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.

Mannosylation of pH-sensitive liposomes promoted cytoplasmic delivery of protein to macrophages: green fluorescent protein (GFP) performed as an endosomal escape tracer.

Conventional non-pH-sensitive liposomes for cytoplasmic delivery of protein suffer from poor efficiency. Here we investigated mannosylated pH-sensitive liposomes (MAN-PSL) for cytoplasmic delivery of protein to macrophages RAW 264.7 using PSL and non-pH-sensitive liposomes for comparison. We characterised the pH-dependent fluorescence of green fluorescent protein (GFP) and encapsulated it in liposomes as an intracellular trafficking tracer. GFP showed a reversed 'S'-shaped pH-fluorescence curve with a dramatic signal loss at acidic pH. GFP stored at 4 °C with light protection showed a half-life of 10 days (pH 5-8). The entrapment efficiency of GFP was dominated by the volume ratio of intraliposomal core to external medium for thin-film hydration. Mannosylation did not affect the pH-responsiveness of PSL. Confocal microscopy elucidated that mannosylation promoted the cellular uptake of PSL. For both these liposomes, the strongest, homogeneously distributed GFP fluorescence in the cytoplasm was found at 3 h, confirming efficient endosomal escape of GFP. Conversely, internalisation of non-pH-sensitive liposomes was slow (peaked at 12 h) and both Nile Red and GFP signals remained weak and punctuated in the cytosol. In conclusion, GFP performed as a probe for endosome escape of liposomal cargo. Mannosylation facilitated the internalisation of PSL without compromising their endosomal escape ability.

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.

CiPerGenesis, A Mutagenesis Approach that Produces Small Libraries of Circularly Permuted Proteins Randomly Opened at a Focused Region: Testing on the Green Fluorescent Protein.

Circularly permuted proteins (cpPs) represent a novel type of mutant proteins with original termini that are covalently linked through a peptide connector and opened at any other place of the polypeptide backbone to create new ends. cpPs are finding wide applications in biotechnology because their properties may be quite different from those of the parental protein. However, the actual challenge for the creation of successful cpPs is to identify those peptide bonds that can be broken to create new termini and ensure functional and well-folded cpPs. Herein, we describe CiPerGenesis, a combinatorial mutagenesis approach that uses two oligonucleotide libraries to amplify a circularized gene by PCR, starting and ending from a focused target region. This approach creates small libraries of circularly permuted genes that are easily cloned in the correct direction and frame using two different restriction sites encoded in the oligonucleotides. Once expressed, the protein libraries exhibit a unique sequence diversity, comprising cpPs that exhibit ordinary breakpoints between adjacent amino acids localized at the target region as well as cpPs with new termini containing user-defined truncations and repeats of some amino acids. CiPerGenesis was tested at the lid region G134-H148 of green fluorescent protein (GFP), revealing that the most fluorescent variants were those starting at Leu141 and ending at amino acids Tyr145, Tyr143, Glu142, Leu141, Lys140, and H139. Purification and biochemical characterization of some variants suggested a differential expression, solubility and maturation extent of the mutant proteins as the likely cause for the variability in fluorescence intensity observed in colonies.

Coordination-Assisted Bioorthogonal Chemistry: Orthogonal Tetrazine Ligation with Vinylboronic Acid and a Strained Alkene.

Bioorthogonal chemistry can be used for the selective modification of biomolecules without interfering with any other functionality that might be present. Recent developments in the field include orthogonal bioorthogonal reactions to modify multiple biomolecules simultaneously. During our research, we observed that the reaction rates for the bioorthogonal inverse-electron-demand Diels-Alder (iEDDA) reactions between nonstrained vinylboronic acids (VBAs) and dipyridyl-s-tetrazines were exceptionally higher than those between VBAs and tetrazines bearing a methyl or phenyl substituent. As VBAs are mild Lewis acids, we hypothesised that coordination of the pyridyl nitrogen atom to the boronic acid promoted tetrazine ligation. Herein, we explore the molecular basis and scope of VBA-tetrazine ligation in more detail and benefit from its unique reactivity in the simultaneous orthogonal tetrazine labelling of two proteins modified with VBA and norbornene, a widely used strained alkene. We further show that the two orthogonal iEDDA reactions can be performed in living cells by labelling the proteasome by using a nonselective probe equipped with a VBA and a subunit-selective VBA bearing a norbornene moiety.

Synthetic green fluorescent protein chromophore analogues with a positive charge at the phenyl-like group.

Synthetic green fluorescent protein (GFP) chromophore analogues with a positive charge at the phenyl-like group have the highly electrophilic amidine carbon, smaller LUMO-HOMO energy gap, red-shifted electronic absorptions and fluorescent emissions, and accelerated E-Z thermoisomerization rates. They are water-labile and their hydrolysis results in ring-opening of the imidazolinone moiety with a half life around 25-37 h in D2O at 25 °C.

Nanoscale click-reactive scaffolds from peptide self-assembly.

BACKGROUND: Due to their natural tendency to self-assemble, proteins and peptides are important components for organic nanotechnology. One particular class of peptides of recent interest is those that form amyloid fibrils, as this self-assembly results in extremely strong, stable quasi-one-dimensional structures which can be used to organise a wide range of cargo species including proteins and oligonucleotides. However, assembly of peptides already conjugated to proteins is limited to cargo species that do not interfere sterically with the assembly process or misfold under the harsh conditions often used for assembly. Therefore, a general method is needed to conjugate proteins and other molecules to amyloid fibrils after the fibrils have self-assembled. RESULTS: Here we have designed an amyloidogenic peptide based on the TTR105-115 fragment of transthyretin to form fibrils that display an alkyne functionality, important for bioorthogonal chemical reactions, on their surface. The fibrils were formed and reacted both with an azide-containing amino acid and with an azide-functionalised dye by the Huisgen cycloaddition, one of the class of "click" reactions. Mass spectrometry and total internal reflection fluorescence optical microscopy were used to show that peptides incorporated into the fibrils reacted with the azide while maintaining the structure of the fibril. These click-functionalised amyloid fibrils have a variety of potential uses in materials and as scaffolds for bionanotechnology. DISCUSSION: Although previous studies have produced peptides that can both form amyloid fibrils and undergo "click"-type reactions, this is the first example of amyloid fibrils that can undergo such a reaction after they have been formed. Our approach has the advantage that self-assembly takes place before click functionalization rather than pre-functionalised building blocks self-assembling. Therefore, the molecules used to functionalise the fibril do not themselves have to be exposed to harsh, amyloid-forming conditions. This means that a wider range of proteins can be used as ligands in this process. For instance, the fibrils can be functionalised with a green fluorescent protein that retains its fluorescence after it is attached to the fibrils, whereas this protein loses its fluorescence if it is exposed to the conditions used for aggregation.

Pd(0)-Catalyzed Direct C-H Functionalization of 2-H-4-Benzylidene Imidazolones: Friendly and Large-Scale Access to GFP and Kaede Protein Fluorophores.

The first one-pot synthesis of N-substituted 2-H-4-benzylidene imidazolones and their subsequent palladium-catalyzed and copper-assisted direct C2-H arylation and alkenylation with aryl- and alkenylhalides are described. This innovative synthesis is step-economical, azide-free, high yielding, highly flexible in the introduction of a variety of electronically different groups, and can be operated on large-scale. Moreover, the method allows direct access to C2-arylated or alkenylated imidazolone-based green fluorescent protein (GFP) and Kaede protein fluorophores, including ortho-hydroxylated models.

Efficient Isotope Editing of Proteins for Site-Directed Vibrational Spectroscopy.

Vibrational spectra contain unique information on protein structure and dynamics. However, this information is often obscured by spectral congestion, and site-selective information is not available. In principle, sites of interest can be spectrally identified by isotope shifts, but site-specific isotope labeling of proteins is today possible only for favorable amino acids or with prohibitively low yields. Here we present an efficient cell-free expression system for the site-specific incorporation of any isotope-labeled amino acid into proteins. We synthesized 1.6 mg of green fluorescent protein with an isotope-labeled tyrosine from 100 mL of cell-free reaction extract. We unambiguously identified spectral features of the tyrosine in the fingerprint region of the time-resolved infrared absorption spectra. Kinetic analysis confirmed the existence of an intermediate state between photoexcitation and proton transfer that lives for 3 ps. Our method lifts vibrational spectroscopy of proteins to a higher level of structural specificity.

Phosphorylation-Mediated Assembly of a Semisynthetic Fluorescent Protein for Label-Free Detection of Protein Kinase Activity.

Protein phosphorylation catalyzed by protein kinases plays a critical role in many intracellular processes, and detecting kinase activity is important in biochemical research and drug discovery. Herein, we developed a novel fluorescent biosensor to detect protein kinase activity based on phosphorylation-mediated assembly of semisynthetic green fluorescent protein (GFP). A chimaera S-peptide composed of the 10th ß-strand of GFP (s10) and a kinase substrate peptide was synthesized. Kinase-catalyzed phosphorylation of the S-peptide can protect its s10 part against cleavage by carboxypeptidase Y (CPY). Then, the peptide can bind the truncated GFP (tGFP, GFP without s10) to assemble intact GFP and recover fluorescence. Unphosphorylated S-peptide would be degraded by CPY, and fluorescent protein assembly could not occur. Thus, the kinase-catalyzed phosphorylation can switch on the fluorescence signal. This platform has been successfully applied to detect the activity of cAMP-dependent protein kinase with a low detection limit of 0.50 mU/µL and its inhibition of H-89 with an IC50 value of 23.4 nM. The feasibility of this method has been further demonstrated by assessment of the kinase activity and inhibition in the cell lysate. Moreover, based on the reverse principle, this method was expanded to detect the activity of protein phosphatase 1. Our method, using semisynthetic GFP as a readout, is facile, sensitive, label-free, and highly versatile, thus showing great potential as a promising platform for protein kinase detection and inhibitor screening.

SpeedyGenes: an improved gene synthesis method for the efficient production of error-corrected, synthetic protein libraries for directed evolution.

The de novo synthesis of genes is becoming increasingly common in synthetic biology studies. However, the inherent error rate (introduced by errors incurred during oligonucleotide synthesis) limits its use in synthesising protein libraries to only short genes. Here we introduce SpeedyGenes, a PCR-based method for the synthesis of diverse protein libraries that includes an error-correction procedure, enabling the efficient synthesis of large genes for use directly in functional screening. First, we demonstrate an accurate gene synthesis method by synthesising and directly screening (without pre-selection) a 747 bp gene for green fluorescent protein (yielding 85% fluorescent colonies) and a larger 1518 bp gene (a monoamine oxidase, producing 76% colonies with full catalytic activity, a 4-fold improvement over previous methods). Secondly, we show that SpeedyGenes can accommodate multiple and combinatorial variant sequences while maintaining efficient enzymatic error correction, which is particularly crucial for larger genes. In its first application for directed evolution, we demonstrate the use of SpeedyGenes in the synthesis and screening of large libraries of MAO-N variants. Using this method, libraries are synthesised, transformed and screened within 3 days. Importantly, as each mutation we introduce is controlled by the oligonucleotide sequence, SpeedyGenes enables the synthesis of large, diverse, yet controlled variant sequences for the purposes of directed evolution.

Cell-free protein synthesis from a release factor 1 deficient Escherichia coli activates efficient and multiple site-specific nonstandard amino acid incorporation.

Site-specific incorporation of nonstandard amino acids (NSAAs) into proteins enables the creation of biopolymers, proteins, and enzymes with new chemical properties, new structures, and new functions. To achieve this, amber (TAG codon) suppression has been widely applied. However, the suppression efficiency is limited due to the competition with translation termination by release factor 1 (RF1), which leads to truncated products. Recently, we constructed a genomically recoded Escherichia coli strain lacking RF1 where 13 occurrences of the amber stop codon have been reassigned to the synonymous TAA codon (rEc.E13.ΔprfA). Here, we assessed and characterized cell-free protein synthesis (CFPS) in crude S30 cell lysates derived from this strain. We observed the synthesis of 190±20 µg/mL of modified soluble superfolder green fluorescent protein (sfGFP) containing a single p-propargyloxy-L-phenylalanine (pPaF) or p-acetyl-L-phenylalanine. As compared to the parent rEc.E13 strain with RF1, this results in a modified sfGFP synthesis improvement of more than 250%. Beyond introducing a single NSAA, we further demonstrated benefits of CFPS from the RF1-deficient strains for incorporating pPaF at two- and five-sites per sfGFP protein. Finally, we compared our crude S30 extract system to the PURE translation system lacking RF1. We observed that our S30 extract based approach is more cost-effective and high yielding than the PURE translation system lacking RF1, ∼1000 times on a milligram protein produced/$ basis. Looking forward, using RF1-deficient strains for extract-based CFPS will aid in the synthesis of proteins and biopolymers with site-specifically incorporated NSAAs.

Direct polymerization of proteins.

We report the synthesis of active polymers of superfolder green fluorescent protein (sfGFP) in one step using Click chemistry. Up to six copies of the non-natural amino acids (nnAAs) p-azido-l-phenylalanine (pAzF) or p-propargyloxy-l-phenylalanine (pPaF) were site-specifically inserted into sfGFP by cell-free protein synthesis (CFPS). sfGFP containing two or three copies of these nnAAs were coupled by copper-catalyzed azide-alkyne cycloaddition to synthesize linear or branched protein polymers, respectively. The protein polymers retained ≥63% of their specific activity (i.e., fluorescence) after coupling. Polymerization of a concentrated solution of triply substituted sfGFP resulted in fluorescent macromolecular particles. Our method can be generalized to synthesize polymers of a protein or copolymers of any two or more proteins, and the conjugation sites can be determined exactly by standard genetic manipulation. Polymers of proteins and small molecules can also be created with this technology to make a new class of scaffolds or biomaterials.

Microcrystals with enhanced emission prepared from hydrophobic analogues of the green fluorescent protein chromophore via reprecipitation.

Certain synthetic analogues of the green fluorescent protein (GFP) chromophore are almost nonfluorescent in dilute solutions but are strongly light-emissive in the solid state, thus exhibiting aggregation-induced emission (AIE) behavior. In the present work, two such hydrophobic derivatives of the GFP chromophore known to be fluorescent in the crystalline state (p-hexyloxy- and p-dodecyloxybenzylideneimidazolinone) were used to prepare aqueous suspensions of particles via a mild solvent-exchange reprecipitation (RP) method. This evolution was monitored at various experimental conditions by UV-vis absorption and fluorescence spectroscopy, fluorescence microscopy, as well as electron transmission and scanning microscopy. Both compounds spontaneously produced platelet-like microcrystals, the size and shape of which were influenced by the experimental conditions. The dodecyl derivative also led to the concomitant formation of nanofibers, a tendency reinforced by addition of poly(acrylic acid) to the RP medium. The photoluminescence properties of the solids produced by RP were compared to pristine microcrystalline powders obtained by crystallization in an organic solvent. Significant differences in the emission properties were found and are discussed. This study illustrates the fact that AIE fluorescence is strongly dependent on the nature of the particles and hence on the preparation methods. Being aware of these variations is important for the preparation and subsequent use of AIE-active compounds as fluorescent materials.

Protein engineering with unnatural amino acids.

Protein engineering has become an extensively used tool in many fields, allowing us to probe protein function, characterize proteins using a range of biophysical techniques, chemically modify proteins and improve protein function for medical and industrial applications. It is now possible to site-specifically incorporate unnatural, or non-canonical, amino acids (uAAs) into proteins, which has had a major impact on protein engineering. In this review, we discuss the recent technical developments in the field and how uAA-protein engineering is becoming an increasingly valuable molecular tool, with the unique chemical functionalities of some uAAs allowing a range of otherwise impossible experiments to be performed. Finally, the impediments that have resulted in a relatively small number of recent studies in which uAA-protein engineering has been used to improve protein function are discussed, alongside some of the recent technical developments that may serve to overcome these obstacles.

Modification of a small ß-barrel protein, to give pseudo-amyloid structures, inhibits amyloid ß-peptide aggregation.

Aggregation of amyloid ß-peptide (Aß) is closely related to the pathogenesis of Alzheimer's disease (AD). Although much effort has been devoted to the construction of molecules that inhibit the aggregation of Aß1-42, high doses are needed for the inhibition of Aß aggregation in many cases. Previously, we reported that designed green fluorescent protein (GFP) analogues that gives pseudo-Aß ß-sheet structures can work as an aggregation inhibitor against Aß. To further test this design strategy, we constructed protein analogues that mimic Aß ß-sheet structures of amyloids by using insulin-like growth factor 2 receptor domain 11 (IGF2R-d11) as a scaffold. A designed protein, named IG11KK, which has a parallel configuration of Aß-like ß sheets, can bind more preferentially to oligomeric Aß1-42 than the monomer. Moreover, IG11KK suppressed the aggregation of Aß1-42 efficiently, even though lower concentrations of IG11KK than Aß were used. The aggregation kinetics of Aß in the presence of the designed proteins revealed that IG11KK can work as an inhibitor not only for the early to middle stages, but also in the latter stage of Aß aggregation owing to its favorable binding to oligomeric structures of Aß. The design strategy using ß-barrel proteins such as IGF2R-d11 and GFP is useful in generating excellent inhibitors of protein misfolding and amyloid formation.

An optimized fluorescent probe for visualizing glutamate neurotransmission.

We describe an intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) with signal-to-noise ratio and kinetics appropriate for in vivo imaging. We engineered iGluSnFR in vitro to maximize its fluorescence change, and we validated its utility for visualizing glutamate release by neurons and astrocytes in increasingly intact neurological systems. In hippocampal culture, iGluSnFR detected single field stimulus-evoked glutamate release events. In pyramidal neurons in acute brain slices, glutamate uncaging at single spines showed that iGluSnFR responds robustly and specifically to glutamate in situ, and responses correlate with voltage changes. In mouse retina, iGluSnFR-expressing neurons showed intact light-evoked excitatory currents, and the sensor revealed tonic glutamate signaling in response to light stimuli. In worms, glutamate signals preceded and predicted postsynaptic calcium transients. In zebrafish, iGluSnFR revealed spatial organization of direction-selective synaptic activity in the optic tectum. Finally, in mouse forelimb motor cortex, iGluSnFR expression in layer V pyramidal neurons revealed task-dependent single-spine activity during running.

[Synthesis of the chromophores of fluorescent proteins and their analogs].

Members of the green fluorescent protein (GFP) family are widely used in experimental biology as genetically encoded fluorescent tags. Chromophores of GFP-like proteins share a common structural core: 3,5-dihydro-4H-imidazol-4-one. This review covers synthetic approaches to 3,5-dihydro-4H-imidazol-4-ones, substituted at different positions. General, as well as specific methods, represented by single examples are considered. The most popular synthetic route to substituted 3,5-dihydro-4H-imidazol-4-ones includes synthesis of azlactones, followed by transformation into N-acyldehydroaminoacids and, finally, cyclization into target heterocycles. Accordingly, the review is divided into three parts: the first part covers syntheses of azlactones, the second part covers main approaches to N-acyldehydroaminoacids, and in the third part we summarize cyclizations of N-acyldehydroaminoacids, as well as all other approaches to 3,5-dihydro-4H-imidazol-4-ones.

Synthesis, photophysical properties, and application of o- and p-amino green fluorescence protein synthetic chromophores.

The o- and p-amino green-fluorescence-protein synthetic chromophores (GFPSCs) were synthesized from the corresponding o- and p-nitro protecting group. Among the four protecting groups of the o-amino group, the o-nitro protecting group is the only choice to synthesize the o-amino GFPSCs. The first singlet excited states of o- and p-amino GFPSCs carry significant charge-transfer character through the mechanism of photoinduced charge transfer (PCT). The o-amino GFPSCs can serve as wavelength-ratiometric fluorescence sensors that selectively recognize Cr(3+) in aqueous medium through a PCT mechanism.