Genetically encoded fluorescent reporters of histone methylation in living cells

We report the design and characterization of two genetically encoded fluorescent reporters of histone protein methylation. The reporters are four-part chimeric proteins consisting of a substrate peptide from the N-terminus of histone H3 fused to a chromodomain (a natural methyllysine-specific recognition domain), sandwiched between a fluorescence resonance energy transfer (FRET)-capable pair of fluorophores, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). Enzymatic methylation by a methyltransferase induces complexation of the methylated substrate peptide to the chromodomain, changing the FRET level between the flanking CFP and YFP domains. Reporters developed using the chromodomains from HP1 and Polycomb respond to enzymatic methylation at the lysine 9 and lysine 27 positions of histone H3, respectively, giving 60% and 28% YFP/CFP emission ratio increases in vitro or in single living cells. These reporters should be useful for studying gene silencing and X-chromosome inactivation with high spatial and temporal resolution in intact cells and may also aid in the search for conjectured histone demethylase activity.     

Synthesis and characterization of a microsphere‐based coating for textiles with potential as an in situ bioactive delivery system

Proteinaceous microspheres have a wide range of biomedical applications, including their use as drug delivery systems. On the other hand, bioactive and antimicrobial textiles are promising substrates for medical care, in particular, as wound‐dressings. This work relates the development of a new process for the functionalization of textiles through the simultaneous formation and linkage of protein‐based microspheres onto textile fibers by sonochemical techniques. The microspheres developed by this process possess antimicrobial properties by themselves, but other may be incorporated by the encapsulation of various pharmaceutical formulations. This new type of microspheres and particularly their fixation onto textile materials encourage the development of textiles that can be used as delivery systems in a simple, fast, and non‐toxic process. Here it is reported the production of microspheres with a combination of bovine serum albumin (BSA), L ‐Cysteine (L ‐Cys), and n‐dodecane, using the ultrasound technology. The size distribution and morphology of the microspheres was determined as a function of several parameters such as irradiation time and BSA and L ‐Cys concentrations. The produced microspheres were analyzed using a laser light scattering size analyzer, an optical microscope and a scanning electron microscope. The new coating of BSA + L ‐Cys microspheres revealed a high stability and excellent antibacterial properties being a promising alternative to design textile‐based bioactive delivery systems with potential application in the development of textile‐based wound‐dressings. Copyright © 2011 John Wiley & Sons, Ltd.     

Sonochemical coating of cotton and polyester fabrics with "antibacterial" BSA and casein spheres

A novel antibacterial coating for cotton and polyester fabrics has been developed by using drug-loaded proteinaceous microspheres made of bovine serum albumin and casein proteins. The microbubbles were created and anchored onto the fabrics (see figure) in a one-step reaction that lasts 3 min. The sonochemically produced "antibacterial fabrics" have been characterized. The efficiency of the sonochemical process in converting the native proteins into microspheres, encapsulating the drug, and coating the fabric has also been studied.     

Detection of mitochondrial caspase activity in real time in situ in live cells.

Apoptosis plays an important role in many physiological and pathological processes. The initiation and execution of the cell death program requires activation of multiple caspases in a stringently temporal order. Here we describe a method that allows real-time observation of caspase activation in situ in live cells based on fluorescent resonance energy transfer (FRET) measurement using the prism and reflector imaging spectroscopy system (PARISS). When a fusion protein consisting of CFP connected to YFP via an intervening caspase substrate that has been targeted to a specific subcellular location is excited with a light source whose wavelength matches the cyan fluorescent protein (CFP) excitation peak, the energy absorbed by the CFP fluorophore is not emitted as fluorescence. Instead, the excitation energy is absorbed by the nearby yellow fluorescent protein (YFP) fluorophore that is covalently linked to CFP through a short peptide containing the caspase substrate. Cleavage of the linker peptide by caspases results in loss of FRET due to the separation of CFP and YFP fluorophores. Using a mitochondrially targeted CFP-caspase 3 substrate-YFP construct (mC3Y), we demonstrate for the first time that there is caspase-3-like activity in the mitochondrial matrix of some cells at very late stage of apoptosis.     

Confocal/TEM overlay microscopy: a simple method for correlating confocal and electron microscopy of cells expressing GFP/YFP fusion proteins.

Genetic manipulation allows simultaneous expression of green fluorescent protein (GFP) and its derivatives with a wide variety of cellular proteins in a variety of living systems. Epifluorescent and confocal laser scanning microscopy (confocal) localization of GFP constructs within living tissue and cell cultures has become routine, but correlation of light microscopy and high resolution transmission electron microscopy (TEM) on components within identical cells has been problematic. In this study, we describe an approach that specifically localizes the position of GFP/yellow fluorescent protein (YFP) constructs within the same cultured cell imaged in the confocal and transmission electron microscopes. We present a simplified method for delivering cell cultures expressing fluorescent fusion proteins into LR White embedding media, which allows excellent GFP/YFP detection and also high-resolution imaging in the TEM. Confocal images from 0.5-microm-thick sections are overlaid atop TEM images of the same cells collected from the next serial ultrathin section. The overlay is achieved in Adobe Photoshop by making the confocal image somewhat transparent, then carefully aligning features within the confocal image over the same features visible in the TEM image. The method requires no specialized specimen preparation equipment; specimens are taken from live cultures to embedding within 8 h, and confocal transmission overlay microscopy can be completed within a few hours.     

Monitoring protein interactions in the living cell through the fluorescence decays of the cyan fluorescent protein.

Using fluorescence lifetime microspectroscopy and imaging techniques, we have studied the fluorescence of cyan fluorescent protein (CFP) transiently expressed in HEK-293 cells, in the presence or absence of its fluorescence resonance energy transfer (FRET) partner, yellow fluorescent protein (YFP). When the two proteins are attached through a 27-amino-acid linker, a 33 % average efficiency of intramolecular energy transfer is accurately determined inside the cell. Additionally, we observe a systematic quenching of the CFP fluorescence with increasing levels of protein expression. This quenching cannot be accounted for by formation of the previously described dimer of GFP-related proteins, since its magnitude is unchanged when the fluorescent proteins carry the mutation A206K shown to dissociate this dimer in vitro. Even when the intracellular protein concentration largely exceeds the in vitro dissociation constant of the dimer, self-association remains undetectable, either between free proteins or intramolecularly within the CFP-YFP construct. Instead, the detailed concentration effects are satisfactorily accounted for by a model of intermolecular, concentration-dependent energy transfer, arising from molecular proximity and crowding. In the case of CFP alone, we suggest that self-quenching could result from a pseudo-homo FRET mechanism between different, spectrally shifted emissive forms of the protein. These phenomena require careful consideration in intracellular FRET studies.     

A novel genetically encoded fluorescent protein as a Cu(I) indicator

We constructed the copper(I)-binding domain of Mycobacterium tuberculosis (Mtb) CDC 1551 (residues 1-162) between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), and formed a novel genetically encoded fluorescent copper(I) responsive protein (PMtb). The sensitivity and selectivity to copper(I) of the PMtb was sought. The experiments showed that the copper(I)-binding domain of the PMtb was highly sensitive and selective towards copper(I).     

Ultrasound‐Assisted One‐pot Synthesis of Bis‐azetidinones in the Presence of Zeolite

Sonochemical method is an innovative task for the sustainable chemical research industry. In this work, an attempt was made to synthesize bis‐azetidin‐2‐ones ( 2a , 2b , 2c , 2d , 2e , 2f , 2g , 2h , 2i , 2j ) by Staudinger reaction ([2 + 2] ketene‐imine cycloaddition reaction) in the presence of zeolite by using both conventional and under ultrasound irradiation. The synthesized compounds have been elucidated on the basis of their elemental analysis and spectral data (IR, ¹H NMR, ¹³C NMR, and mass spectra) to evaluate its performance obtained under ultrasonic waves. It was observed that complete conversion to azetidin‐2‐ones occurred in 1–2 h by sonochemical method and in 15–46 h by conventional method. Finally, it has been observed that 2‐azetidinones synthesis using sonochemical method is an energy efficient and environmentally friendly technique over the conventional method.     

Covalent Attachment of Cyclic TAT Peptides to GFP Results in Protein Delivery into Live Cells with Immediate Bioavailability

The delivery of free molecules into the cytoplasm and nucleus by using arginine‐rich cell‐penetrating peptides (CPPs) has been limited to small cargoes, while large cargoes such as proteins are taken up and trapped in endocytic vesicles. Based on recent work, in which we showed that the transduction efficiency of arginine‐rich CPPs can be greatly enhanced by cyclization, the aim was to use cyclic CPPs to transport full‐length proteins, in this study green fluorescent protein (GFP), into the cytosol of living cells. Cyclic and linear CPP–GFP conjugates were obtained by using azido‐functionalized CPPs and an alkyne‐functionalized GFP. Our findings reveal that the cyclic‐CPP–GFP conjugates are internalized into live cells with immediate bioavailability in the cytosol and the nucleus, whereas linear CPP analogues do not confer GFP transduction. This technology expands the application of cyclic CPPs to the efficient transport of functional full‐length proteins into live cells.     

Preparation of molecular imprinted polymer with quaternary ammonium groups as recognition sites for separation of pig cyclophilin 18 and bovine serum albumin

We introduce a new type of molecular imprinted polymer (MIP) with immobilized assistant recognition polymer chains (ARPCs) to create effective recognition sites. In this work, cloned pig cyclophilin 18 (pCyP18) and BSA were used as templates, respectively. The template protein was selectively assembled with ARPCs from the library which consists of numerous limited length polymer chains with randomly distributed recognition sites of the quaternary ammonium cationic groups and immobilizing sites. The assemblies of protein and ARPCs were adsorbed by macroporous microspheres and immobilized by cross‐linking polymerization. After removing the templates, the two kinds of synthesized MIPs were used to adsorb cloned pCyP18 and BSA from protein mixtures respectively and both showed high selectivity. It confirms that this new method is suitable to separate proteins of both low and high molecular weight. The extended experiment on adsorption of natural pCyP18 from cytosol shows that the obtained MIP using cloned protein as template can be used to enrich natural protein of low content.     

BSA‐imprinted synthetic receptor for reversible template recognition

A novel approach to the manufacturing of protein‐responsive imprints on a home‐made chitosan substrate was established together with m‐aminophenylboronic acid (APBA) as a functional monomer. The produced polymers were characterized using both (1) equilibrium adsorption assays and (2) high performance liquid chromatography analysis. Results confirmed that the synthesized BSA‐MIP (molecularly imprinted polymer) has a high affinity towards its template compared to the determined control proteins. The produced BSA‐MIP featured largely in its good adsorption reversibility, especially in competitive binding assays, which is of great biological significance in separations. Non‐specific binding was reduced to almost zero in a BSA/BHb competitive binding event. An excellent HPLC profile of template recognition was found for BSA‐MIP, even under harsh mobile phase conditions. In the present work, the adopted trapped‐template‐release method permits recovery of bound BSA [1]. The strategy of making an artificial protein‐receptor with high adsorption affinity and reversibility is promising in on‐line isolation of target protein from complicated biological environments.     

Are the Fluorescent Properties of the Cyan Fluorescent Protein Sensitive to Conditions of Oxidative Stress?

The modifications induced by reactive oxygen species (ROS) on fluorescent proteins (FPs) may have important implications for live cell fluorescence imaging. Using quantitative γ-radiolysis, we have studied the ROS-induced biochemical and photophysical perturbations on recombinant cyan fluorescent protein (CFP). After oxidation by the ˙OH radical, the protein displays a modified RP-HPLC elution profile, while the CFP fluorescence undergoes pronounced decreases in intensity and lifetime, without changes in its excitation and emission spectra. Meanwhile, the Förster resonant energy transfer (FRET) between the single W⁵⁷ and the chromophore remains unperturbed. These results rule out a direct oxidation of the CFP chromophore and of W⁵⁷ as well as major changes in the protein 3D structure, but show that new fluorescent forms associated to a higher level of dynamic quenching have been generated. Thus, strict in situ controls are required when CFP is to be used for FRET studies in situations of oxidative activity, or under strong illumination.     

Protein Denaturation Through the Use of Magnetic Molecularly Imprinted Polymer Nanoparticles

The inhibition of the protein function for therapeutic applications remains challenging despite progress these past years. While the targeting application of molecularly imprinted polymer are in their infancy, no use was ever made of their magnetic hyperthermia properties to damage proteins when they are coupled to magnetic nanoparticles. Therefore, we have developed a facile and effective method to synthesize magnetic molecularly imprinted polymer nanoparticles using the green fluorescent protein (GFP) as the template, a bulk imprinting of proteins combined with a grafting approach onto maghemite nanoparticles. The hybrid material exhibits very high adsorption capacities and very strong affinity constants towards GFP. We show that the heat generated locally upon alternative magnetic field is responsible of the decrease of fluorescence intensity.     

Antibody modified Bovine Serum Albumin microspheres for targeted delivery of anticancer agent Gemcitabine

Inhibition of the EGFR signaling pathway is one of the attractive therapeutic targets for pancreatic cancer as recent studies demonstrated that EGFR is over‐expressed in pancreatic cancer. In this article we have demonstrated the design of targeted drug delivery system containing Bovine Serum Albumin (BSA) microspheres as delivery vehicle, gemcitabine as anticancer drug and anti‐EGFR (epidermal growth factor receptor) monoclonal antibody as targeting agent. The conjugated BSA microspheres were characterized by several physico‐chemical techniques such as scanning electron microscope, optical microscopy, fluorescent microscopy etc. Administration of these BSA microspheres containing gemcitabine and anti‐EGFR (BSA‐Gem‐EGFR) shows significant inhibition of pancreatic cancer cells (AsPC1) compared to the cells treated with only BSA microspheres, BSA with gemcitabine (BSA‐Gem), and free gemcitabine. This strategy could be used as a generalized approach for the treatment of pancreatic cancer along with other cancers which overexpress EGFR on cell surface. Copyright © 2012 John Wiley & Sons, Ltd.     

Slow exchange in the chromophore of a green fluorescent protein variant

Green fluorescent protein and its mutants have become valuable tools in molecular biology. They also provide systems rich in photophysical and photochemical phenomena of which an understanding is important for the development of new and optimized variants of GFP. Surprisingly, not a single NMR study has been reported on GFPs until now, possibly because of their high tendency to aggregate. Here, we report the (19)F nuclear magnetic resonance (NMR) studies on mutants of the green fluorescent protein (GFP) and cyan fluorescent protein (CFP) labeled with fluorinated tryptophans that enabled the detection of slow molecular motions in these proteins. The concerted use of dynamic NMR and (19)F relaxation measurements, supported by temperature, concentration- and folding-dependent experiments provides direct evidence for the existence of a slow exchange process between two different conformational states of CFP. (19)F NMR relaxation and line shape analysis indicate that the time scale of exchange between these states is in the range of 1.2-1.4 ms. Thermodynamic analysis revealed a difference in enthalpy (Delta)H(0) = (18.2 +/- 3.8) kJ/mol and entropy T(Delta)S(0) = (19.6 +/- 1.2) kJ/mol at T = 303 K for the two states involved in the exchange process, indicating an entropy-enthalpy compensation. The free energy of activation was estimated to be approximately 60 kJ/mol. Exchange between two conformations, either of the chromophore itself or more likely of the closely related histidine 148, is suggested to be the structural process underlying the conformational mobility of GFPs. The possibility to generate a series of single-atom exchanges ("atomic mutations") like H --> F in this study offers a useful approach for characterizing and quantifying dynamic processes in proteins by NMR.     

An Inorganic Biopolymer Polyphosphate Controls Positively Charged Protein Phase Transitions

Polyphosphate (PolyP) is one of the most compact inorganic polyanionic biopolymers that participates in various physiological processes. However, the mechanism of the interaction between polyP and proteins remains poorly understood. Herein, we report that polyP can interact with positively charged green fluorescent protein, +36GFP, resulting in liquid–liquid phase separation (LLPS) by intermolecular electrostatic interactions in cells. Upon nutrient deprivation, genetically engineered Citrobacter freundii accumulates intracellular polyP at a rate of 210 μm min^?1, resulting in the compartmentation of +36GFP at the cell poles within 1 h. Medium chain‐length polyP (60‐mer) could induce the formation of +36GFP coacervates in vitro at a protein concentration as low as 200 nm , which is of the same magnitude as native proteins. In contrast, shorter polyP (14‐mer) could not induce LLPS under the same conditions. This may offer a general approach to manipulate protein–protein interactions through LLPS.     

Preparation and characterization of monodisperse CdS quantum dot‐polymer microspheres

A novel and effective method for the preparation of monodisperse CdS quantum dot‐polymer microspheres was proposed. The monodisperse hollow polymer microspheres were firstly swelled in chloroform. Then, the reaction precursor composed of CdO and sulfur, was impregnated into the hollow polymer microspheres. Subsequently, the CdS quantum dots were synthesized directly within the polymer microspheres by thermal decomposition. The morphology, structure, and fluorescence properties of CdS quantum dot‐polymer microspheres were studied by scanning electron microscope, transmission electron microscope, fluorescence microscope, and flow cytometry. The results indicate that the fluorescent CdS quantum dots are successfully synthesized in the monodisperse hollow polymer microspeheres, which provide very strong fluorescence intensity, and offer excellent photostability due to the compact structure of the polymer matrix. These CdS quantum dot‐polymer microspheres have potential applications in biotechnology and biomedicine. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 751–755, 2010     

Design and synthesis of intramolecular charge transfer-based fluorescent reagents for the highly-sensitive detection of proteins

Novel fluorescent molecular probes possessing both a hydroxystyryl and a cyanopyranyl moieties were designed and synthesized to detect the proteins via noncovalent bonding. These fluorescent probes indicated very weak fluorescence emission in the absence of protein. On the other hand, the fluorescence spectra of these probes showed a large Stokes shift and dramatic increase of fluorescence intensity, and red emission was observed after addition of BSA. These fluorescence spectral changes upon binding proteins were caused by the ICT process. Fluorescence intensities of the probes were plotted as a function of protein concentrations. A good linear relationship was observed up to 1000 microg/mL of protein, and the detection limit was found to be 100 ng/mL at the given assay conditions. Similar results were observed for the measurements of not only BSA but also other proteins (BGG, etc.). The responses of these probes to various nonprotein substances (inorganic salts, chelating agents, etc.) were observed, the fluorescence intensity did not change before and after the addition of foreign substances, and correct protein monitoring was successful using these fluorescent probes. To demonstrate the application of these probes, proteins after the separation using SDS-PAGE were stained in the medium containing 1, and the imaging of the proteins in the gel was successful. The experimental results clearly showed that these probes are good protein indicators for easy and highly sensitive detection.     

Nanobody‐Conjugated Nanotubes for Targeted Near‐Infrared In Vivo Imaging and Sensing

Fluorescent nanomaterials such as single‐walled carbon nanotubes (SWCNTs) have many advantages in terms of their photophysics, but it is difficult to target them to specific locations in living systems. In contrast, the green fluorescent protein (GFP) has been genetically fused to proteins in many cells and organisms. Therefore, GFP can be seen not only as a fluorophore but as a universal target/handle. Here, we report the conjugation of GFP‐binding nanobodies to DNA‐wrapped SWCNTs. This approach combines the targeting capabilities of GFP‐binding nanobodies and the nonbleaching near‐infrared fluorescence (850–1700 nm) of SWCNTs. These conjugates allow us to track single Kinesin‐5‐GFP motor proteins in developing embryos of Drosophila melanogaster. Additionally, they are sensitive to the neurotransmitter dopamine and can be used for targeted sensing of dopamine in the nm regime.