomerization. Complementary spectroscopic techniques as a table-top toolset are thus essential to study photochemistry in physiological environments., (© 2022 Wiley-VCH GmbH.)

We report the results of a computational study of the mechanism of the light-induced chemical reaction of chromophore hydration in the fluorescent protein Dreiklang, responsible for its switching from the fluorescent ON-state to the dark OFF-state. We explore the relief of the charge-transfer excited-state potential energy surface in the ON-state to locate minimum energy conical intersection points with the ground-state energy surface. Simulations of the further evolution of model systems allow us to characterize the ground-state reaction intermediate tentatively suggested in the femtosecond studies of the light-induced dynamics in Dreiklang and finally to arrive at the reaction product. The obtained results clarify the details of the photoswitching mechanism in Dreiklang, which is governed by the chemical modification of its chromophore.

To date, fluorescent protein biosensors are widely used in research. In vivo, they can be applied to dynamically monitor several physiological parameters in various subcellular compartments. Redox-sensitive green fluorescent protein 2 (roGFP2) senses the glutathione redox potential via a disulfide bridge formed between neighboring beta-strands of its beta-barrel structure. As changes in redox state affect both excitation maxima of roGFP2 oppositely, sensor responses are ratiometric. The reaction mechanism of roGFP2 is well characterized and involves an intermediate S-glutathionylation step. Thus, roGFP2 is also used in enzymatic in vitro assays, e.g., assessing glutaredoxin kinetics. In addition to the fluorescent read-out, the roGFP2 redox state can also be determined by differential migration on a non-reducing SDS-PAGE. This read-out mode may be beneficial in some applications, e.g., if mass-spectrometric analysis of posttranslational cysteine modifications is desired. Here, we describe a protocol for gel-based fluorescent read-out of the roGFP2 redox state, as well as modification of free cysteines by maleimide-based reagents., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2022 The Protein Society.)

The Green Fluorescent Protein (GFP) and its analogues have been widely used as fluorescent biomarkers in cell biology. Yet, the chromophore responsible for the fluorescence of the GFP is not emissive when isolated in solution, outside the protein environment. The most accepted explanation is that the quenching of the fluorescence results from the rotation of the aryl-alkene bond and from the Z/E isomerization. Over the years, many efforts have been performed to block these torsional rotations, mimicking the environment inside the protein β-barrel, to restore the emission intensity. Molecule rigidification through chemical modifications or complexation, or through crystallization, is one of the strategies used. This review presents an overview of the strategies developed to achieve highly emissive GFP chromophore by hindering the torsional rotations.

Photoswitchable fluorescent proteins (FPs) have become indispensable tools for studying life sciences. mSAASoti FP, a biphotochromic FP, is an important representative of this protein family. We created a series of mSAASoti mutants in order to obtain fast photoswitchable variants with high brightness. K145P mSAASoti has the highest molar extinction coefficient of all SAASoti mutants studied; C21N/K145P/M163A switches to the dark state 36 times faster than mSAASoti, but it lost its ability to undergo green-to-red photoconversion. Finally, the C21N/K145P/F177S and C21N/K145P/M163A/F177S variants demonstrated a high photoswitching rate between both green and red forms.

The potential of nucleic acid therapeutics to treat diseases by targeting specific cells has resulted in its increasing number of uses in clinical settings. However, the major challenge is to deliver bio-macromolecules into target cells and/or subcellular locations of interest ahead in the development of delivery systems. Although, supercharged residues replaced protein 36 + GFP can facilitate itself and cargoes delivery, its efficiency is still limited. Therefore, we combined our recent progress to further improve 36 + GFP based delivery efficiency. We found that the penetration efficacy of 36 + GFP protein was significantly improved by fusion with CPP-Dot1l or treatment with penetration enhancer dimethyl sulfoxide (DMSO) in vitro . After safely packaged with plasmid DNA, we found that the efficacy of in vitro and in vivo transfection mediated by 36 + GFP-Dot1l fusion protein is also significantly improved than 36 + GFP itself. Our findings illustrated that fusion with CPP-Dot1l or incubation with DMSO is an alternative way to synergically promote 36 + GFP mediated plasmid DNA delivery in vitro and in vivo .

We report the different properties of two types of red fluorescent proteins (RFP), undescribed species, extracted from two octocorals, Scleronephthya sp. 1 (S. sp. 1) and S. sp, 2 (Alcyonacea, Nephtheidae). S. sp. 1, named Alc-Orange, emits strong green emission at 492 nm and weak red emission at 590 and 630 nm when excited at 449 and 574 nm, respectively. S. sp. 2, LS-Red, emits strong deep red at 642 nm and weak green at 480 and 510 nm when excited at 574 nm and 434 nm, respectively. LS-Red has a very large Stokes shift of about 208 nm emitting at 642 nm when excited at 434 nm. Interestingly, LS-Red shows some emissions at 480 (blue emission), 514 (green emission), 563 (orange emission), and 642 nm (deep red emission) continuously at pH 7.5, which means multicolored fluorescence protein by one excitation at 434 nm. In pH dependence of fluorescence of Alc-Orange (pH 13 to 3.5), no relation between 'green and red FPs' was observed, whereas LS-Red showed the interconversion between 'green and red forms' depending on pH (11.5 to 4.5)., (© 2022 John Wiley & Sons Ltd.)

We present a multifaceted approach to effectively probe complex local protein environments utilizing the vibrational reporter unnatural amino acid (UAA) 4-cyano-l-phenylalanine (pCNPhe) in the model system superfolder green fluorescent protein (sfGFP). This approach combines temperature-dependent infrared (IR) spectroscopy, X-ray crystallography, and molecular dynamics (MD) simulations to provide a molecular interpretation of the local environment of the nitrile group in the protein. Specifically, a two-step enantioselective synthesis was developed that provided an 87% overall yield of pCNPhe in high purity without the need for chromatography. It was then genetically incorporated individually at three unique sites (74, 133, and 149) in sfGFP to probe these local protein environments. The incorporation of the UAA site-specifically in sfGFP utilized an engineered, orthogonal tRNA synthetase in E. coli using the Amber codon suppression protocol, and the resulting UAA-containing sfGFP constructs were then explored with this approach. This methodology was effectively utilized to further probe the local environments of two surface sites (sites 133 and 149) that we previously explored with room temperature IR spectroscopy and X-ray crystallography and a new interior site (site 74) featuring a complex local environment around the nitrile group of pCNPhe. Site 133 was found to be solvent-exposed, while site 149 was partially buried. Site 74 was found to consist of three distinct local environments around the nitrile group including nonspecific van der Waals interactions, hydrogen-bonding to a structural water, and hydrogen-bonding to a histidine side chain.

The unique tripeptide structure of green fluorescent protein (GFP), a Ser-Tyr-Gly motif, generates the mature chromophore in situ to define the emission profiles of GFP. Here, we describe the rational design and discovery of a biomimetic fluorescent emitter, MBP, by mimicking the key structure of the Ser-Tyr-Gly motif. Through systematically tailoring the tripeptide, a family of four chromophores were engineered, while only MBP exhibited bright fluorescence in different fluid solvents with highly enhanced quantum yields. Distinct to previous hydrogen-bonding-induced fluorescence quenching of GFP chromophore analogues, the emission of MBP was only slightly decreased in protic solvents. Heteronuclear multiple bond correlation techniques demonstrated the fundamental mechanism for enhanced fluorescence emission owing to the synergy of the formation of the intramolecular hydrogen-bonding-ring structure and the self-restricted effect, which was further illustrated via theoretical calculations. This work puts forward an extraordinary approach toward highly emissive biomimicking fluorophores, which gives new insights into the emission mechanisms and photophysics of GFP-like chromophores.

The chromophore (CRO) of fluorescent proteins (FPs) is embedded in a complex environment that is a source of specific interactions with the CRO. Understanding how these interactions influence FPs spectral properties is important for a directed design of novel markers with desired characteristics. In this work, we apply computational chemistry methods to gain insight into one-, two- and three-photon absorption (1PA, 2PA, 3PA) tuning in enhanced green fluorescent protein (EGFP). To achieve this goal, we built EGFP models differing in: i) number and position of hydrogen-bonds (h-bonds) donors to the CRO and ii) the electric field, as approximated by polarizable force field, acting on the CRO. We find that h-bonding to the CRO's phenolate oxygen results in stronger one- and multiphoton absorption. The brighter absorption can be also achieved by creating more positive electric field near the CRO's phenolate moiety. Interestingly, while individual CRO - environment h-bonds usually enhance 1PA and 2PA, it takes a few h-bond donors to enhance 3PA. Clearly, response of the absorption intensity to many-body effects depends on the excitation mechanism. We further employ symmetry-adapted perturbation theory (SAPT) to reveal excellent (2PA) and good (3PA) correlation of multiphoton intensity with electrostatic and induction interaction energies. This points to importance of accounting for mutual CRO - environment polarization in quantitative calculations of absorption spectra in FPs., (© 2022 Wiley-VCH GmbH.)

Precisely quantifying the magnitude, direction, and biological functions of electric fields in proteins has long been an outstanding challenge in the field. The most widely implemented experimental method to measure such electric fields at a particular residue in a protein has been through changes in p K a of titratable residues. While many computational strategies exist to predict these values, it has been difficult to do this accurately or connect predicted results to key structural or mechanistic features of the molecule. Here, we used experimentally determined p K a values of the fluorophore in superfolder green fluorescent protein (GFP) with amino acid mutations made at position Thr 203 to evaluate the p K a prediction ability of molecular dynamics (MD) simulations using a polarizable force field, AMOEBA. Structure ensembles from AMOEBA were used to calculate p K a values of the GFP fluorophore. The calculated p K a values were then compared to trajectories using a conventional fixed charge force field (Amber03 ff). We found that the position of water molecules included in the p K a calculation had opposite effects on the p K a values between the trajectories from AMOEBA and Amber03 force fields. In AMOEBA trajectories, the inclusion of water molecules within 35 Å of the fluorophore decreased the difference between the predicted and experimental values, resulting in calculated p K a values that were within an average of 0.8 p K a unit from the experimental results. On the other hand, in Amber03 trajectories, including water molecules that were more than 5 Å from the fluorophore increased the differences between the calculated and experimental p K a values. The inaccuracy of p K a predictions determined from Amber03 trajectories was caused by a significant stabilization of the deprotonated chromophore's free energy compared to the result in AMOEBA. We rationalize the cutoffs for explicit water molecules when calculating p K a to better predict the electrostatic environment surrounding the fluorophore buried in GFP. We discuss how the results from this work will assist the prospective prediction of p K a values or other electrostatic effects in a wide variety of folded proteins.

Genetically encoded visible fluorescent proteins (VFPs) are a key tool used to visualize cellular processes. However, compared to synthetic fluorophores, VFPs are photophysically complex. This photophysical complexity includes the presence of non-emitting, dark proteins within the ensemble of VFPs. Quantitative fluorescence microcopy approaches that rely on VFPs to obtain molecular insights are hampered by the presence of these dark proteins. To account for the presence of dark proteins, it is necessary to know the fraction of dark proteins ( f dark ) in the ensemble. To date, f dark has rarely been quantified, and different methods to determine f dark have not been compared. Here, we use and compare two different methods to determine the f dark of four commonly used VFPs: EGFP, SYFP2, mStrawberry, and mRFP1. In the first, direct method, we make use of VFP tandems and single-molecule two-color coincidence detection (TCCD). The second method relies on comparing the bright state fluorescence quantum yield obtained by photonic manipulation to the ensemble-averaged fluorescence quantum yield of the VFP. Our results show that, although very different in nature, both methods are suitable to obtain f dark . Both methods show that all four VFPs contain a considerable fraction of dark proteins. We determine f dark values between 30 and 60% for the different VFPs. The high values for f dark of these commonly used VFPs highlight that f dark has to be accounted for in quantitative microscopy and spectroscopy.

HBDI-like chromophores represent a novel set of biomimetic switches mimicking the fluorophore of the green fluorescent protein that are currently studied with the hope to expand the molecular switch/motor toolbox. However, until now members capable of absorbing visible light in their neutral (i. e. non-anionic) form have not been reported. In this contribution we report the preparation of an HBDI-like chromophore based on a 3-phenylbenzofulvene scaffold capable of absorbing blue light and photoisomerizing on the picosecond timescale. More specifically, we show that double-bond photoisomerization occurs in both the E-to-Z and Z-to-E directions and that these can be controlled by irradiating with blue and UV light, respectively. Finally, as a preliminary applicative result, we report the incorporation of the chromophore in an amphiphilic molecule and demonstrate the formation of a visible-light-sensitive nanoaggregated state in water., (© 2022 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

The accumulation of β-sheet-rich protein aggregates, amyloid fibrils, accompanies severe pathologies (Alzheimer's, Parkinson's diseases, ALS, etc.). The high amyloidogenicity of proteins with a native β-barrel structure, and the amyloidogenic peptides ability to form a universal cylindrin-like oligomeric state were proven. The mechanisms for the proteins' transformation from this state to a fibrillar one are still not fully understood. We defined the structural rearrangements of the amyloidogenic β-barrel superfolder GFP (sfGFP) prior to fibrillogenesis using its tryptophan and chromophore fluorescence. We characterized the early intermediate "native-like" state preserving the integrity of the sfGFP β-barrel scaffold despite the partial distortion of the three β-strands closing it. The interaction between the "melted" regions of the protein leads to the assembly of high molecular weight complexes, which are not dynamic structures but are less stable and less cytotoxic than mature amyloids. Additional contacts of sfGFP monomers facilitate the global reorganization of its structure and stabilization of the second intermediate state in which the β-barrel opens and some of the native α-helices and disordered regions refold into non-native β-strands, which, along with native β-strands, form an amyloid fiber. Reported sfGFP structural transformations may occur during the fibrillogenesis of other β-barrel proteins, and the identified intermediate states are likely universal. Thus sfGFP can be used as a sensing platform to develop therapeutic agents inhibiting amyloidogenesis through interaction with protein intermediates and destroying low-stable aggregates formed at the early stages of fibrillogenesis., (Copyright © 2022. Published by Elsevier B.V.)

The application of action spectroscopy in connection with determination of the S 0 to S 1 band origin in the GFP anion model chromophore (deprotonated HBDI) is discussed. We specifically address the consequences of the lowest vibrational levels in S 1 being located behind a potential-energy barrier that inhibits internal conversion to the S 0 electronic ground state. Action spectroscopy based on consecutive absorption of two photons together with internal conversion will as a consequence reveal an apparent band origin that is significantly blue-shifted.

Optical fluorescence microscopy plays a pivotal role in the exploration of biological structure and dynamics, especially on live specimens. Progress in the field relies, on the one hand, on technical advances in imaging and data processing and, on the other hand, on progress in fluorescent marker technologies. Among these, genetically encodable fluorescent proteins (FPs) are invaluable tools, as they allow facile labeling of live cells, tissues or organisms, as these produce the FP markers all by themselves after introduction of a suitable gene. Here we cover FP markers from the GFP family of proteins as well as tetrapyrrole-binding proteins, which further complement the FP toolbox in important ways. A broad range of FP variants have been endowed, by using protein engineering, with photophysical properties that are essential for specific fluorescence microscopy techniques, notably those offering nanoscale image resolution. We briefly introduce various advanced imaging methods and show how they utilize the distinct properties of the FP markers in exciting imaging applications, with the aim to guide researchers toward the design of powerful imaging experiments that are optimally suited to address their biological questions., (Creative Commons Attribution license.)

Fluorescent proteins have become routine tools for biological imaging. However, their nanosecond lifetimes on the excited state present computational hurdles to a full understanding of these photoactive proteins. In this work, we simulate approximately 0.5 nanoseconds of ab initio molecular dynamics to elucidate steric and electronic features responsible for fluorescent protein behavior. Using green fluorescent protein (GFP) and Dronpa2─widely used fluorescent proteins with contrasting functionality─as case studies, we leverage previous findings in the gas phase and solution to explore the deactivation mechanisms available to these proteins. Starting with ground-state analyses, we identify steric (the distribution of empty pockets near the chromophore) and electronic (electric fields exerted on chromophore moieties) factors that offer potential avenues for rational design. Picosecond timescale simulations on the excited state reveal that the chromophore can access twisted structures in Dronpa2, while the chromophore is largely confined to planarity in GFP. We couple ab initio multiple spawning (AIMS) and enhanced sampling simulations to discover and characterize conical intersection seams that facilitate internal conversion, which is a rare event in both systems. Our AIMS simulations correctly capture the relative fluorescence profiles of GFP and Dronpa2 within the first few picoseconds, and we attribute the diminished fluorescence intensity of Dronpa2, relative to GFP, to flexible chromophore intermediates on the excited state. Furthermore, we predict that twisted chromophore intermediates produce red-shifted intensities in the Dronpa2 fluorescence spectrum. If confirmed experimentally, this spectroscopic signature would provide valuable insights when screening and developing novel fluorescent proteins.

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.

Cyclic adenosine monophosphate (cAMP) is a canonical intracellular messenger playing diverse roles in cell functions. In neurons, cAMP promotes axonal growth during early development, and mediates sensory transduction and synaptic plasticity after maturation. The molecular cascades of cAMP are well documented, but its spatiotemporal profiles associated with neuronal functions remain hidden. Hence, we developed a genetically encoded cAMP indicator based on a bacterial cAMP-binding protein. This indicator "gCarvi" monitors [cAMP] i at 0.2 to 20 µM with a subsecond time resolution and a high specificity over cyclic guanosine monophosphate (cGMP). gCarvi can be converted to a ratiometric probe for [cAMP] i quantification and its expression can be specifically targeted to various subcellular compartments. Monomeric gCarvi also enables simultaneous multisignal monitoring in combination with other indicators. As a proof of concept, simultaneous cAMP/Ca 2+ imaging in hippocampal neurons revealed a tight linkage of cAMP to Ca 2+ signals. In cerebellar presynaptic boutons, forskolin induced nonuniform cAMP elevations among boutons, which positively correlated with subsequent increases in the size of the recycling pool of synaptic vesicles assayed using FM dye. Thus, the cAMP domain in presynaptic boutons is an important determinant of the synaptic strength.

Nanomedicine emerged some decades ago with the hope to be the solution for most unmet medical needs. However, tracking materials at nanoscale is challenging to their reduced size, below the resolution limit of most conventional techniques. In this context, we propose the use of direct stochastic optical reconstruction microscopy (dSTORM) to study time stability and cell trafficking after transfection of oligopeptide end-modified poly(β-aminoester) (OM-pBAE) nanoparticles. We selected different combinations of cationic end oligopeptides (arginine - R; histidine - H; and lysine - K) among polymer libraries, since the oligopeptide combination demonstrated to be useful for different applications, such as vaccination and gene silencing. We demonstrate that their time evolution as well as their cell uptake and trafficking are dependent on the oligopeptide. This study opens the pave to broad mechanistic studies at nanoscale that could enable a rational selection of specific pBAE nanoparticles composition after determining their stability and cell trafficking., (© 2022 The Authors. ChemMedChem published by Wiley-VCH GmbH.)

Spectrally diverse fluorescent proteins (FPs) provide straightforward means for multiplexed imaging of biological systems. Among FPs fitting standard color channels, blue FPs (BFPs) are characterized by lower brightness compared to other spectral counterparts. Furthermore, available BFPs were not systematically characterized for imaging in cultured mammalian cells and common model organisms. Here we introduce a pair of new BFPs, named Electra1 and Electra2, developed through hierarchical screening in bacterial and mammalian cells using a novel dual-expression vector. We performed systematic benchmarking of Electras against state-of-art BFPs in cultured mammalian cells and demonstrated their utility as fluorescent tags for structural proteins. The Electras variants were validated for multicolor neuroimaging in Caenorhabditis elegans, zebrafish larvae, and mice in comparison with one of the best in the class BFP mTagBFP2 using one-photon and two-photon microscopy. The developed BFPs are suitable for multicolor imaging of cultured cells and model organisms in vivo. We believe that the described dual-expression vector has a great potential to be adopted by protein engineers for directed molecular evolution of FPs., (© 2022. The Author(s).)

The advancement of super-resolution imaging (SRI) relies on fluorescent proteins with novel photochromic properties. Using light, the reversibly switchable fluorescent proteins (RSFPs) can be converted between bright and dark states for many photocycles and their emergence has inspired the invention of advanced SRI techniques. The general photoswitching mechanism involves the chromophore cis - trans isomerization and proton transfer for negative and positive RSFPs and hydration-dehydration for decoupled RSFPs. However, a detailed understanding of these processes on ultrafast timescales (femtosecond to millisecond) is lacking, which fundamentally hinders the further development of RSFPs. In this review, we summarize the current progress of utilizing various ultrafast electronic and vibrational spectroscopies, and time-resolved crystallography in investigating the on/off photoswitching pathways of RSFPs. We show that significant insights have been gained for some well-studied proteins, but the real-time "action" details regarding the bidirectional cis - trans isomerization, proton transfer, and intermediate states remain unclear for most systems, and many other relevant proteins have not been studied yet. We expect this review to lay the foundation and inspire more ultrafast studies on existing and future engineered RSFPs. The gained mechanistic insights will accelerate the rational development of RSFPs with enhanced two-way switching rate and efficiency, better photostability, higher brightness, and redder emission colors.

Genetically encoded potassium indicators lack optimal binding affinity for monitoring intracellular dynamics in mammalian cells. Through structure-guided design and genome mining of potassium binding proteins, we developed green fluorescent potassium indicators with a broad range of binding affinities. KRaION1 (K + ratiometric indicator for optical imaging based on mNeonGreen 1), based on the insertion of a potassium binding protein, Kbp, from E. coli (Ec-Kbp) into the fluorescent protein mNeonGreen, exhibits an isotonically measured K d of 69 ± 10 mM (mean ± standard deviation used throughout). We identified Ec-Kbp's binding site using NMR spectroscopy to detect protein-thallium scalar couplings and refined the structure of Ec-Kbp in its potassium-bound state. Guided by this structure, we modified KRaION1, yielding KRaION1/D9N and KRaION2, which exhibit isotonically measured K d 's of 138 ± 21 and 96 ± 9 mM. We identified four Ec-Kbp homologues as potassium binding proteins, which yielded indicators with isotonically measured binding affinities in the 39-112 mM range. KRaIONs functioned in HeLa cells, but the K d values differed from the isotonically measured case. We found that, by tuning the experimental conditions, K d values could be obtained that were consistent in vitro and in vivo . We thus recommend characterizing potassium indicator K d in the physiological context of interest before application.

The de novo computational design of proteins with predefined three-dimensional structure is becoming much more routine due to advancements both in force fields and algorithms. However, creating designs with functions beyond folding is more challenging. In that regard, the recent design of small beta barrel proteins that activate the fluorescence of an exogenous small molecule chromophore (DFHBI) is noteworthy. These proteins, termed mini fluorescence activating proteins (mFAPs), have been shown to increase the brightness of the chromophore more than 100-fold upon binding to the designed ligand pocket. The design process created a large library of variants with different brightness levels but gave no rational explanation for why one variant was brighter than another. Here, we use quantum mechanics and molecular dynamics simulations to investigate how molecular flexibility in the ground and excited states influences brightness. We show that the ability of the protein to resist dihedral angle rotation of the chromophore is critical for predicting brightness. Our simulations suggest that the mFAP/DFHBI complex has a rough energy landscape, requiring extensive ground-state sampling to achieve converged predictions of excited-state kinetics. While computationally demanding, this roughness suggests that mFAP protein function can be enhanced by reshaping the energy landscape toward conformations that better resist DFHBI bond rotation.

Chemistry can be described as the movement of nuclei within molecules and the concomitant instantaneous change in electronic structure. This idea underpins the central chemical concepts of potential energy surfaces and reaction coordinates. To experimentally capture such chemical change therefore requires methods that can probe both the nuclear and electronic structure simultaneously and on the time scale of atomic motion. In this Account, we show how time-resolved photoelectron imaging can do exactly this and how it can be used to build a detailed and intuitive understanding of the electronic structure and excited-state dynamics of chromophores. The chromophore of the photoactive yellow protein (PYP) is used as a case study. This chromophore contains a para-substituted phenolate anion, where the substituent, R, can be viewed as an acrolein derivative. It is shown that the measured photoelectron angular distribution can be directly related to the electronic structure of the para-substituted phenolate anion. By incrementally considering differing R groups, it is also shown that these photoelectron angular distributions are exquisitely sensitive to the conformational flexibility of R and that when R contains a π-system the excited states of the chromophore can be viewed as a linear combination of the π* molecular orbitals on the phenolate (π Ph *) and the R substituent (π R *). Such Hückel treatment shows that the S 1 state of the PYP chromophore has predominantly π R * character and that it is essentially the same as the chromophore of the green fluorescent protein (GFP). The S 1 excited-state dynamics of the PYP chromophore probed by time-resolved photoelectron imaging clearly reveals both structural (nuclear) dynamics through the energy spectrum and electronic dynamics through the photoelectron angular distributions. Both motions can be accurately assigned using quantum chemical calculations, and these are consistent with the intuitive Hückel treatment presented. The photoactive protein chromophores considered here are examples of where a chemists' intuitive Hückel view for ground-state chemistry appears to be transferable to the prediction of photochemical excited-state reactivity. While elegant and insightful, such models have limitations, including nonadiabatic dynamics, which is present in a related PYP chromophore, where a fraction of the S 1 state population forms a nonvalence (dipole-bound) state of the anion.

GFP-like chromoproteins (CPs) with non-fluorescence ability have been used as bioimaging probes. Existing CPs have voids in the optical absorption window which limits their extensibility. The development of new CP color is therefore ongoing. Here, we cloned CPs from the jellyfish, Olindias formosa, and developed a completely non-fluorescent monomeric red CP, R-Velour, with an absorption peak at 528 nm. To analyze the photophysical properties from a structural aspect, we determined the crystal structure of R-Velour at a 2.1 Å resolution. R-Velour has a trans-chromophore similar to the green fluorescence protein, Gamillus, derived from the same jellyfish. However, in contrast to the two coplanar chromophoric rings in Gamillus, R-Velour has a large torsion inducing non-fluorescence property. Through site-directed mutagenesis, we surveyed residues surrounding the chromophore and found a key residue, Ser155, which contributes to the generation of four-color variants with the bathochromic and hypsochromic shift of the absorption peak, ranging from 506 to 554 nm. The recently proposed spectrum shift theory, based on the Marcus-Hush model, supports the spectrum shift of these mutants. These findings may support further development of R-Velour variants with useful absorption characteristics for bioimaging, including fluorescence lifetime imaging and photoacoustic imaging., (© 2022 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Transition metals such as zinc and copper are essential in numerous life processes, and both deficiency and toxic overload of these metals are associated with various diseases. Fluorescent metal sensors are powerful tools for studying the roles of metal ions in the physiology and pathology of biological systems. Green fluorescent protein (GFP) and its derivatives are highly utilized for protein-based sensor design, but application to anaerobic systems is limited because these proteins require oxygen to become fluorescent. Bacteriophytochrome-based monomeric near-infrared fluorescent proteins (miRFPs) covalently bind a bilin cofactor, which can be added exogenously for anaerobic cells. miRFPs can also have emission wavelengths extending to >700 nm, which is valuable for imaging applications. Here, we evaluated the suitability of miRFP670 and miRFP709 as platforms for single fluorescent protein metal ion sensors. We found that divalent metal ions like Zn 2+ , Co 2+ , Ni 2+ , and Cu 2+ can quench from ∼6-20% (Zn 2+ , Co 2+ , and Ni 2+ ) and up to nearly 90% (Cu 2+ ) of the fluorescence intensity of pure miRFPs and have similar impacts in live Escherichia coli cells expressing miRFPs. The presence of a 6× histidine tag for purification influences metal quenching, but significant Cu 2+ -induced quenching and a picomolar binding affinity are retained in the absence of the His 6 tag in both cuvettes and live bacterial cells. By comparing the Cu 2+ and Cu + -induced quenching results for miRFP670 and miRFP709 and through examining absorption spectra and previously reported crystal structures, we propose a surface metal binding site near the biliverdin IXα chromophore.

The spectral properties of fluorescent proteins (FPs) depend on the protein environment of the chromophore (CRO). A deeper understanding of the CRO - environment interactions in terms of FPs' spectral characteristics will allow for a rational design of novel markers with desired properties. Here, we are taking a step towards achieving this important goal. With the time-dependent density functional theory (TDDFT), we calculate one- and two-photon absorption (OPA and TPA) spectra for 5 green FPs (GFPs) and 3 yellow FPs (YFPs) differing in amino acid sequence. The goal is to reveal the roles of: (i) electrostatic interactions, (ii) hydrogen-bonds (h-bonds) and (iii) h-bonds together with distant electrostatic field in absorption spectra tuning. Our results point to design hypothesis towards FPs optimised for TPA-based applications. Both h-bonds and electrostatic interactions co-operate in enhancing TPA cross-section ( σ T P A ) for the S 0 → S 1 transition in GFPs. Furthermore, it seems that details of h-bonds network in the CRO's vicinity influences σ T P A response to CRO - environment electrostatic interactions in YFPs. We postulate that engineering FPs with more hydrophilic CRO's environment can lead to greater σ T P A . We also find that removing h-bonds formed with the CRO's phenolate leads to TPA enhancement for transition to higher excited states than S 1 . Particularly Y145 and T203 residues are important in this regard., (© 2022 Wiley-VCH GmbH.)

Many membrane proteins function as dimers or larger oligomers, including transporters, channels, certain signaling receptors, and adhesion molecules. In some cases, the interactions between individual proteins may be weak and/or dependent on specific lipids, such that detergent solubilization used for biochemical and structural studies disrupts functional oligomerization. Solubilized membrane protein oligomers can be captured in lipid nanodiscs, but this is an inefficient process that can produce stoichiometrically and topologically heterogeneous preparations. Here, we describe a technique to obtain purified homogeneous membrane protein dimers in nanodiscs using a split GFP (sGFP) tether. Complementary sGFP tags associate to tether the coexpressed dimers and control both stoichiometry and orientation within the nanodiscs, as assessed by quantitative Western blotting and negative-stain EM. The sGFP tether confers several advantages over other methods: it is highly stable in solution and in SDS-PAGE, which facilitates screening of dimer expression and purification by fluorescence, and also provides a dimer-specific purification handle for use with GFP nanobody-conjugated resin. We used this method to purify a Frizzled-4 homodimer and a Frizzled-4/low-density lipoprotein receptor-related protein 6 heterodimer in nanodiscs. These examples demonstrate the utility and flexibility of this method, which enables subsequent mechanistic molecular and structural studies of membrane protein pairs., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The development of bright fluorescent proteins (FPs) emitting beyond 600 nm continues to be of interest both from a fundamental perspective in understanding protein-chromophore interactions and from a practical perspective as these FPs would be valuable for cellular imaging. We previously reported ultrafast spectral observations of the excited-state dynamics in mPlum resulting from interconversion between direct hydrogen bonding and water-mediated hydrogen bonding between the chromophore acylimine carbonyl and the Glu16 side chain. Here, we report temperature-dependent steady-state and time-resolved fluorescence measurements of mPlum and its E16H variant, which does not contain a side-chain permitting hydrogen bonding with the acylimine carbonyl. Lowering the temperature of the system freezes interconversion between the hydrogen-bonding states, thus revealing the spectral signatures of the two states. Analysis of the temperature-dependent spectra assuming Boltzmann populations of the two states yields a 205 cm -1 energy difference. This value agrees with the predictions from a quantum mechanics/molecular mechanics study of mPlum (198 cm -1 ). This study demonstrates the first use of cryogenic spectroscopy to quantify the energetics and timescales of FP chromophore structural states that were only previously obtained from computational methods and further confirms the importance of acylimine hydrogen-bonding dynamics to the fluorescence spectral shifts of red FPs.

Genetically encoded monomeric blue-to-red fluorescent timers (mFTs) change their fluorescent color over time. mCherry-derived mFTs were used for the tracking of the protein age, visualization of the protein trafficking, and labeling of engram cells. However, the brightness of the blue and red forms of mFTs are 2-3- and 5-7-fold dimmer compared to the brightness of the enhanced green fluorescent protein (EGFP). To address this limitation, we developed a blue-to-red fluorescent timer, named mRubyFT, derived from the bright mRuby2 red fluorescent protein. The blue form of mRubyFT reached its maximum at 5.7 h and completely transformed into the red form that had a maturation half-time of 15 h. Blue and red forms of purified mRubyFT were 4.1-fold brighter and 1.3-fold dimmer than the respective forms of the mCherry-derived Fast-FT timer in vitro. When expressed in mammalian cells, both forms of mRubyFT were 1.3-fold brighter than the respective forms of Fast-FT. The violet light-induced blue-to-red photoconversion was 4.2-fold less efficient in the case of mRubyFT timer compared to the same photoconversion of the Fast-FT timer. The timer behavior of mRubyFT was confirmed in mammalian cells. The monomeric properties of mRubyFT allowed the labeling and confocal imaging of cytoskeleton proteins in live mammalian cells. The X-ray structure of the red form of mRubyFT at 1.5 Å resolution was obtained and analyzed. The role of the residues from the chromophore surrounding was studied using site-directed mutagenesis.

The past decades have witnessed an explosion of de novo protein designs with a remarkable range of scaffolds. It remains challenging, however, to design catalytic functions that are competitive with naturally occurring counterparts as well as biomimetic or nonbiological catalysts. Although directed evolution often offers efficient solutions, the fitness landscape remains opaque. Green fluorescent protein (GFP), which has revolutionized biological imaging and assays, is one of the most redesigned proteins. While not an enzyme in the conventional sense, GFPs feature competing excited-state decay pathways with the same steric and electrostatic origins as conventional ground-state catalysts, and they exert exquisite control over multiple reaction outcomes through the same principles. Thus, GFP is an "excited-state enzyme". Herein we show that rationally designed mutants and hybrids that contain environmental mutations and substituted chromophores provide the basis for a quantitative model and prediction that describes the influence of sterics and electrostatics on excited-state catalysis of GFPs. As both perturbations can selectively bias photoisomerization pathways, GFPs with fluorescence quantum yields (FQYs) and photoswitching characteristics tailored for specific applications could be predicted and then demonstrated. The underlying energetic landscape, readily accessible via spectroscopy for GFPs, offers an important missing link in the design of protein function that is generalizable to catalyst design.

We describe an engineered violet fluorescent protein from the lancelet Branchiostoma floridae (bfVFP). This is the first example of a GFP-like fluorescent protein with a stable fluorescent chromophore lacking an imidazolinone ring; instead, it consists of oxidized tyrosine 68 flanked by glycine 67 and alanine 69. bfVFP contains the simplest chromophore reported in fluorescent proteins and was generated from the yellow protein lanFP10A2 by two synergetic mutations, S148H and C166I. The chromophore structure was confirmed crystallographically and by high-resolution mass spectrometry. The photophysical characteristics of bfVFP (323/430 nm, quantum yield 0.33, and E c 14,300 M -1  cm -1 ) make it potentially useful for multicolor experiments to expand the excitation range of available FP biomarkers and Förster resonance energy transfer with blue and cyan fluorescent protein acceptors., (© 2021 The Protein Society.)

Strategic incorporation of a meta-dimethylamino (-NMe 2 ) group on the conformationally locked green fluorescent protein (GFP) model chromophore (m-NMe 2 -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-NMe 2 -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 H 2 O and ROH emissions in various H 2 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 -NMe 2 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-NMe 2 group twisting coordinate upon electronic excitation was corroborated by experiments on control samples without the meta-NMe 2 group or with both meta-NMe 2 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., (© 2021 American Society for Photobiology.)

Optical methods to modulate microtubule dynamics show promise for reaching the micron- and millisecond-scale resolution needed to decrypt the roles of the cytoskeleton in biology. However, optical microtubule stabilisers are under-developed. We introduce "STEpos" as GFP-orthogonal, light-responsive epothilone-based microtubule stabilisers. They use a novel styrylthiazole photoswitch in a design to modulate hydrogen-bonding and steric effects that control epothilone potency. STEpos photocontrol microtubule dynamics and cell division with micron- and second-scale spatiotemporal precision. They substantially improve potency, solubility, and ease-of-use compared to previous optical microtubule stabilisers, and the structure-photoswitching-activity relationship insights in this work will guide future optimisations. The STEpo reagents can contribute greatly to high-precision research in cytoskeleton biophysics, cargo transport, cell motility, cell division, development, and neuroscience., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

The photophysics of green fluorescent protein (GFP) and red Kaede fluorescent protein (rKFP) are defined by the intrinsic properties of the light-absorbing chromophore and its interaction with the protein binding pocket. This work deploys photodissociation action spectroscopy to probe the absorption profiles for a series of synthetic GFP and rKFP chromophores as the bare anions and as complexes with the betaine zwitterion, which is assumed as a model for dipole microsolvation. Electronic structure calculations and energy decomposition analysis using Symmetry-Adapted Perturbation Theory are used to characterize gas-phase structures and complex cohesion forces. The calculations reveal a preponderance for coordination of betaine to the phenoxide deprotonation site predominantly through electrostatic forces. Calculations using the STEOM-DLPNO-CCSD method are able to reproduce absolute and relative vertical excitation energies for the bare anions and anion-betaine complexes. On the other hand, treatment of the betaine molecule with a point-charge model, in which the charges are computed from some common electron density population analysis schemes, show that just electrostatic and point-charge induction interactions are unable to account for the betaine-induced spectral shift. The present methodology could be applied to investigate cluster forces and optical properties in other gas-phase ion-zwitterion complexes.

mNeonGreen, an engineered green fluorescent protein (GFP) derived from lancelet, is one of the most brightly fluorescent homologs of Aequorea victoria jellyfish GFP (avGFP) yet reported. In this work, we investigated whether this bright fluorescence might be retained in homologs of mNeonGreen with modified chromophore structures and altered fluorescent hues. We found mNeonGreen to be generally less tolerant than avGFP to chromophore modification by substitution of the key chromophore-forming tyrosine residue with other aromatic amino acids. However, we were ultimately successful in creating a variant, designated as NeonCyan1, with a tryptophan-derived cyan fluorescent protein (CFP)-type chromophore, and two additional mutants with distinct spectral hues. Structural, computational, and photophysical characterization of NeonCyan1 and its variants provided insight into the factors that control the fluorescence emission color. Though not recommended as replacements for contemporary CFP variants, we demonstrate that NeonCyan1 variants are potentially suitable for live cell imaging applications., (© The Author(s) 2022. Published by Oxford University Press.)

Two-dimensional covalent organic frameworks (2D-COFs) are a class of crystalline porous organic polymers that consist of covalently linked, two-dimensional sheets that can stack together through noncovalent interactions. Here we report the synthesis of a novel COF, called PyCOFamide, which has an experimentally observed pore size that is greater than 6 nm in diameter. This is among the largest pore size reported to date for a 2D-COF. PyCOFamide exhibits permanent porosity and high crystallinity as evidenced by the nitrogen adsorption, powder X-ray diffraction, and high-resolution transmission electron microscopy. We show that the pore size of PyCOFamide is large enough to accommodate fluorescent proteins such as Superfolder green fluorescent protein and mNeonGreen. This work demonstrates the utility of noncovalent structural reinforcement in 2D-COFs to produce larger and persistent pore sizes than previously possible.

The viral polyprotein Gag plays a central role for HIV-1 assembly, release and maturation. Proteolytic processing of Gag by the viral protease is essential for the structural rearrangements that mark the transition from immature to mature, infectious viruses. The timing and kinetics of Gag processing are not fully understood. Here, fluorescence lifetime imaging microscopy and single virus tracking are used to follow Gag processing in nascent HIV-1 particles in situ. Using a Gag polyprotein labelled internally with eCFP, we show that proteolytic release of the fluorophore from Gag is accompanied by an increase in its fluorescence lifetime. By tracking nascent virus particles in situ and analyzing the intensity and fluorescence lifetime of individual traces, we detect proteolytic cleavage of eCFP from Gag in a subset (6.5%) of viral particles. This suggests that for the majority of VLPs, Gag processing occurs with a delay after particle assembly.

Solvatofluorochromism, a solvation effect on the fluorescence color of an organic dye, is a property generally limited to fluid solutions. We demonstrate herein the concept of solid-state solvatofluorochromism by using an organogelator ( 1 -SG), which consists of a solvatofluorochromic green fluorescence protein (GFP) chromophore ( 1 ) and a sugar gelator (SG). While 1 -SG could be located in the liquid phase or in the fibrous solid matrix of the SG gel, our results show that the one in the solid matrix but near the liquid interface has superior fluorescence stability and quantum efficiency as well as solvatofluorochromicity than the one in the liquid phase. In addition, the phenomenon of fluorescence turn-on occurs when the gel is formed in protic solvents. These features have been applied to perform multicolor fluorescence patterning, chemical vapor sensing, data encryption and decryption, and real-time fluorescence cell imaging.

Fluorescent proteins (FPs) have become ubiquitous tools for biological research and concomitantly they are intriguing molecules that are amenable to study with a wide range of experimental and theoretical tools. This perspective explores the connection between the engineering of improved FPs and basic ideas from physical chemistry that explain their properties and drive the molecular design of brighter and more photostable variants. We highlight some of the progress and the many knowledge gaps in understanding the relationship between FP brightness and photostability. We also explore some of the pertinent remaining questions and suggest ways in which physical chemists might further examine the physical basis of brightness and photostability in these systems.

Protein conformational switches are widely used in biosensing. They are often composed of an input domain (which binds a target ligand) fused to an output domain (which generates an optical readout). A central challenge in designing such switches is to develop mechanisms for coupling the input and output signals via conformational changes. Here, we create a biosensor in which binding-induced folding of the input domain drives a conformational shift in the output domain that results in a sixfold green-to-yellow ratiometric fluorescence change in vitro and a 35-fold intensiometric fluorescence increase in cultured cells. The input domain consists of circularly permuted FK506 binding protein (cpFKBP) that folds upon binding its target ligand (FK506 or rapamycin). cpFKBP folding induces the output domain, an engineered green fluorescent protein (GFP) variant, to replace one of its β-strands (containing T203 and specifying green fluorescence) with a duplicate β-strand (containing Y203 and specifying yellow fluorescence) in an intramolecular exchange reaction. This mechanism employs the loop-closure entropy principle, embodied by the folding of the partially disordered cpFKBP domain, to couple ligand binding to the GFP color shift. This study highlights the high-energy barriers present in GFP folding which cause β-strand exchange to be slow and are also likely responsible for the shift from the β-strand exchange mechanism in vitro to ligand-induced chromophore maturation in cells. The proof-of-concept design has the advantages of full genetic encodability and potential for modularity. The latter attribute is enabled by the natural coupling of binding and folding and circular permutation of the input domain, which theoretically allows different binding domains to be compatible for insertion into the GFP surface loop.

Tracking and mapping the nascent RNA molecules in cells is essential for deciphering embryonic development and neuronal differentiation. Here, we utilized 4-thiouridine (s 4 U) as a metabolic tag to label nascent RNA and developed a fluorescence imaging method based on the DNA-templated oxidative amination (DTOA) reaction of s 4 U. The DTOA reaction occurred between amine-modified DNA and s 4 U-containing RNA with high sequence specificity and chemical selectivity. Target nascent mRNAs in HeLa cells, including those encoding green fluorescent proteins (GFPs) and endogenous BAG-1, were thus lit up selectively by DTOA-based fluorescence in situ hybridization (DTOA FISH). We believe the DTOA conjugation chemistry shown in this study could be generally applied to investigate the spatial distribution of nascent transcription dynamics in cellular processes.

Infertility affects 10-15% of families worldwide. However, the pathogenesis of female infertility caused by abnormal early embryonic development is not clear. A recent study showed that poly(A)binding protein nuclear 1-like (PABPN1L) recruited BTG anti-proliferation factor 4 (BTG4) to mRNA 3'-poly(A) tails and was essential for maternal mRNA degradation. Here, we generated a PABPN1L-antibody and found "ring-like" PABPN1L aggregates in the cytoplasm of MII oocytes. PABPN1L-EGFP proteins spontaneously formed "ring-like" aggregates in vitro. This phenomenon is similar with CCR4-NOT catalytic subunit, CCR4-NOT transcription complex subunit 7 (CNOT7), when it starts deadenylation process in vitro. We constructed two mouse model (Pabpn1l-/- and Pabpn1l  tm1a/tm1a) simulating the intron 1-exon 2 abnormality of human PABPN1L and found that the female was sterile and the male was fertile. Using RNA-Seq, we observed a large-scale up-regulation of RNA in zygotes derived from Pabpn1l-/- MII oocytes. We found that 9222 genes were up-regulated instead of being degraded in the Pabpn1l-♀/+♂zygote. Both the Btg4 and CCR4-NOT transcription complex subunit 6 like (Cnot6l) genes are necessary for the deadenylation process and Pabpn1l-/- resembled both the Btg4 and Cnot6l knockouts, where 71.2% genes stabilized in the Btg4-♀/+♂ zygote and 84.2% genes stabilized in the Cnot6l-♀/+♂zygote were also stabilized in Pabpn1l-♀/+♂ zygote. BTG4/CNOT7/CNOT6L was partially co-located with PABPN1L in MII oocytes. The above results suggest that PABPN1L is widely associated with CCR4-NOT-mediated maternal mRNA degradation and PABPN1L variants on intron 1-exon 2 could be a genetic marker of female infertility., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The in vivo function of cell-derived extracellular vesicles (EVs) is challenging to establish since cell-specific EVs are difficult to isolate and differentiate. We, therefore, created an EV reporter using truncated CD9 to display enhanced green fluorescent protein (EGFP) on the EV surface. CD9truc-EGFP expression in cells did not affect EV size and concentration but enabled co-precipitation of EV markers TSG101 and ALIX from the cell-conditioned medium by anti-GFP immunoprecipitation. We then created a transgenic mouse where CD9truc-EGFP was inserted in the inverse orientation and double-floxed, ensuring irreversible Cre recombinase-dependent EV reporter expression. We crossed the EV reporter mice with mice expressing Cre ubiquitously (CMV-Cre), in cardiomyocytes (αMHC-MerCreMer) and renal tubular epithelial cells (Pax8-Cre), respectively. The CD9truc-EGFP positive mice showed Cre-dependent EGFP expression, and plasma CD9truc-EGFP EVs were immunoprecipitated only from CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxαMHC-Cre mice, but not in CD9truc-EGFPxPax8-Cre and CD9truc-EGFP negative mice. In urine samples, CD9truc-EGFP EVs were detected by immunoprecipitation only in CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxPax8-Cre mice, but not CD9truc-EGFPxαMHC-Cre and CD9truc-EGFP negative mice. In conclusion, our EV reporter mouse model enables Cre-dependent EV labeling, providing a new approach to studying cell-specific EVs in vivo and gaining a unique insight into their physiological and pathophysiological function., (© 2022. The Author(s).)

Multiple myeloma (MM) is a cancer of bone marrow (BM) plasma cells, which is increasingly treatable but still incurable. In 90% of MM patients, severe osteolysis results from pathological interactions between MM cells and the bone microenvironment. Delineating specific molecules and pathways for their role in cancer supportive interactions in the BM is vital for developing new therapies. Very Late Antigen 4 (VLA4, integrin α 4 β 1 ) is a key player in cell-cell adhesion and signaling between MM and BM cells. We evaluated a VLA4 selective near infrared fluorescent probe, LLP2A-Cy5, for in vitro and in vivo optical imaging of VLA4. Furthermore, two VLA4-null murine 5TGM1 MM cell (KO) clones were generated by CRISPR/Cas9 knockout of the Itga4 (α 4 ) subunit, which induced significant alterations in the transcriptome. In contrast to the VLA4 +  5TGM1 parental cells, C57Bl/KaLwRij immunocompetent syngeneic mice inoculated with the VLA4-null clones showed prolonged survival, reduced medullary disease, and increased extramedullary disease burden. The KO tumor foci showed significantly reduced uptake of LLP2A-Cy5, confirming in vivo specificity of this imaging agent. This work provides new insights into the pathogenic role of VLA4 in MM, and evaluates an optical tool to measure its expression in preclinical models., (© 2022. The Author(s).)

Undergraduate laboratory courses are essential to teaching core principles in STEM. This course, Quantitative Biological Methods, provides a unique approach to teaching molecular biology research techniques to students, in a laboratory that is delivered in a sequence that parallels standard biomedical research laboratory protocols. Students attend a lecture where they are taught the essential principles of biomedical research, and a lab where they learn to use laboratory equipment, perform experiments, and purify and quantify DNA and proteins. The course begins with an introduction to laboratory safety, pipetting, centrifugation, spectrophotometry, and other basic laboratory techniques. Next, the lab focuses on the purification and analysis of glutathione S-transferase (GST) fused to green fluorescent protein (GFP) from an Escherichia coli lysate. Students study this GST-GFP fusion protein and perform protein quantification, enzyme assays, chromatography, fluorescent detection, normalization, SDS-PAGE, and western blotting. Students then learn recombinant DNA technology using the GST-GFP vector that was the source of the fusion protein in the prior labs, and perform ligation, transformation of E. coli cells, blue/white screening, DNA purification via a miniprep, PCR, DNA quantification, restriction enzyme digestion, and agarose gel electrophoresis. Students write laboratory reports to demonstrate an understanding of the principles of the laboratory methods, and they must present and critically analyze their data. The lab methods described herein aim to emphasize the core molecular biology principles and techniques, prepare students for work in a biomedical research laboratory, and introduce students to both GST and GFP, two versatile laboratory proteins., (© 2021 The Authors. Biochemistry and Molecular Biology Education published by Wiley Periodicals LLC on behalf of International Union of Biochemistry and Molecular Biology.)