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Genetically encoded fluorescent indicators for imaging intracellular potassium ion concentration
摘要： Potassium ion (K+) homeostasis and dynamics play critical roles in biological activities. Here we describe three genetically encoded K+ indicators. KIRIN1 (potassium (K) ion ratiometric indicator) and KIRIN1-GR are F?rster resonance energy transfer (FRET)-based indicators with a bacterial K+ binding protein (Kbp) inserting between the fluorescent protein FRET pairs mCerulean3/cp173Venus and Clover/mRuby2, respectively. GINKO1 (green indicator of K+ imaging) is a single fluorescent protein-based K+ indicator constructed by insertion of Kbp into enhanced green fluorescent protein (EGFP). These indicators are suitable for detecting K+ at physiologically relevant concentrations in vitro and in cells. KIRIN1 enabled imaging of cytosolic K+ depletion in live cells and K+ efflux and reuptake in cultured neurons. GINKO1, in conjunction with red fluorescent Ca2+ indicator, enable dual-color imaging of K+ and Ca2+ dynamics in neurons and glial cells. These results demonstrate that KIRIN1 and GINKO1 are useful tools for imaging intracellular K+ dynamics.
关键词： FRET-based sensors,potassium ion imaging,single fluorescent protein sensors,intracellular K+ dynamics,genetically encoded indicators
更新于2025-11-21 11:24:58
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Green Fluorescent Protein-Based Glucose Indicators Report Glucose Dynamics in Living Cells
摘要： Glucose is the most important energy source for living animals. Here, we developed a series of single fluorescent protein (FP)-based glucose indicators, named as "Green Glifons", to understand the hierarchal and mutual relationships between molecules involved in energy metabolism. Three indicators showed a different EC50 for glucose (50 μM, 600 μM and 4,000 μM), producing a ~7-fold change in fluorescence intensity in response to glucose. The indicators could visualize glucose dynamics in the cytoplasm, plasma membrane, nucleus and mitochondria of living HeLa cells and in vivo, in the pharyngeal muscle of C. elegans and could measure murine blood glucose levels. Finally, the indicators were applicable to dual-color imaging, revealing the dynamic interplay between glucose and Ca2+ in mouse pancreatic MIN6 m9 β cells. We propose that these indicators will facilitate and contribute to in vivo and multi-color imaging of energy metabolism.
关键词： biosensors,artificial sweeteners,dual-color imaging,C. elegans,live cell imaging,glucose,blood glucose level,fluorescent protein
更新于2025-11-21 11:24:58
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Does the wavelength dependent photoisomerization process of the p?coumaric acid come out from the electronic state dependent pathways?
摘要： Similar to the anion photoactive yellow protein (PYP) chromophore, the neutral form of the PYP chromophore was also found to exhibit a the wavelength-dependent photoisomerization quantum yield. The isomerization quantum yield increases with the increasing excitation energy on the S1 state, while decreases when being excited to the S2 state. Does this wavelength dependent product yield come out from the specific reaction pathways of the S1 and S2 states? This would mean that, the relaxation pathway of the S2 state is distinct from that of the S1 state and does not involve twisting motion. Does it break Kasha's rule by exhibiting a direct transition from the S2 state to the ground state? The underlying mechanism needs further in. In this article, we employed the on-the-fly dynamics simulations and static electronic structure calculations to reveal the deactivation mechanism of the neutral form of the PYP chromophore. Our results indicated that the C_C twisting motion dominates the S1 state decay process. In contrast, for the decay process of the S2 state, an ultrafast transition from the S2 to the S1 state through a planar conical intersection is observed, and the excess energy activates a new reaction channel to the ground state characterized by a puckering distortion of the ring. This pathway competes with the photoisomerization channel. No direct transition from S2 to S0 is observed, hence Kasha's rule is valid for this process. Our calcualtions can provide a reasonable explanation of the wavelength-dependent isomerization quantum yield of neutral PYP chromophore, and we hope it can provide theoretical foundations for comparing the effect of protonation state on the dynamcal behaviors of PYP chromophore.
关键词： Wavelength dependent,Photoisomerization,Fluorescent protein,Nonadiabatic process
更新于2025-09-23 15:23:52
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Ethyl violet–bovine serum albumin fluorescent protein nanovessels target to lysosomes and mitochondria
摘要： Aim: Organelles are essential in maintaining homeostasis of mammalian cells. Monitoring the morphology and dynamics of organelles is of significance in cell state determination and disease diagnosis. Materials & methods: We describe here a new material called ethyl violet–bovine serum albumin fluorescent protein nanovessel (EV–BSA FPN). Upon heating, BSA was denatured to form higher polyhedral structures, which was prone to EV binding. These dye–protein hybrid materials were red fluorescence emissive upon excitation. Results: EV–BSA FPNs can be readily internalized by mammalian cells and dual localized in lysosomes and mitochondria. Besides, EV–BSA FPN can serve as carriers and efficiently deliver drug into cells. Conclusion: EV–BSA FPNs can be dual function fluorescent vessels for both dual-organelle imaging and drug delivery.
关键词： dual-organelle localization,fluorescent protein nanovessel,lysosomes and mitochondria imaging,ethyl violet,drug carrier
更新于2025-09-23 15:23:52
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Detection of the conformational changes of <i>Discosoma</i> red fluorescent proteins adhered on silver nanoparticles-based nanocomposites <i>via</i> surface-enhanced Raman scattering
摘要： Description of the relationship between protein structure and function remains a primary focus in molecular biology, biochemistry, protein engineering and bioelectronics. Regardless the targeted application, the current strategies on revealing the relationship between protein structure and function lead to exposure and interaction of proteins with non-biological organic and inorganic solid surfaces. Proper description of the underlying mechanisms will certainly unveil the fundamental protein-adsorption problem and add value to the effort of record and quantification of the conformational changes of the protein native state when interacting with solid surfaces. To that end the application of physics-based diagnostic methods is suitable and highly demanded. Raman spectroscopy appears the most frequently used method for the study of biomolecule recognition, and ultra-sensitive analysis, down to a single molecule. However, to tackle the sensitivity limitations of Raman spectroscopy imposed by the small Raman cross sections, the biological systems should be coupled with metallic nanostructures. The scattering efficiency can be thus increased by several orders of magnitude due to the activation of localized surface plasmon resonance (LSPR) that induces strong enhancement of the electromagnetic (EM) field in the vicinity of the metallic surface. This enables to largely extend the application of Raman spectroscopy in molecular spectroscopy, biomolecule recognition, and ultra-sensitive analysis, down to a single molecule. Besides the sensing properties, the strong EM enhancement can be exploited to probe protein conformational changes under photoexcitation, including real-time monitoring. Therefore, since its discovery in the late 70s, the Surface-Enhanced Raman Scattering (SERS) has proven to be a very powerful and reliable analytical tool for chemo- and bio-sensing, due to the strong enhancement of the vibrational signatures of analytes in different chemical environments. In this context, a lot of resources and time have been employed in the effort to develop plasmonic substrates based on metallic nanostructures aiming at a further increase of the EM enhancement for the realization of noninvasive, highly-sensitive, and large-scale optical sensors. A large variety of metallic nanostructure morphologies and arrangements (nanosphere, nanotriangles, nanodisks, nanorods, nanocubes, etc.) and different coupling geometries (dimers, trimers, arrays, etc.) have been developed up to date for SERS platforms. However, their conversion to macroscopic plasmonic substrates relies generally on the NPs volunteer arrangement on dielectric surfaces (mainly through applying chemical methods), thus often resulting in non-uniform distribution on large areas, without a well-defined control of the spacing between the metallic nanostructures and the probed molecules, high point-to-point variability, scarce reproducibility and stability under irradiation conditions (due to photothermal and photodegradation processes). To overcome the limitations in producing solid SERS substrates various physical approaches, like thermal evaporation, combined nanoimprint lithography-shadow evaporation, gas aggregation source (GAS), pulsed laser deposition (PLD), low-energy ion beam synthesis (LE-IBS), and plasma-based deposition processes, have been proposed in the literature. It is generally acknowledged that the silver nanoparticles (AgNPs) realize the best nanoscale antenna in the visible range for amplifying local electronic and vibrational signals, thus providing unique molecular information in the optical far-field regime. Indeed, compared to gold nanoparticles, the AgNPs offer the advantage of stronger plasmonic enhancement because of lower interference between intraband and interband electronic transitions. Moreover, the use of AgNPs covers another aspect of the relationship between protein structure and function which concerns the biological activity of the AgNPs. Because of their antimicrobial properties, the AgNPs have the potential to impact human health and environment. The biological activity of AgNPs goes both ways, through the activity of ionic silver (Ag+) and through direct contact with the AgNPs resulting in protein denaturation at different cell locations; specifically sensible are those enzymes of the respiratory chain and transport channels. Therefore, there exists a recognized need to address the relationship between protein structure and function from two distinctly different vantage points: (i) quantification of the conformational changes of proteins by using the antenna effect of AgNPs and (ii) analysis of the conformational changes of proteins induced by the AgNPs extreme chemical and biological activities. The intent of this work is to bring additional insight into the mechanisms of adsorption of proteins on solid surfaces through quantification of the conformational changes of proteins adhered on AgNPs-based nanocomposites via SERS. We focus on the wild-type Discosoma recombinant red fluorescent protein (DsRed), belonging to the family of naturally fluorescent proteins (FPs). The strong interest toward the FP family originates from their application in molecular biology as reporters of gene expression, as noninvasive markers in molecular biology and other singular events of cell activity. Potential use of the FPs extends toward therapeutics, tissue regeneration, bioelectronics and protein engineering. The most widely characterized member of this family is the green fluorescent protein (GFP). The lately cloned from reef coral Discosoma sp. DsRed protein possesses the longest yet reported, for a wild-type spontaneously fluorescent protein, excitation and emission maxima at 558 nm and 583 nm, respectively. Owing to its high fluorescence yield the red fluorescent DsRed protein has become important both as a model for understanding fluorescent proteins and as a tool for biomedical research. The DsRed protein and its engineered derivatives have found broad use in cell and molecular biology including fluorescence microscopy as a marker, fluorescence correlation spectroscopy (FCS) and fluorescence activated cell sorting (FACS). Recently, the DsRed was found suitable for rational design of ultra-stable and reversible photoswitches for super-resolution imaging. Moreover, it has been hypothesized that FPs from reef-building corals operate as part of an adaptive mechanism to optically interact and to regulate the symbiotic relationship between corals and photosynthetic algae. Structural rearrangements near the chromophore influence the maturation speed and brightness of the DsRed variants. It is therefore essential to examine the conformational transitions that affect the protein’s ability to transfer optical excitation energy. Studies of the conformational changes of DsRed protein have been reported in the literature but the DsRed Raman fingerprints were investigated only by recurring to chemically synthesized model chromophores. However, the later differ from the wild-type DsRed protein for the absence of the α-helix and β-sheets that naturally surround the chromophore and for the different extensions of the conjugated π-system. The choice of chemically synthesized model chromophores is explained by the complications brought by the presence of immature green species in the solution created as a photoproduct of the red ones, thus often resulting in an unclear or incomplete band assignment. The novelty of this work lays down the point that we work with the wild-type DsRed protein in its native state and not with DsRed model chromophore. All reported experimental studies in the literature were performed in solution. No information on the DsRed protein structural and conformational changes can be found when the DsRed protein is adhered on a solid substrate and irradiated by light. The lack of information on the above discussed issues motivated this study focusing on the investigation of the interaction of wild-type DsRed proteins with AgNPs-based plasmonic substrates. Our approach involves analysis of dehydrated DsRed protein layers in link with natural conditions during drying. To perform the SERS study on the conformational changes of DsRed proteins adhered on AgNPs-based nanocomposites we have elaborated, by plasma process, highly uniform and reproducible plasmonic substrates composed of a single layer of AgNPs coated by a silica layer. Focus was made on the possibility to well control, on a large scale, the AgNPs size distribution and interparticle distances. The resulting uniformity of hot-spot distribution guarantees the reproducibility and stability of this plasmonic sensor. Subsequently, we show how the enhanced EM field in the vicinity of the AgNPs could be employed to detect the presence and identify the conformational changes of proteins, adsorbed and adhered to the plasmonic substrate, during optical irradiation. The achieved enhancement of the electromagnetic field in the vicinity of the AgNPs is as high as 105. This very strong enhancement factor allowed detecting Raman signals from discontinuous layers of DsRed issued from solution with protein concentration of only 80 nM. Three different conformations of the DsRed proteins after adhesion and dehydration on the plasmonic substrates were identified. It was found that the DsRed chromophore structure of the adsorbed proteins undergoes optically assisted chemical transformations when interacting with the optical beam, which leads to reversible transitions between the three different conformations. The proposed time-evolution scenario endorses the dynamical character of the relationship between protein structure and function. It also confirms that the conformational changes of proteins with strong internal coherence, like DsRed proteins, are reversible.
关键词： plasmonic substrate,protein conformation,surface-enhanced Raman scattering,plasma deposition process,Discosoma red-fluorescent protein DsRed,Silver nanoparticles
更新于2025-09-23 15:22:29
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[Methods in Molecular Biology] Autophagy Volume 1880 (Methods and Protocols) || Correlative Light and Electron Microscopy to Analyze LC3 Proteins in Caenorhabditis elegans Embryo
摘要： In this chapter, we present a protocol to perform correlative light and electron microscopy (CLEM) on Caenorhabditis elegans embryos. We use a specific fixation method which preserves both the GFP fluorescence and the structural integrity of the samples. Thin sections are first analyzed by light microscopy to detect GFP-tagged proteins, then by transmission electron microscopy (TEM) to characterize the ultrastructural anatomy of cells. The superimposition of light and electron images allows to determine the subcellular localization of the fluorescent protein. We have used this method to characterize the roles of autophagy in the phagocytosis of apoptotic cells in C. elegans embryos. We analyzed in apoptotic cell and phagocytic cell the localization of the two homologs of LC3/GABARAP proteins, namely, LGG-1 and LGG-2.
关键词： LC3-associated phagocytosis,High-pressure freezing,Freeze substitution,LGG-2,Green fluorescent protein,GMA resin,LGG-1
更新于2025-09-23 15:22:29
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Detection of Metastasis in a Patient-derived Orthotopic Xenograft (PDOX) Model of Undifferentiated Pleomorphic Sarcoma with Red Fluorescent Protein
摘要： Background/Aim: Undifferentiated pleomorphic sarcoma (UPS) is a common soft tissue sarcoma and highly recalcitrant. We have previously developed patient-derived orthotopic xenograft (PDOX) mouse models of UPS and other major sarcoma types. Unlike PDOX models of other cancer types, it has been difficult to demonstrate metastasis in the sarcoma PDOX models. Materials and Methods: To visualize metastasis at high resolution in the UPS PDOX model, established tumor fragments were implanted in transgenic nude mice expressing red fluorescent protein (RFP) for one passage. The tumors acquired RFP-expressing stroma from transgenic host. UPS tumor with RFP stromal cells were harvested and implanted orthotopically in non-transgenic nude mice. After six weeks of UPS tumor growth in the PDOX model, the primary tumor was imaged non-invasively and lung, liver, and spleen were resected and imaged ex-vivo in order to visualize the presence of RFP, with a FluorVivo? imaging system and FV1000? confocal laser microscope, respectively. Results: The primary tumor was imaged non-invasively. Confocal microscopy visualized the presence of RFP in the lung and liver indicating metastases in these organs. This is the first report of metastasis in a sarcoma PDOX model. Conclusion: This study should prove very useful to screen for anti-metastatic drugs for the PDOX donor patients and to understand the metastatic process in sarcoma.
关键词： PDOX,patient-derived orthotopic xenograft,red fluorescent protein,soft-tissue,stromal cell,Undifferentiated pleomorphic sarcoma,metastasis
更新于2025-09-23 15:22:29
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A Recombinant Fluorescent Peptidomimetic Tracer for Immunodetection of Imidaclothiz
摘要： Peptidomimetic and anti-immunocomplex peptides, which could be readily isolated from phage display library, have shown a great potential for small molecule immunoassay development because they typically improve the sensitivity and avoid the use of chemical haptens as coating or tracers antigens. However, the phage borne peptides are unconventional immunoassay reagent, which greatly limits their use in commercial applications and requires secondary reagents for detection. In order to overcome these limitations, we used C2-15, a peptidomimetic of imidaclothiz, as a model peptide fused to emerald green fluorescent protein (EmGFP) at the N terminus (C2-15-EmGFP) and C terminus (EmGFP-C2-15) to generate novel fluorescent peptide tracers. Both recombinant fluorophores reacted with similar affinity to the anti-imidaclothiz monoclonal antibody 1E7, but due to its higher expression C2-15-EmGFP was chosen to develop a competitive magnetic separation fluorescence immunoassay (MSFIA). After a competitive step with the analyte, the C2-15-EmGFP/antibody complex bound to the magnetic beads was separated with a magnet, and due to the fast dissociation of the peptide-antibody interaction, the fluorescence signal was detected following the spontaneous dissociation of the complex in fresh buffer. The concentration of imidaclothiz causing the 50% inhibitory concentration (IC50) was 11.00 ng mL-1, and the MSFIA performed with excellent recovery and good correlation with high-performance liquid chromatography in different matrices.
关键词： Magnetic nanoparticles,Emerald green fluorescent protein,Recombinant peptide,Imidaclothiz,Peptidomimetic
更新于2025-09-23 15:21:01
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Side chain torsion dictates planarity and ionizability of Green fluorescent protein’s chromophore leading to spectral perturbations.
摘要： Spectral characteristics of Fluorescent Proteins (FP) are well studied and through protein engineering, several FP variants constituting entire visible spectrum have been created. One of the most common mechanisms attributed to spectral shifts in FP are excited state proton transfer (ESPT), hydroxyl moiety protonation and deprotonation, along with chromophore Cis-Trans isomerism. Most widely studied FPs are those derived from avGFP (Aequorea victoria GFP) and Dsred (Discosoma coral). Apart from the above mechanism, certain interacting residues are said to play a vital role in altering the proton transfer pathway shift. The torsional changes of wild avGFP, S65T avGFP, and Dsred has been studied to van der Waals packing in Dsred is more compact than that of avGFP, thus creating a low solvent occupiable environment and reduced solvent interactions having higher red spectral hypothesize the torsional landscape and altered residual interactions are prominent factors for the spectral shifts. Through our 200 ns molecular dynamics investigation we prospect that leading to numerous spectral variants. Similarly, the hydrogen-bonded networks solely cannot dictate the energy landscape of FPs. Non-bonded interactions also can create secondary harmonic shifts by dipole-dipole inductions. Side chain contacts tend to alter the topological and torsional geometry, thereby disturbing the chromophore’s planarity. Side chain torsional variations have almost been unaccounted for their distortions in FP’s. We comprehend the inter-residual contact distance and the geometrical descriptors.
关键词： Green fluorescent protein,spectral perturbations,Sidechain torsional variations,Residual accessible area,Chromophore planarity
更新于2025-09-23 15:21:01
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Impact of external amino acids on fluorescent protein chromophore biosynthesis revealed by molecular dynamics and mutagenesis studies
摘要： The precise positioning of catalytic amino acids against the substrate in an enzyme active site is a crucial factor in biocatalysis. Biosynthesis of the chromophores of fluorescent proteins (FPs) is an autocatalytic process that must conform to these requirements. Here, we show that, in addition to the internal amino acid residues in the proximity of the chromophore, chromophore biosynthesis is influenced by the remote amino acids exposed on the outer surface of the β-barrel structure of the FP. It has been shown earlier that chromophore biosynthesis of the red FP from Zoanthus sp. (zoan2RFP) proceeds via an immature green state. At the same time, the green state is the final stage of chromophore biosynthesis of green FP (zoanGFP), which is highly homologous to zoan2RFP. It was also shown that a single N66D substitution in the chromophore-forming sequence of zoanGFP might trigger the synthesis of the red chromophore. However, in this case, the synthesis of the red chromophore is incomplete and occurs only at elevated temperatures. Here, we tried to uncover additional structural determinants that govern the biosynthesis of the red chromophore. A comparison of zoanGFP and zoan2RFP revealed intrabarrel amino acid differences at five positions. Exhaustive substitutions of these five positions in zoanGFP-N66D gave rise to zoanGFPmut with the same intrabarrel amino acid composition as zoan2RFP. zoanGFPmut showed only partial green-to-red chromophore transformation at elevated temperatures. To elucidate the extra factors that can affect red chromophore biosynthesis, we performed comparative molecular dynamics simulations of zoan2RFP and zoanGFPmut. The simulations revealed several external amino acids that might influence the arrangement and flexibility of the chromophore-surrounding amino acid residues in these proteins. Mutagenesis experiments confirmed the crucial role of these residues in red chromophore biosynthesis. The obtained zoanGFPmut2 exhibited complete green-to-red transformation, suggesting that the mutated amino acids exposed on the surface of the β-barrel contribute to red chromophore biosynthesis.
关键词： autocatalysis,chromophore,protein engineering,fluorescent protein,molecular dynamics simulation,long-range interaction
更新于2025-09-23 15:19:57