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Fluorescent Proteins as Biomarkers and Biosensors: Throwing Color Lights on Molecular and Cellular Processes
Abstract
Green fluorescent protein (GFP) from jellyfish Aequorea victoria is the most extensively studied and widely used in cell biology protein. GFP-like proteins constitute a fast growing family as several naturally occurring GFP-like proteins have been discovered and enhanced mutants of Aequorea GFP have been created. These mutants differ from wild-type GFP by conformational stability, quantum yield, spectroscopic properties (positions of absorption and fluorescence spectra) and by photochemical properties. GFP-like proteins are very diverse, as they can be not only green, but also blue, orange-red, far-red, cyan, and yellow. They also can have dual-color fluorescence (e.g., green and red) or be non-fluorescent. Some of them possess kindling property, some are photoactivatable, and some are photoswitchable. This review is an attempt to characterize the main color groups of GFP-like proteins, describe their structure and mechanisms of chromophore formation, systemize data on their conformational stability and summarize the main trends of their utilization as markers and biosensors in cell and molecular biology.
... It is intrinsically fluorescent, emitting a brilliant green light when exposed to ultraviolet or blue light, due to a chromophore formed from a maturation reaction of three specific aa at the center of the protein (Ser65, Tyr66, and Gly67) [4,5]. An enhanced GFP variant (eGFP) was used in the present work, which contained substitution of Phe64 to Leu, which improves folding at 37 °C, and substitution of Ser65 to Thr that makes the protein 35 times brighter than the wild-type GFP [5,6]. The eGFP variant was obtained from the construction of a library of mutant GFP molecules using an oligo-directed, codon-based mutagenesis method [7], one of the several protein design techniques presented in the lecture course. ...
... It is intrinsically fluorescent, emitting a brilliant green light when exposed to ultraviolet or blue light, due to a chromophore formed from a maturation reaction of three specific aa at the center of the protein (Ser65, Tyr66, and Gly67) [4,5]. An enhanced GFP variant (eGFP) was used in the present work, which contained substitution of Phe64 to Leu, which improves folding at 37 • C, and substitution of Ser65 to Thr that makes the protein 35 times brighter than the wild-type GFP [5,6]. The eGFP variant was obtained from the construction of a library of mutant GFP molecules using an oligo-directed, codonbased mutagenesis method [7], one of the several protein design techniques presented in the lecture course. ...
... GFP-like proteins are widely used as quantitative genetically encoded markers for studying protein-protein interactions and cell tracking [12,13]. One of the most interesting aspects of GFP over other fluorescent tags is that the chromophore forms spontaneously and without accessory co-factors, substrates, or enzymes; it only requires the presence of oxygen during maturation [6], which means that the gene could be taken directly from A. victoria and expressed in other organisms as the Gram-negative bacterium Escherichia coli while still maintaining fluorescence. ...
Protein Engineering is a highly evolved field of engineering aimed at developing proteins for specific industrial, medical, and research applications. Here, we present a practical teaching course to demonstrate fundamental techniques used to express, purify and analyze a recombinant protein produced in Escherichia coli—the enhanced green fluorescent protein (eGFP). The methodologies used for eGFP production were introduced sequentially over six laboratory sessions and included (i) bacterial growth, (ii) sonication (for cell lysis), (iii) affinity chromatography and dialysis (for eGFP purification), (iv) bicinchoninic acid (BCA) and fluorometry assays for total protein and eGFP quantification, respectively, and (v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for qualitative analysis. All groups were able to isolate the eGFP from the cell lysate with purity levels up to 72%. Additionally, a mass balance analysis performed by the students showed that eGFP yields up to 46% were achieved at the end of the purification process following the adopted procedures. A sensitivity analysis was performed to pinpoint the most critical steps of the downstream processing.
... The green fluorescent protein (GFP; 27 kDa) is the most commonly used indicator for successful gene transfection and expression in tissues such as skeletal muscle. The gene encoding GFP was identified in the jellyfish Aequorea victoria and produces a protein with fluorescent excitation and emission that can be viewed under the microscope or through in vitro and in vivo live imaging (130,131). One of the advantages of using GFP is its production of a fluorescent signal in the intracellular space allowing for direct observation and identification of successfully transfected cells over time (Fig. 2C). ...
... The advancement of fluorescent bioimaging in intact organelles, live cells, and whole organisms has led to the development of GFP-like proteins. These GFP-like proteins from Anthozoa species have fluorescent spectra positions for blue, orange-red, far-red, cyan, and yellow (130). In skeletal muscle, proteins that are conjugated (tagged) with GFP or GFP-like proteins can be used in localization studies performed using fusion proteins to determine if they are targeted to subcellular structures such as neuromuscular junctions or mitochondria (54,123,124,132). ...
The strategy of gene delivery into skeletal muscles has provided exciting avenues in identifying new potential therapeutics towards muscular disorders and addressing basic research questions in muscle physiology through overexpression and knockdown studies. In vivo electroporation methodology offers a simple, rapidly effective technique for the delivery of plasmid DNA into post-mitotic skeletal muscle fibers and the ability to easily explore the molecular mechanisms of skeletal muscle plasticity. The purpose of this review is to describe how to robustly electroporate plasmid DNA into different hindlimb muscles of rodent models. Further, key parameters (e.g., voltage, hyaluronidase, plasmid concentration) which contribute to the successful introduction of plasmid DNA into skeletal muscle fibers will be discussed. In addition, details on processing tissue for immunohistochemistry and fiber cross-sectional area (CSA) analysis will be outlined. The overall goal of this review is to provide the basic and necessary information needed for successful implementation of in vivo electroporation of plasmid DNA and thus open new avenues of discovery research in skeletal muscle physiology.
... 1,2,4-Trisubstituted imidazolinones are major contributors to the fluorescence of FPs, which are useful tools for the study of gene regulation, protein localization, and for live imaging of intermolecular interactions. [19,20]. Several synthetic methodologies were developed for the synthesis of imidazolinone analogs because of their great importance ( Figure 2). ...
... Conducting the reaction for 10 minutes or less afforded the desired product in lower yield and gave large amount of unreacted starting materials (entries 22-23). Neat 230 (conv) 5 33 Reaction time higher than 20 minutes led to consumption of starting material but resulted in more degradation products (entries [18][19]. Carrying out the reaction for 20 minutes resulted in significant increase in the yield of 2a (73%, entry 20). ...
We have developed a solvent- and catalyst-free cyclization reaction for the synthesis of 1-arylidenamino-2,4-disubstituted-2-imidazoline-5-ones under neat conditions. The new synthetic procedure is efficient and green in nature. All synthesized compounds were obtained in good yields (70–78%) and have been characterized by ¹H and ¹³C NMR spectroscopy. The structure of compound 2d was characterized crystallographically using a single crystal X-Ray diffractometer. Compound 2d was found to crystallize in the triclinic space group P-1 with Z = 2. Moreover, the Frontier Molecular Orbitals (FMOs), the global chemical reactivity descriptors, and the molecular Electrostatic Potential (MEP) of 2d have been calculated at B3LYP/6-31G(d,p) level of theory.
... Green fluorescent protein (GFP) is one of the most extensively studied and widely used fluorescent proteins in biochemistry and cell biology [1][2][3] . Due to intrinsic fluorescence, fast and complete maturation, good toleration in fusion proteins and convenience of visualization with standard filter sets on conventional fluorescence instruments, GFP has shown great usefulness not only for studying protein dynamics and functions but also for the detection of ions, metabolite messengers and protein interactions in live cells and in vivo 4 . ...
Fluorescent RNAs (FRs) provide an attractive approach to visualizing RNAs in live cells. Although the color palette of FRs has been greatly expanded recently, a green FR with high cellular brightness and photostability is still highly desired. Here we develop a fluorogenic RNA aptamer, termed Okra, that can bind and activate the fluorophore ligand ACE to emit bright green fluorescence. Okra has an order of magnitude enhanced cellular brightness than currently available green FRs, allowing the robust imaging of messenger RNA in both live bacterial and mammalian cells. We further demonstrate the usefulness of Okra for time-resolved measurements of ACTB mRNA trafficking to stress granules, as well as live-cell dual-color superresolution imaging of RNA in combination with Pepper620, revealing nonuniform and distinct distributions of different RNAs throughout the granules. The favorable properties of Okra make it a versatile tool for the study of RNA dynamics and subcellular localization.
... Fluorescent proteins (FPs) are widely used optical markers in molecular and cellular biology [1,2]. FPs are useful for tracking the target functions in research applications such as in Förster or fluorescence resonance energy transfer (FRET) [3,4], optogenetics [5], chemogenetics [6,7], subcellular localization [8], in vivo imaging [9,10], and genome editing [11]. ...
The monomeric red fluorescent protein DsRed (mDsRed) is widely used as an optical probe for multicolor applications in flow cytometry or fluorescence microscopy. Understanding the structure and dynamics of mDsRed provides fundamental information for its practical applications. The mDsRed crystal structure has been reported, but the structural dynamics have not been fully elucidated. Herein, the crystal structure of mDsRed was determined at 2.9 Å resolution, and the molecular flexibility was analyzed. mDsRed contains a solvent-accessible hole between the β7-strand and β9-α10 loop, which is connected to the chromophore. A partial disorder was present in the electron density map of the tyrosine-ring group of the mDsRed chromophore, indicating a flexible conformation of the chromophore. The refined mDsRed chromophore displayed a cis-conformation with a nonplanar configuration between the tyrosine and imidazoline rings of the chromophore. Temperature factor analysis indicated that the β-barrel fold of mDsRed is rigid, while the loops at the top and bottom of the β-barrel are relatively flexible. The β-barrel surface of mDsRed was closer to the native conformation compared with the previously reported Zn-bound state of mDsRed. These structural findings extend our understanding of the molecular flexibility of mDsRed.
... Column-I shows the spectra of original/wild-types and column-II shows the spectra of mutants. Solid lines indicate emission spectra whereas dotted lines indicate absorption spectra(Stepanenko et al., 2008). ...
... Relative fluorescence units (RFUs) were calculated using a LAS X relative fluorescence calculator using a 200 × 200-µm square as a background measurement for the fluorescence intensity of the worm. F(t) = fluorescence channel/region of interest (ROI); F(0) = fluorescence channel/background (Bkg), and K is set to 1 as normalized EGFP (Stepanenko et al., 2008): ...
The short lifespan of Caenorhabditis elegans enables the efficient investigation of probiotic interventions affecting stress and longevity involving the potential therapeutic value of Lactococcus lactis and Leuconostoc mesenteroides isolated from organic basil. The lactic acid bacteria were cultured from the produce collected from a local grocery store in Tulsa, Oklahoma, and then identified through 16S rDNA sequencing and biochemical tests. To dive deep into this analysis for potential probiotic therapy, we used fluorescent reporters that allow us to assess the differential induction of multiple stress pathways such as oxidative stress and the cytoplasmic, endoplasmic reticulum, and the mitochondrial unfolded protein response. This is combined with the classic health span measurements of survival, development, and fecundity, allowing a wide range of organismal observations of the different communities of microbes supported by probiotic supplementation with Lactococcus lactis and Leuconostoc mesenteroides. These strains were initially assessed in relation to the Escherichia coli feeding strain OP50 and the C. elegans microbiome. The supplementation showed a reduction in the median lifespan of the worms colonized within the microbiome. This was unsurprising, as negative results are common when probiotics are introduced into healthy microbiomes. To further assess the supplementation potential of these strains on an unhealthy (undifferentiated) microbiome, the typical axenic C. elegans diet, OP50, was used to simulate this single-species biome. The addition of lactic acid bacteria to OP50 led to a significant improvement in the median and overall survival in simulated biomes, indicating their potential in probiotic therapy. The study analyzed the supplemented cultures in terms of C. elegans’ morphology, locomotor behavior, reproduction, and stress responses, revealing unique characteristics and stress response patterns for each group. As the microbiome’s influence on the health span gains interest, the study aims to understand the microbiome relationships that result in differential stress resistance and lifespans by supplementing microbiomes with Lactococcus lactis and Leuconostoc mesenteroides isolated from organic basil in C. elegans.
... If aging is proven to have significant effects on accelerating AD progression compared to the loss of SIRT1, the progression of AD would likely be more of a time-reliant process including the accumulations of pathway errors, the weakening of repairment systems, expression of genes through time and the buildup of DNA damages [25][26][27][28]. Therefore, on the one hand, more focus should be put on comprehensive anti-aging measures to find solutions to AD; On the other hand, the result suggests that the effects of aging in AD mice model studies are significant and that mice models with early expression of AD pathological markers could bring unignorable distortions [29,30]. In that case, mice models with AD progression through natural aging should be utilized more widely in research henceforth. ...
Alzheimer’s (AD) is a neurodegenerative disease closely linked to aging. However, mouse models with early onset AD are widely utilized, which may cause data deviations since AD normally occurs in aged subjects. In this work, we devised transgenic mouse models with SIRT1, Aβ42 regulated at the first month after birth and 19th months old via Tet-Off/CREer-LoxP systems. Aging/AD/SIRT1-interacted biomarkers were tracked throughout life. Based on the models, the effects of SIRT1, APP, and aging on AD progression are differentiated through temporal manipulation. The possible results of equal or more significant effects of SIRT1 deficiency on AD progression compared to aging hint the presence of pathology distinctly related to AD, which might lie in induced increased neuron death signals and unprotected mitochondria from blocked P53, triggered by loss of SIRT-1 control over apoptotic factors including P53 and FOXO3a. The data obtained from our work can reveal the magnitude of deviation resulting from using early-onset AD models rather than aging mice, hence serving as a reference to possible bias. The raised pathology about SIRT1 and uncontrolled apoptotic factors could serve as a potential target for AD study.
... The Green Fluorescent Protein (GFP) is a light-producing protein from the jellyfish Aequora Victoria [5] with a bright green, fluorescent emission. In 2008, the Nobel Prize in chemistry was attributed to Osamu Shimomura, Martin Chalfie and Roger Tsien for the work developed on the discovery and isolation of the GFP [6]. ...
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.
... At present, we are in the state where we are harnessing the natural scaffold and functions of photoreceptors with minimal engineering such as random or site-directed mutagenesis to optimize or alter its performance Taslimi et al., 2016;Zoltowski et al., 2009). With the advent of modern biotechnology such as enzyme directed evolution (Chen and Arnold, 1991;Herwig et al., 2017;Yang et al., 2019), and de novo protein design (Huang et al., 2016;Kuhlman et al., 2003), together with the aid of artificial intelligence and advanced algorithms for protein structure prediction (Senior et al., 2020;Yang et al., 2020b), it is highly envisioned that we can push forward optogenetics to a more synthetic and diversified level by engineering existing photoreceptors into variants of distinct wavelength specificity, (like how variants Green Fluorescent Proteins were developed (Stepanenko et al., 2008)), or entirely new photoreceptors with novel modes of action. These will in turn, enrich the assortment of optogenetic toolkits and promote the development of microbial synthetic biology and for a more sustainable world. ...
Chemical induction is one of the most common modalities used to manipulate gene expression in living systems. However, chemical induction can be toxic or expensive that compromise the economic feasibility when it comes to industrial-scale synthetic biology applications. These complications have driven the pursuit of better induction systems. Optogenetics technique can be a solution as it not only enables dynamic control with unprecedented spatiotemporal precision but also is inexpensive and eco-friendlier. The optogenetic technique harnesses natural light-sensing modules that are genetically encodable and re-programmable in various hosts. By further engineering these modules to connect with the microbial regulatory machinery, gene expression and protein activity can be finely tuned simply through light irradiation. Recent works on applying optogenetics to microbial synthetic biology have yielded remarkable achievements. To further expand the usability of optogenetics, more optogenetic tools with greater portability that are compatible with different microbial hosts need to be developed. This review focuses on non-opsin optogenetic systems and the current state of optogenetic advancements in microbes, by showcasing the different designs and functions of optogenetic tools, followed by an insight into the optogenetic approaches used to circumvent challenges in synthetic biology.
... The QY obtained for the SpyTag-ZsYellow was much lower than the respective fluorescent protein [29]. This could be due to a possible substitution of a key amino acid in the fusion protein that interacts with the chromophore and, although it provided greater ionic stability (hypsochromic shift, Supplementary Material Figure S3), it also caused a very significant decrease in the quantum yield [30]. ...
Mycotoxins are low molecular weight toxic compounds, which can cause severe health problems in animals and humans. Immunoassays allow rapid, simple and cost-effective screening of mycotoxins. Sandwich assays with a direct readout provide great improvement in terms of selectivity and sensitivity, compared to the widely used competitive assay formats, for the analysis of low molecular weight molecules. In this work, we report a non-competitive fluorescence anti-immune complex (IC) immunoassay, based on the specific recognition of HT-2 toxin with a pair of recombinant antibody fragments, namely antigen-binding fragment (Fab) (anti-HT-2 (10) Fab) and single-chain variable fragment (scFv) (anti-IC HT-2 (10) scFv). The SpyTag and SpyCatcher glue proteins were applied for the first time as a bioconjugation tool for the analysis of mycotoxins. To this aim, a SpyTag-mScarlet-I (fluorescent protein) and scFv-SpyCatcher fusion proteins were constructed, produced and fused in situ during the assay by spontaneous Tag-Catcher binding. The assay showed an excellent sensitivity with an EC50 of 4.8 ± 0.4 ng mL ⁻¹ and a dynamic range from 1.7 ± 0.3 to 13 ± 2 ng mL ⁻¹ , an inter-day reproducibility of 8.5% and a high selectivity towards HT-2 toxin without cross-reactivity with other Fusarium toxins. The bioassay was applied to the analysis of the toxin in an oat reference material and in oat samples, with a LOD of 0.6 µg kg ⁻¹ , and the results were validated by analysing a certificate reference material and by HPLC–MS/MS.
Graphical abstract
... Therefore, our group constructed a vector called pAAV-Sico-Red that continuously expresses a fluorescent protein of an additional wavelength to distinguish knockdown cells that have lost the fluorescent signal (Jung et al., 2016). However, in actual in vivo experiments, the two fluorescent proteins showed differences in stability (Stepanenko et al., 2008), and since expression is induced by different promoters (EF1α and CMV), it was difficult to control the timing and degree of expression. Moreover, since this system used two fluorescence wavelengths, it showed limitations in utilizing multiple immunostaining techniques. ...
Background
RNA interference (RNAi) is a powerful technique to effectively silence or knock down gene function in mammalian cells. For better cell-type RNAi experiments in vivo, AAV vector-based RNA interference systems need to be improved.
New Method: In this study, we developed an AAV vector (CREon shRNA) that expressed CRE-dependent short hairpin RNA (shRNA) and fluorescent proteins simultaneously.
Results
We verified the Cre-dependent knockdown efficiency in both TREK-1 expressing HEK293T cells and endogenous TREK-1 endogenous TREK-1 PC3 cells using the newly developed CREon shRNA vector. Next, we packaged this TREK-1 CREon vector with AAV and injected it into the hippocampus of the brain together with a synapsin or GFAP promoter-driven CRE virus, confirming that it works well cell-selectively even in vivo. Finally, this viral vector was applied to an animal model of LPS-induced depression to determine whether behavioral changes occurred.
Comparison with existing methods: With the existing pSico or pAAV-Sico-Red vectors, expression of fluorescent protein disappears when shRNA is conditionally activated by CRE recombinase, but our Creon shRNA vector showed simultaneous expression of both shRNA and fluorescent protein. Thus, it offers the advantage of allowing easy visual distinction of knocked-down cells.
Conclusion
The newly improved CREon shRNA vector can be used as a novel research tool for conditional shRNA, and may be useful for various in vivo studies such as cancer and neurobiology.
... The intense peak at 423.5 nm corroborated the presence of dityrosine and diphenylalanine cross bridges, formed as a result of glutaraldehyde cross linking [95]. A prominent peak at 468 nm corresponded to the coupling of Trp indole ring and a neighboring His imidazole ring [96]. The low-intensity peaks at 442− 445 nm, 454− 457 nm, and 462− 468 nm in MCH/HA possibly reflected inorganic ion-amino acid functional group interactions. ...
This study was conducted to implement fish collagen hydrolysate as a substrate for synthesizing a nanophasic hydroxyapatite (HA) coating that would display superior osteoinductivity than HA derived from collagen (C/HA) and to comprehend the contribution of secondary structure on HA synthesis. Collagen type I was isolated from mullet skin and fragmented to obtain collagen hydrolysate (MCH). CD, FTIR, XRD, DLS and MS studies revealed that the MCH peptides oriented in polyproline-II conformation and coiled to form a mimic-helix with a packing distance of 0.885 nm. Multiple helices self-assembled into a quasi-fibrillar network with a diameter of 931 nm. The MCH was suspended in HA-nucleating solution to form MCH/HA nanocomposite crystals, which exhibited HA-specific planes in XRD with crystal orientation towards the quasi-fibrillar axis and a % crystallinity of 96.04. MCH/HA crystals were plate-shaped with an average size of 55.4 nm and a Ca/P ratio of 1.77 with moderate resilience (9.9 GPa) and high wettability. Consequently, osteoblasts cultured on MCH/HA coated-Ti surface proliferated faster and expressed 1.2-2 times higher levels of osteogenic differentiation markers when compared to C/HA-coated and uncoated surfaces. The study identified MCH/HA to be a superior coating material, by virtue of the unique quasi-fibrillar architecture of MCH.
... Several possible functions of green FPs-for example, the regeneration of coelenterazine in bioluminescent organisms-are associated with their ability to act as electron donors for biologically significant oxidants [115]. It should be noted that the unique ability of GFP-like proteins to fluoresce in the visible region of the spectrum determines the wide application of fluorescent biomarkers and biosensors developed on their basis for solving numerous fundamental and practical problems of cell biology and medicine via fluorescent methods [116][117][118][119]. ...
Insoluble protein aggregates with fibrillar morphology called amyloids and β-barrel proteins both share a β-sheet-rich structure. Correctly folded β-barrel proteins can not only function in monomeric (dimeric) form, but also tend to interact with one another-followed, in several cases, by formation of higher order oligomers or even aggregates. In recent years, findings proving that β-barrel proteins can adopt cross-β amyloid folds have emerged. Different β-barrel proteins were shown to form amyloid fibrils in vitro. The formation of functional amyloids in vivo by β-barrel proteins for which the amyloid state is native was also discovered. In particular, several prokaryotic and eukaryotic proteins with β-barrel domains were demonstrated to form amyloids in vivo, where they participate in interspecies interactions and nutrient storage, respectively. According to recent observations, despite the variety of primary structures of amyloid-forming proteins, most of them can adopt a conformational state with the β-barrel topology. This state can be intermediate on the pathway of fibrillogenesis ("on-pathway state"), or can be formed as a result of an alternative assembly of partially unfolded monomers ("off-pathway state"). The β-barrel oligomers formed by amyloid proteins possess toxicity, and are likely to be involved in the development of amyloidoses, thus representing promising targets for potential therapy of these incurable diseases. Considering rapidly growing discoveries of the amyloid-forming β-barrels, we may suggest that their real number and diversity of functions are significantly higher than identified to date, and represent only "the tip of the iceberg". Here, we summarize the data on the amyloid-forming β-barrel proteins, their physicochemical properties, and their biological functions, and discuss probable means and consequences of the amyloidogenesis of these proteins, along with structural relationships between these two widespread types of β-folds.
... ProteinFluorescent proteins can be inserted into a cell line so that as a protein is expressed it fluoresces[ 270] . HaloTag and SNAP-tag Protein Self-labeling protein tags such as HaloTag and SNAP-tag ...
The nucleus, central to cellular activity, relies on both direct mechanical input as well as its molecular transducers to sense external stimuli and respond by regulating intra-nuclear chromatin organization that determines cell function and fate. In mesenchymal stem cells of musculoskeletal tissues, changes in nuclear structures are emerging as a key modulator of their differentiation and proliferation programs. In this review we will first introduce the structural elements of the nucleoskeleton and discuss the current literature on how nuclear structure and signaling are altered in relation to environmental and tissue level mechanical cues. We will focus on state-of-the-art techniques to apply mechanical force and methods to measure nuclear mechanics in conjunction with DNA, RNA, and protein visualization in living cells. Ultimately, combining real-time nuclear deformations and chromatin dynamics can be a powerful tool to study mechanisms of how forces affect the dynamics of genome function.
... Our first proposal utilizes genetically engineered fluorescent bacteria to produce a signal that will be transmitted to the on-body reader. The use of fluorescent proteins, a technique introduced to these bacterial strains in the 2000s, has led to the development of many applications, including biological sensors, tracking-visualization systems, and many other sophisticated biological devices [4,5]. With the emergence of synthetic biology, control of MC as well as the control of fluorescent signals from bacteria can be achieved with high precision and programmability for many ...
Although molecular communication systems have been shown to bear great potential for many useful in-body applications, they require the intervention, action, or input of an out-of-body actor. From an Internet of Bio-Nano Things perspective, a successful overall network aims to bring together the two links belonging to the in-body and out-of-body networks for end-to-end communications. For most applications, the uplink from the in-body sensor is more significant since it provides the multi-scalar connection required to relay the information sensed and carried by the molecular communication system to a macro-scale smart terminal. This article proposes two different mechanisms to sense the output of the molecular communication system and transmit the information to an on-body reader. Each mechanism involves different genetically engineered bacteria and specific antenna designs. An experimental setup is provided to demonstrate each proposed concept. The results constitute a proof of concept to detect the in-body bacterial activity from the on-body reader.
... Fluorescent proteins (FPs) have become the most important fluorescent probes for live cell imaging (for extensive reviews of FPs see [193][194][195]) and have been successfully used for imaging in plants (see review [83]). Due to their relatively small size (22)(23)(24)(25)(26)(27)(28), they can often be fused to other proteins without interfering with their target's cellular function. ...
Plant cell wall-derived biomass serves as a renewable source of energy and materials with increasing importance. The cell walls are biomacromolecular assemblies defined by a fine arrangement of different classes of polysaccharides, proteoglycans, and aromatic polymers and are one of the most complex structures in Nature. One of the most challenging tasks of cell biology and biomass biotechnology research is to image the structure and organization of this complex matrix, as well as to visualize the compartmentalized, multiplayer biosynthetic machineries that build the elaborate cell wall architecture. Better knowledge of the plant cells, cell walls, and whole tissue is essential for bioengineering efforts and for designing efficient strategies of industrial deconstruction of the cell wall-derived biomass and its saccharification. Cell wall-directed molecular probes and analysis by light microscopy, which is capable of imaging with a high level of specificity, little sample processing, and often in real time, are important tools to understand cell wall assemblies. This review provides a comprehensive overview about the possibilities for fluorescence label-based imaging techniques and a variety of probing methods, discussing both well-established and emerging tools. Examples of applications of these tools are provided. We also list and discuss the advantages and limitations of the methods. Specifically, we elaborate on what are the most important considerations when applying a particular technique for plants, the potential for future development, and how the plant cell wall field might be inspired by advances in the biomedical and general cell biology fields.
... The intense, sharp peak at 423-424nm depicted the presence of dityrosine cross bridges in both samples, a result of glutaraldehyde cross linking [48,49]. A prominent peak at 468nm in both spectra corresponded to the presence of a tryptophan aromatic ring coupled with the imidazole ring of a neighboring histidine [50]. Trp produced its distinct peak at around 490nm. ...
This study had a two-fold objective: To utilize collagen hydrolysate for synthesizing a nanoscale Hydroxyapatite (HA) coating that would act as a superior osteoblast adhesion/proliferation agent compared to collagen-derived HA (C/HA) and to comprehend the significant role played by structural constraints on HA nucleation. Collagen was extracted from pacu skin with a high yield of 65.3% (w/w of tissue). It was digested by collagenase and the hydrolysate (CH) was purified with a high yield of 0.68g/g of collagen. The CH peptides had a mass of 6kDa, a predominant PP-II conformation and formed self-assembling hierarchical structures at physiological pH with dimensions of 842.2±229nm. The HA synthesized on CH (CH/HA) displayed higher yield when compared to C/HA. Structural analysis of CH/HA revealed that the PP-II peptides coiled to form mimic-helical moieties with reduced intermolecular packing distance of 0.9nm. The mimic helices cross-linked to form a vast quasi-fibrillar network that was comparatively smaller than collagen fibrils but exhibited enhanced stability and greater dynamicity. CH/HA displayed intense calcium-carboxyl interactions, sharper diffraction planes, smaller size of 48±6.2nm and a Ca/P ratio closer to 1.69 when compared to C/HA along with displaying serrated edge blooming crystals. Because of the small size, the CH/HA nanocrystals displayed significantly better osteoblast adhesion than C/HA and reduced the doubling time of cells. Overall, the results indicated that CH based nanocomposites displayed suitable morphological characteristics and cellular response for potential application as implant and bone graft coating material.
Graphical abstract
... The development of advanced techniques to assess new antibacterial agents targeting the metabolic activities of pathogens is critical, as conventional growth-based methods require long exposure times, cannot be used for non-culturable microbes, and are unable to discriminate between bactericidal and bacteriostatic effects (Figure 1) (Christensen et al., 1985;Hartree, 1972;Ishiki et al., 2018;Manina et al., 2015;Mosmann, 1983;Richards et al., 2020;Tao et al., 2017;Walker and Keevil, 1994). Technologies exist to assess the metabolic activities of biofilms labeled with isotope or fluorescent substrates, such as singlecell Raman spectromicroscopy and adenosine triphosphate (ATP) bioluminescence (Lee et al., 2017;Stepanenko et al., 2008;Takenaka, 1994;Wang et al., 2020). However, none of these techniques allow realtime monitoring of the metabolic activities of cells in biofilms without labeling. ...
Summary:
Concerns regarding increased antibiotic resistance arising from the emergent properties of biofilms, especially those containing pathogens, have spurred interest in the discovery of novel antibiotic agents and techniques to directly estimate metabolic activity in biofilms. Although conventional methods, such as crystal violet staining, electrochemical impedance spectroscopy, bioluminescence, and quartz crystal microbalance, are frequently used to quantify biofilm formation, real-time quantitative assessment of metabolic activity in label-free biofilms remains a challenge. Production of electrical current via extracellular electron transport (EET) has recently been found in pathogens and appears to correlate with their metabolic activity. Accordingly, monitoring the production of electrical currents as an indicator of cellular metabolic activity in biofilms represents a new direction for research aiming to assess and screen the effects of antimicrobials on biofilm activity. In this review, we reviewed EET-capable pathogens and the methods to monitor biofilm activity to discuss advantages of using the capability of pathogens to produce electrical currents and effective combination of these methods. Moreover, we discuss differences between EET by pathogenic and environmental bacteria and open questions for the physiological roles of EET in biofilm pathogens. The present limitations and possible future directions of in situ biofilm metabolic activity assessment for large-scale screening of antimicrobials are also discussed.
... FPs emit strong fluorescence even at low concentrations. Therefore, they are not only used as biosensor markers [8], but also as screening probes to monitor the expression of membrane proteins with low expression levels [9]. Beyond life sciences, fluorescent proteins have been used for breeding green pigs and cats, and genetically modified fluorescent fishes are already found in home aquariums [10]. ...
The fluorescence of fluorescent proteins (FPs) is quenched when they are exposed to certain transition metals, which makes them promising receptor materials for metal biosensors. In this study, we report the spectroscopic analysis of metal-induced fluorescence quenching of the fluorescent protein ZsGreen from Zoanthus sp. The fluorescence of ZsGreen was reduced to 2%, 1%, and 20% of its original intensity by Fe2+, Fe3+, and Cu2+, respectively. Metal titration experiments indicated that the dissociation constants of Fe2+, Fe3+, and Cu2+ for ZsGreen were 11.5, 16.3, and 68.2 μM, respectively. The maximum binding capacities of ZsGreen for Fe2+, Fe3+, and Cu2+ were 103.3, 102.2, and 82.9, respectively. Reversibility experiments indicated that the fluorescence of ZsGreen, quenched by Fe2+ and Fe3+, could be recovered, but only to about 15% of its original intensity, even at a 50-fold molar excess of EDTA. In contrast, the fluorescence quenched by Cu2+ could be recovered up to 89.47% of its original intensity at a Cu2+: EDTA ratio of 1:5. The homology model of ZsGreen revealed that the protein does not share any metal-binding sites with previously reported FPs, suggesting that ZsGreen contains unprecedented binding sites for fluorescence quenching metal ions.
... Lack of co-diffusion suggests that this complex is not strongly fluorescent (e.g., due to self-quenching) but still primarily recognized by the Nb. Fluorescence modulation upon aggregation or mis-folding of FPs and their chromophores has been described before (Camacho et al., 2018;Ge et al., 2017;Jung et al., 2005b;Kruitwagen et al., 2015;Stepanenko et al., 2008). Upon binding, Nb could partially disassemble the dark complex and release a few molecules from the complex that become fluorescent but are not bound to a Nb. ...
Advanced fluorescence microscopy studies require specific and monovalent molecular labelling with bright and photostable fluorophores. This necessity led to the widespread use of fluorescently labelled nanobodies against commonly employed fluorescent proteins. However, very little is known how these nanobodies influence their target molecules. Here, we tested commercially available nanobodies and observed clear changes of the fluorescence properties, mobility and organisation of green fluorescent protein (GFP) tagged proteins after labelling with the anti-GFP nanobody. Intriguingly, we did not observe any co-diffusion of fluorescently-labelled nanobodies with the GFP-labelled proteins. Our results suggest significant binding of the nanobodies to a non-emissive, likely oligomerized, form of the fluorescent proteins, promoting disassembly into monomeric form after binding. Our findings have significant implications on the application of nanobodies and GFP labelling for studying dynamic and quantitative protein organisation in the plasma membrane of living cells using advanced imaging techniques.
... In our laboratory, we investigate OPA and TPA processes in fluorescent proteins, which became a valuable tool in modern in vivo experiments. [18][19][20] Hence, in the present contribution, we focus on removing the artifacts in simulated OPA and TPA spectra of FPs models based on the TDDFT/PE level of theory. The PE model was brought to our attention since the ESO problem is not really observed or at least not so profound with the less advanced EE. 16,[21][22][23] However, missing the polarization effects was shown to severely influence the quality of both OPA and TPA spectra. ...
The multiscale calculations involving excited states may suffer from the electron spill-out (ESO) problem. This seems to be especially the case when the environment of the core region, described with the electronic structure method, is approximated by a polarizable force field. The ESO effect often leads to incorrect physical character of electronic excitations, spreading outside the quantum region, which, in turn, results in erroneous absorption spectra. In this work, we investigate means to remove the artifacts in one-photon absorption (OPA) and two-photon absorption (TPA) spectra of green and yellow fluorescent protein representatives. This includes (i) using different basis sets, (ii) extending the core subsystem beyond the chromophore, (iii) modification of polarization interaction between the core region and its environment, and (iv) including the Pauli repulsion through effective core potentials (ECPs). Our results clearly show that ESO is observed when diffuse functions are used to assemble the multielectron wave function regardless of the exchange–correlation functional used. Furthermore, extending the core region, thus accounting for exchange interactions between the chromophore and its environment, leads to even more spurious excited states. Also, damping the interactions between the core subsystem and the polarizable force field is hardly helpful. In contrast, placing ECPs in the position of sites creating the embedding potential leads to the removal of artificious excited states that presumably should not be observed in the OPA and TPA spectra. We prove that it is a reliable and cost-effective approach for systems where the covalent bond(s) between the core region and its environment must be cut.
... While TCS mitochondrial toxicity could lead to extracellular acidification which could affect ASAP2 fluorescence, TCS-induced extracellular acidification likely occurs on a longer timescale (Ajao et al., 2015) than the 15 min exposure assessed in the current study. Also, if TCS were acidifying the extracellular space around the RBL mast cells and Jurkat T cells within the experimental exposure time (15 min), overcoming the buffering of the BT solution, a decrease in ASAP2 fluorescence would have occurred (Stepanenko et al., 2008). Together, these data suggest that TCS does not affect mast cell or T cell PMP but, instead TCS lowers cytosolic pH. ...
Triclosan (TCS) is an antimicrobial agent that was effectively banned by the FDA from hand soaps in 2016, hospital soaps in 2017, and hand sanitizers in 2019; however, TCS can still be found in a few products. At consumer-relevant, non-cytotoxic doses, TCS inhibits the functions of both mitochondria and mast cells, a ubiquitous cell type. Via the store-operated Ca²⁺ entry mechanism utilized by many immune cells, mast cells undergo antigen-stimulated Ca²⁺ influx into the cytosol, for proper function. Previous work showed that TCS inhibits Ca²⁺ dynamics in mast cells, and here we show that TCS also inhibits Ca²⁺ mobilization in human Jurkat T cells. However, the biochemical mechanism behind the Ca²⁺ dampening has yet to be elucidated. Three-dimensional super-resolution microscopy reveals that TCS induces mitochondrial swelling, in line with and extending the previous finding of TCS inhibition of mitochondrial membrane potential via its proton ionophoric activity. Inhibition of plasma membrane potential (PMP) by the canonical depolarizer gramicidin can inhibit mast cell function. However, use of the genetically encoded voltage indicators (GEVIs) ArcLight (pH-sensitive) and ASAP2 (pH-insensitive), indicates that TCS does not disrupt PMP. In conjunction with data from a plasma membrane-localized, pH-sensitive reporter, these results indicate that TCS, instead, induces cytosolic acidification in mast cells and T cells. Acidification of the cytosol likely inhibits Ca²⁺ influx by uncoupling the STIM1/ORAI1 interaction that is required for opening of plasma membrane Ca²⁺ channels. These results provide a mechanistic explanation of TCS disruption of Ca²⁺ influx and, thus, of immune cell function.
... Recently, there has been increasing interest in determining Φ f in scattering media such as biological tissues [2,3], crystalline materials [4,5], quantum dots [6], printing products [7], fluorescent biomarkers and biosensors [8], fluorescent fabrics [9], etc. ...
The fluorescence quantum yield is a measure of the efficiency of photon emission and quantifies the luminescent performance of a given sample. The determination of fluorescence quantum yields, particularly in scattering media, is relevant in the areas of materials science, technology and photonics. It is equally crucial when studying fluorescent bioanalytical probes and biological systems either for medical applications, physiological analyses or the interpretation of optical signals in nature. This type of determination represents a challenge since light scattering introduces an appreciable complexity in the measurements. Hence, the use of experimentally accurate methods and the understanding of their basis and principles is indispensable for obtaining reliable results. In addition, light re-absorption processes are usually very significant in these systems and the experimental quantum yields normally differ from the true quantum yields of the fluorophore. The first purpose of this work is to provide a clear and comprehensive compilation of the various optical methods that can be used for the determination of quantum yields in scattering media. A second purpose is to present the correction models to account for light re-absorption processes, applicable in each case. The advantages and disadvantages of each methodology are comparatively discussed, the difference between experimental and true quantum yield is clarified and it is explained which should be used depending on the case. Several examples previously published in literature are illustrated. The methods presented here are adequate for the study of very diverse samples such as suspensions, solid powders, films, animal tissues and even plant material.
... the discovery of new antimicrobial molecules and the development of easy and effective techniques for their assessment against bacterial colonization is in great demand [13]. Currently, to check the impact of antimicrobial drugs, pathogens' metabolic activity assay is subjected to microscopic observations, which require gene engineering for expressing the fluorescent protein and complicated interpretations [14]. Among electrochemical analyses for the pathogen's detection, electrochemical impedance spectroscopy (EIS) has a strong advantage in terms of sensitivity and simplicity, but it does not clearly correlate with metabolic activity and mainly has the benefit of cellular adhesion's detection on the electrode surface [15]. ...
The development of a simple and direct assay for quantifying microbial metabolic activity is important for identifying antibiotic drugs. Current production capabilities of environmental bacteria via the process called extracellular electron transport (EET) from the cell interior to the exterior is well investigated in mineral-reducing bacteria and have been used for various energy and environmental applications. Recently. the capability of human pathogens for producing current has been identified in different human niches, which was suggested to be applicable for drug assessment, because the current production of a few strains correlated with metabolic activity. Herein, we report another strain, a highly abundant pathogen in human oral polymicrobial biofilm, Corynebacterium matruchotii, to have the current production capability associated with its metabolic activity. It showed the current production of 50 nA/cm 2 at OD 600 of 0.1 with the working electrode poised at +0.4 V vs. a standard hydrogen electrode in a three-electrode system. The addition of antibiotics that suppress the microbial metabolic activity showed a significant current decrease (>90%), establishing that current production reflected the cellular activity in this pathogen. Further. the metabolic fixation of atomically labeled 13 C (31.68% ± 2.26%) and 15 N (19.69% ± 1.41%) confirmed by high-resolution mass spectrometry indicated that C. matruchotii cells were metabolically active on the electrode surface. The identified electrochemical activity of C. matruchotii shows that this can be a simple and effective test for evaluating the impact of antibacterial compounds, and such a method might be applicable to the polymicrobial oral biofilm on electrode surfaces, given four other oral pathogens have already been shown the current production capability.
... In this, when these cells are exposed to any toxic molecule, this interferes with the metabolic process of this cell and substantially decreases bioluminescence in these cells. This confirms that these luminescent yeast cells recognize the chemicals toxic to eukaryotic cells, which were previously not assessed by prokaryotic biosensors in the sample (Stepanenko et al., 2008). Another group of researchers developed a biosensor by using mutant S. cerevisiae strains to assess the carcinogens as well as mutagens pertaining to the environment. ...
Microbial populations like bacteria and fungi are used as sensing elements by converting biological response into electric signals. Fungal-based biosensors are advantageous over bacteria as they have a high binding capacity and more specificity. Natural bioluminescent properties in fungi make them suitable candidate for preparing fungal-based biosensors. Common fungal-based biosensors reported are electrical methods, growth rate, colorimetry, luminescence, fluorescence, etc. Recent advancements in the field of molecular biology, such as insertion of new coding sequences or modification of fungal genome, offer more precise, accurate, and cost-effective procedures. Many yeast- and mold-based biosensors are utilized for environmental and eco-toxicity analysis. In this chapter, we explore the importance and aspects of fungi-based biosensors for a sensitive and rapid method to assess environmental samples.
... NOTE: In general, it is recommended to electroporate a vector carrying a fluorescence marker as additional transfection control. Dependent on the chosen marker and its chromophore maturation the fluorescence will be visible within around 24-48 h post transfection 18 . ...
Electroporation is a common method for transfection with different kinds of molecules by electrical permeabilization of the plasma membrane. With the increasing use of organoids as a culturing method for primary patient material in the last years, efficient transfer methods of components for genetic engineering in this 3D culture system are in need. Especially for organoids, the efficiency of genetic manipulations depends on a successful transfection. Thus, this protocol was developed to facilitate the electroporation of organoids and to prove its universal functionality in different entities. Human colorectal, pancreatic, hepatic and gastric cancer organoids were successfully electroporated with small and large plasmids in comparison. Based on GFP encoding vectors, the transfection efficiency was determined by FACS. No extensive preparation of the
cells or special, cost-intensive electroporation buffers are necessary, and the protocol can be performed within one day.
... On the other hand, the cnidarian lipid composition may vary considerably depending on their diet or symbiotic association with unicellular algae (Papina et al., 2003;De Souza et al., 2007;Dunn et al., 2012;Leone et al., 2013). In this framework, there could be positive aspects to an sea full of jellyfish (called "Jellyfish Apocalypse" by Lee et al., 2014) as scientists have looked into using jellyfish in several ways: age reduction powers in one type of jellyfish (Rich, 2012) (life-cycle reversal in Turritopsis dohrnii (Weismann, 1883) medusa (Piraino et al., 1996;Martell et al., 2016), proteins used for disease control purposes (Hsieh et al., 2001), and biomarkers used in medical diagnostics (Stepanenko et al., 2008). On the other hand, jellyfish as new food sources must be fully characterised from nutritional, nutraceutical, biochemical, microbial and toxicological point of view. ...
The rise in water temperature in the Mediterranean Sea, and associated migrations of temperate marine biota, are occurring in the context of a global warming causing an expansion of the tropical jellyfish range, exacerbating jellyfish outbreaks linked to coastal development, nutrient loading, and overfishing. The gelatinous component of plankton is considered as ‘the dark side of ecology’ capable of appearing and disappearing at unpredictable times. In the last decade an increasingly high number of gelatinous plankton blooms are occurring and this makes us wonder if ‘a Mediterranean Sea full of jellyfish is a probable future’. The reasons for rising jellyfish blooms are, probably, manifold. Current studies are aimed to highlight how climatic change is interacting with the Mediterranean ecosystem favouring entrance, abundances and success of alien species and triggering ‘regime shifts’ such as from fish to jellyfish. Jellyfish damage the economic success of power plants, fish farms, tourism, and affect fisheries consuming larvae of commercial fish species. On the other hand, several studies were also taken into account on uses for jellyfish as biofuels and foods but more experimentation is needed to improve the first encouraging results.
... Prospects for the use of extrinsic fluorophores to measure neuronal metabolism and function are promising. Because many available biomarkers can be conjugated to a fluorescent marker and given the very distinctive lifetimes of different fluorophores, future approaches may provide intensive or ratiometric fluorescent readouts (Stepanenko et al. 2008). ...
Retinal function has long been studied with psychophysical methods in humans, whereas detailed functional studies of vision have been conducted mostly in animals owing to the invasive nature of physiological approaches. There are exceptions to this generalization, for example, the electroretinogram. This review examines exciting recent advances using in vivo retinal imaging to understand the function of retinal neurons. In some cases, the methods have existed for years and are still being optimized. In others, new methods such as optophysiology are revealing novel patterns of retinal function in animal models that have the potential to change our understanding of the functional capacity of the retina. Together, the advances in retinal imaging mark an important milestone that shifts attention away from anatomy alone and begins to probe the function of healthy and diseased eyes.
Fluorescent proteins (FPs) have transformed cell biology through their use in fluorescence microscopy, enabling precise labeling of proteins via genetic fusion. A key advancement is altering primary sequences to customize their photophysical properties for specific imaging needs. A particularly notable family of engineered mutants is constituted by Reversible Switching Fluorescent Proteins (RSFPs), i.e. variant whose optical properties can be toggled between a bright and a dark state, thereby adding a further dimension to microscopy imaging. RSFPs have strongly contributed to the super-resolution (nanoscopy) revolution of optical imaging that has occurred in the last 20 years and afforded new knowledge of cell biochemistry at the nanoscale. Beyond high-resolution applications, the flexibility of RSFPs has been exploited to apply these proteins to other non-conventional imaging schemes such as photochromic fluorescence resonance energy transfer (FRET). In this work, we explore the origins and development of photochromic behaviors in FPs and examine the intricate relationships between structure and photoswitching ability. We also discuss a simple mathematical model that accounts for the observed photoswitching kinetics. Although we review most RSFPs developed over the past two decades, our main goal is to provide a clear understanding of key switching phenotypes and their molecular bases. Indeed, comprehension of photoswitching phenotypes is crucial for selecting the right protein for specific applications, or to further engineer the existing ones. To complete this picture, we highlight in some detail the exciting applications of RSFPs, particularly in the field of super-resolution microscopy.
Recent Trends in Livestock Innovative Technologies explores the most recent developments and developing trends in the livestock farming industry. The book delves into the application of innovative technologies in various aspects of livestock production, management, and health through edited chapters.
The book starts with an outline of the difficulties the livestock sector faces and the necessity for technological solutions to these difficulties. Subsequent chapters cover innovations in this area. Key topics include:
Advances in genetics and breeding methods: Contributing authors stress the possible impact of issues like marker-assisted selection, genomic selection, and gene editing on the future of animal breeding.
Precision livestock farming: The use of sensor technologies, data analytics, and automation to monitor and control livestock production systems more effectively. The authors examine how these technologies enable real-time monitoring of environmental variables, animal activity, and health, which enhances production, animal welfare, and resource use.
The management of feed and nutrition in livestock production: The book explores cutting-edge feed formulations, precise feeding systems, and alternative feed sources that can increase feed efficiency, lessen negative effects on the environment, and improve animal health.
Fresh methods for illness prevention and management, such as the use of vaccines, diagnostics, and biosecurity measures.
Social and ethical issues related to the adoption of cutting-edge livestock technologies. The authors attempt to give a fair assessment of the advantages and drawbacks of these technologies, and address concerns about animal welfare, environmental sustainability, and public perception of current farming practices.
Recent Trends in Livestock Innovative Technologies is an informative resource for researchers, professionals, and policymakers interested in staying up-to-date with the advancements and future directions of the livestock industry.
This chapter provides the most practical and sustainable protocols in the synthesis of a wide range of important molecules. Several activation strategies such as classical heating, mechanochemistry, microwave activation, and sonochemistry under both solvent‐ and catalyst‐free reaction conditions undoubtedly lead to highly efficient synthetic processes with simple operation, waste reduction, excellent atom economy, gram‐scale synthetic applicability, and avoidance of toxic solvents/catalyst, which are greatly desirable for potential industrial purposes.
This chapter is dedicated to Fluorescent Proteins, divided according to their spectral characteristics into Blue, Cyan, Yellow, Green, Orange, Red, and Infrared Fluorescent Proteins. More than 90 Fluorescent Proteins are mentioned, of which the chapter provides information on the molecular structure of the chromophore and the spectral behavior, with particular attention to the problems associated with their use in Flow Cytometry.
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.
Fluorescence spectroscopy and microscopy have been used extensively in diverse areas of both scientific research and industrial applications. Particularly, fluorescence microscopy is one of the most sought-after imaging techniques in biological research field. With the advent of many novel technologies, method of fluorescence imaging has grown manifolds. Our ability to visualize and resolve samples at different length scales using fluorescence imaging has improved significantly with a wide variety of microscopic techniques that are currently available. We have now access to various kinds of microscopes starting from basic epi-fluorescence microscope to highly advanced super resolution imaging techniques like photoactivated localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), etc. With such superresolution microscopic techniques, we now have the ability to detect and resolve the signals at the level of individual molecules. Overall fluorescence microscopic techniques have contributed immensely to the advancement of many biological research fields.
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 (σTPA ) for the S0→S1 transition in GFPs. Furthermore, it seems that details of h‐bonds network in the CRO's vicinity influences σTPA response to CRO – environment electrostatic interactions in YFPs. We postulate that engineering FPs with more hydrophilic CRO's environment can lead to greater σTPA . We also find that removing h‐bonds formed with the CRO's phenolate leads to TPA enhancement for transition to higher excited states than S1. Particularly Y145 and T203 residues are important in this regard.
Structural and dynamic behaviors of the green fluorescent protein dimer from jellyfish Clytia gregaria (cgGFP) were investigated by means of molecular dynamics (MD) simulation. Both neutral and ionic forms of the chromophore, p-hydroxybenzylideneimidazolinone (GYS) were considered. The partial atomic charges of the chromophore were derived by BCC and RESP approaches. The structures were compared between the anionic and neutral cgGFP, and between the two subunits (Sub A and Sub B) of the protein dimer. The observed fluorescence intensity and anisotropy decays were further analyzed with theoretical expressions by employing the atomic coordinates of neutral cgGFP obtained by MD simulation. It was assumed that the fluorescence quenching of GYSA and GYSB is ascribed to HB formations between heteroatoms of GYSs and nearby amino acids. Excellent agreement between the observed and calculated intensity decays, and the observed and calculated anisotropy decays were obtained with RESP1 model. The agreements were better in RESP model than those in BCC one. Mean quenching constants of GYSA and GYSB were 0.27 and 0.59 ns⁻¹ overall MD snapshots with RESP1. Mean value of square of direction cosine between the two transition moments of GYSs was 0.74, and that of square of orientation factor was 0.53, and the FRET rates from GYSA to GYSB, and from GYSB to GYSA were 0.87 and 1.87 ns⁻¹.
Fluorescent proteins (FP) can be applied as biomarkers and biosensors in the industrial and medical fields, but their large-scale use, especially for new industrial applications, is limited due to their low stability. Hence, the discovery of additives capable of preserving the activity of FP at room temperature and under stress conditions can help to expand and facilitate their commercial use. With this goal, we evaluated the application of 1-alkyl-3-methylimidazolium chloride-based ([Cnmim]Cl) ionic liquids (ILs) as additives to preserve the activity of Enhanced Green Fluorescent Protein (EGFP) at different storage times and under unfavorable conditions. All [Cnmim]Cl solutions (at 0.100 mol L⁻¹) were able to preserve EGFP fluorescence for longer than the phosphate-saline buffer (PBS) and NaCl solutions, increasing its fluorescence manifestation from 1 to 3 months. [Cnmim]Cl with shorter to medium cationic alkyl chains were the most effective in preserving EGFP fluorescence. [Cnmim]Cl also protected EGFP activity in the presence of the surfactant SDS, the acid guanidine hydrochloride, and H2O2. Therefore, [Cnmim]Cl can be added to aqueous solutions to preserve EGFP fluorescence activity at room temperature for longer storage times and to reduce the negative impact of denaturing agents on EGFP. Therefore, there is a massive potential for the application of ILs as additives to preserve FP in the long-term without refrigeration and under unfavorable conditions, and this is fundamental to enable expansion of FP in industrial and commercial applications.
Epidermal growth factor receptor variant III is a mutant variant of EGFR that has a deletion on its DNA encoding extracellular ligan-binding domain. EGFRvIII is only found in cancer cells and not in normal cells, make it an ideal target as a biomarker for antibody-based cancer therapy. This study performed the expression and characterization of EGFRvIII extracellular domain conjugated with a blue fluorescent protein (BFP) as a fusion protein in Escherichia coli periplasmic space. Endoxylanase signal peptide was employed to guide the recombinant protein through the membrane. IPTG as inducer was added into expression medium with various concentrations of 0; 0.1; 0.25; 0.5; 1 mM, followed by periplasmic extraction using the hypertonic solution. Total proteins and periplasmic proteins were characterized using SDS-PAGE and slot blot analyses. Extracellular domain of EGFRvIII-BFP fusion protein was detected using confocal fluorescence microscopy. This study showed that the ∼43 kDa target protein was successfully expressed on E. coli NiCo21(DE3) periplasmic space with optimum IPTG concentration of 0.1 mM and and showed a blue fluorescence color under the microscope..
We previously generated transgenic pigs with enhanced growth rate and reduced nutrient loss. However, the composition of their gut microbiome is unknown. In this study, we successfully generated EGFP marker-free transgenic (MF-TG) pigs with high expression levels of microbial β-glucanase, xylanase, and phytase in the parotid gland. We collected intestinal contents from the ileum, cecum and colon of five MF-TG and five wild-type (WT) sows and investigated the gut microbiome of the transgenic pigs via metagenomic analysis. Results showed that the levels of probiotics, such as Lactobacillus reuteri and Streptococcus, were more abundant in the cecum of the MF-TG pigs and higher than those of WT pigs. By contrast, the levels of harmful microorganisms, such as Campylobacter, Chlamydia trachomatis, and Campylobacter fetus, and various unidentified viruses, were higher in the cecum of the WT pigs than those of the MF-TG pigs. By comparing unigenes and the eggNOG database, we found that the microorganisms in the colon of the MF-TG pigs had high fractional abundance in DNA (cytosine-5)-methyltransferase 1 and serine-type D-Ala-D-Ala carboxypeptidase, whereas the aspartate carbamoyltransferase regulatory subunit and outer membrane protein pathways were enriched in the WT pigs. Moreover, the microorganisms in the cecum of the MF-TG pigs were active in GlycosylTransferase Family 8 (GT8), Glycoside Hydrolase Family 13 (GH13), and Glycoside Hydrolase Family 32 (GH32). Furthermore, the levels of numerous carbohydrases, such as glucan 1,3-beta-glucosidase, xylan 1,4-beta-xylosidase and exo-1,3-1,4-glucanase, were higher in the cecum of the MF-TG pigs than those of the WT pigs. The results indicated that intestinal microbes can change adaptively to the secretion of transgenic enzymes, thereby forming a benign cooperation with their host. This cooperation could be beneficial for improving feed efficiency.
Somatic hybridisation in the carrot, as in other plant species, enables the development of novel plants with unique characteristics. This process can be induced by the application of electric current to isolated protoplasts, but such electrofusion requires an effective hybrid cell identification method. This paper describes the non-toxic fluorescent protein (FP) tagging of protoplasts which allows discrimination of fusion components and identification of hybrids in real-time during electrofusion. One of four FPs: cyan (eCFP), green (sGFP), yellow (eYFP) or the mCherry variant of red FP (RFP), with a fused mitochondrial targeting sequence, was introduced to carrot cell lines of three varieties using Agrobacterium-mediated transformation. After selection, a set of carrot callus lines with either GFP, YFP or RFP-labelled mitochondria that showed stable fluorescence served as protoplast sources. Various combinations of direct current (DC) parameters on protoplast integrity and their ability to form hybrid cells were assessed during electrofusion. The protoplast response and hybrid cell formation depended on DC voltage and pulse time, and varied among protoplast sources. Heterofusants (GFP + RFP or YFP + RFP) were identified by detection of a dual-colour fluorescence. This approach enabled, for the first time, a comprehensive assessment of the carrot protoplast response to the applied electric field conditions as well as identification of the DC parameters suitable for hybrid formation, and an estimation of the electrofusion success rate by performing real-time observations of protoplast fluorescence.
We systematically investigate an impact of the size and content of a quantum (QM) region, treated at density functional theory (DFT) level, in embedding calculations on one- (OPA) and two-photon absorption (TPA) spectra of the following fluorescent proteins (FPs) models: Aequorea victoria green fluorescent protein with neutral (avGFP-n) and anionic (avGFP-a) chromophore as well as Citrine FP. We find that amino acid (a.a.) residues as well as water molecules h-bonded to the chromophore usually boost both OPA and TPA processes intensity. The presence of hydrophobic a.a. residues in the quantum region also non-negligibly affects both absorption spectra but decreases absorption intensity. We conclude that to reach a quantitative description of OPA and TPA spectra in multiscale modelling of FPs the quantum region should consist of a chromophore and most of a.a. residues and water molecules in radius of 0.30-0.35 nm (ca. 200–230 atoms) when the remaining part of the system is approximated by the electrostatic point-charges. The optimal size of the QM region can be reduced to 80-100 atoms by utilizing more advanced polarizable embedding model. We also find components of the QM region that are specific to a FP under study. We propose that F165 a.a. residue is important in tuning TPA spectrum of avGFP-n but not other investigated FPs. In case of Citrine, Y203 and M69 a.a. residues must definitely be part of the QM subsystem. Furthermore, we find that long-range electrostatic interactions between QM region and the rest of the protein cannot be neglected even for the most extensive QM regions (ca. 350 atoms).
Green fluorescent protein (GFP) is a useful biomolecule in biotechnology; however, its price makes its widespread application prohibitive. To overcome this issue, recently, the use of aqueous two-phase systems (ATPS) for GFP purification was proposed as an alternative platform to reduce processing costs. Aligned with this goal, this study performed bioprocess modelling coupled with economic analysis using the software Biosolve to evaluate the potential and commercial applicability of ATPS for GFP purification. This work analysed a collection of fourteen ATPS to discriminate through production costs while also incorporating the concept of product purity into the calculations. The two best systems (a PEG-based and an ionic liquid (IL)-based ATPS) were placed in a full bioprocess at different scale models (1 to 100 L) to elucidate the viability of applying ATPS at large scale. Although the results showed that the PEG-based ATPS exhibit the lowest costs (between USD 3.5x10³.g⁻¹ at 1 L and USD 0.33x10³.g⁻¹ at 100 L), for further developments, the inclusion of an ATPS granting a higher purity is desired for the development of simpler bioprocesses. Therefore, as a third approach in this work, a sensitivity analysis was performed to determine the impact of varying different model parameters (recovery yield, material costs discount and production titre), to elucidate the circumstances under which the IL-based system can overcome the production costs of the traditional PEG-based ATPS. The results indicate that the best cost-effectiveness approach is to improve the production titre (although it can affect all ATPS studied), as an increase from 1.33 to 3.8 g/L is enough for the IL-based ATPS to be less expensive than the traditional system at all analysed scales. This study demonstrates that ATPS can greatly reduce GFP manufacturing costs, which can potentially help to popularize new applications of fluorescent proteins that are currently mostly restricted to research kits due to their high prices.
The green fluorescent protein (GFP) is extensively used as a biomarker for fluorescence biological imaging. The chromophore in GFP is only fluorescent when confined into the β–barrel of the protein. Similarly, synthetic analogues of the fluorophore of GFP are usually non-emissive in solution, due to free rotation around the aryl-alkene bond and (Z/E)-isomerization of the double bond. Here, the synthesis and characterization of three analogues of the fluorophore of GFP are reported. The introduction of more electron donating substituents induces a red-shift in the absorption and emission. The fluorophores are more emissive in the solid state than in solution, and a study of their crystal structure reveals that the (Z/E)-isomerization is efficiently blocked in the crystals.
Canlı bünyesinde meydana gelen reaksiyonlar sonucunda kimyasal enerjiden görünür ışık üretilmesine ve buna bağlı olarak meydana gelen ışımaya biyolüminesans ışıma denir. Biyolminesans gösteren organizmaların sentezledikleri lusiferaz enzimler ve kimyasal dönüşümlerini katalizledikleri ilgili substratların oluşturdukları reaksiyonlar neticesinde biyolüminesans ışıma meydana gelmektedir. Farklı canlı türlerinde çeşitli lusiferaz enzimleri bulunmaktadır. Lusiferaz enzimlerden seçilecek olan birini kodlayan reporter gen, cDNA aracılığıyla herhangi bir proteini kodlayan genle kaynaştırılmak suretiyle, ilgili proteinin lokasyonu veya etkileşimleri in vivo olarak izlenebilmektedir. İlgilenilen virüs, bakteri, parazit ve maya türlerine aktarılan lusiferaz enzim genleri sayesinde, bu türlerin oluşturdukları enfeksiyonların seyir süreçleri izlenebilmektedir. İzleme düzeneği, ilgili denek hayvana lusiferaz geninin aktarılması, hayvana substratın enjekte edilmesi ve CCD kamera (foton-elektron etkileşimli kamera) ile izlemenin yapılması basamaklarından oluşmaktadır. Özellikle protein-protein etkileşim çalışmalarında kullanılan BRET (biyolüminesans ışımaya dayalı rezonans enerji transferi) tekniği ile biyolüminesans ve floresan ışımalar bir arada izlenebilmektedir. Diğer protein saptama/izleme teknikleri ile kıyaslandığında in vivo biyolüminesans görüntüleme denek hayvana girişimde bulunmayı gerektirmeyen, basit, ucuz ve oldukça elverişli bir tekniktir. Bu çalışmada biyolüminesans ışımanın temel prensipleri, biyolüminesans ışıma üreten enzim-substrat sistemleri ve biyolüminesans ışımaya dayalı çeşitli in vivo izleme düzenekleri hakkında genel bilgiler verilmiş ve bu konularla ilgili önemli çalışmaların sonuçları derlenmiştir.
Mercury ion (Hg2+) is a universal pollutant and its detection is crucial for public health care. In this study, we developed a novel fluorescent biosensor by construction of a protein fusion between the mercury‐sensing transcription factor MerR and enhanced yellow fluorescent protein (EYFP). Hg2+–induced conformational change of MerR was transduced into fluorescence signal. Fluorescence intensity of the biosensor protein decreased with increasing concentrations of Hg2+ and a linear response was obtained in the range of 0.5‐40 nM. The limit of detection (LOD) was 0.5 nM, which was much lower than the maximum allowed level in water. The biosensor specificity was highly dependant on type and concentration of metal ion. The biosensor exhibited high specificity in a mixture of metal ions at 0.5 nM concentration. However, the interference effect was more pronounced at 40 nM concentration of metal ions. The measurement was completed in less than 1 min with no need for sample preparation or pre‐incubation steps. The biosensor achieved accurate and reliable detection in the spiked drinking water sample, as validated by the inductively coupled plasma optical emission spectrometry (ICP‐OES). This article is protected by copyright. All rights reserved
The mammalian protein TAP/p115 and its yeast homologue Uso1p have an essential role in membrane traffic (Nakajima et al., 1991; Waters et al., 1992; Sztul et al., 1993; Rabouille et al., 1995). To inquire into the site and mechanism of TAP/p115 action, we aimed to localize it and to identify domains required for its function. We show that in interphase cells, TAP/p115 localizes predominantly to the Golgi and to peripheral structures that represent vesicular tubular clusters (VTCs) involved in ER to Golgi transport. Using BFA/ nocodazole treatments we confirm that TAP/p115 is present on ER to Golgi transport intermediates. TAP/ p115 redistributes to peripheral structures containing ERGIC-53 during a 15°C treatment, suggesting that it is a cycling protein. Within the Golgi, TAP/p115 is associated with pleiomorphic structures on the cis side of the cis-Golgi cisterna and the cis-most cisterna, but is not detected in more distal compartments of the Golgi.
TAP/p115 binds the cis-Golgi protein GM130, and the COOH-terminal acidic domain of TAP/p115 is required for this interaction. TAP/p115 interaction with GM130 occurs only in the Golgi and is not required for TAP/p115 association with peripheral VTCs. To examine whether interaction with GM130 is required to recruit TAP/p115 to the Golgi, TAP/p115 mutants lacking the acidic domain were expressed and localized in transfected cells. Mutants lacking the GM130-binding domain showed normal Golgi localization, indicating that TAP/p115 is recruited to the Golgi independently of its ability to bind GM130. Such mutants were also able to associate with peripheral VTCs. Interestingly, TAP/p115 mutants containing the GM130-binding domain but lacking portions of the NH2-terminal region were restricted from the Golgi and localized to the ER. The COOH-terminal domain required for GM130 binding and the NH2-terminal region required for Golgi localization appear functionally relevant since expression of TAP/p115 mutants lacking either of these domains leads to loss of normal Golgi morphology.
The photophysics and photochemistry taking place in
the DsRed protein, a recently cloned red fluorescent protein from a
coral of the Discosoma genus, are investigated here by
means of ensemble and single-molecule time-resolved detection and
spectroscopic measurements. Ensemble time-resolved data reveal that
25% of the immature green chromophores are present in tetramers
containing only this immature form. They are responsible for the weak
fluorescence emitted at 500 nm. The remaining 75% of the immature
green chromophores are involved in a fluorescence resonance energy
transfer process to the red species. The combination of time-resolved
detection with spectroscopy at the single-molecule level reveals, on
543-nm excitation of individual DsRed tetramers, the existence of a
photoconversion of the red chromophore emitting at 583 nm and decaying
with a 3.2-ns time constant into a super red one emitting at 595 nm and
for which the decay time constant ranges between 2.7 and 1.5 ns. The
phenomenon is further corroborated at the ensemble level by the
observation of the creation of a super red form and a blue absorbing
species on irradiation with 532-nm pulsed light at high excitation
power. Furthermore, single-molecule experiments suggest that a similar
photoconversion process might occur in the immature green species on
488-nm excitation.
Amyloid fibrils are assemblies of misfolded proteins and are associated with pathological conditions such as Alzheimer's disease and the spongiform encephalopathies. In the amyloid diseases, a diverse group of normally soluble proteins self-assemble to form insoluble fibrils. X-ray fibre diffraction studies have shown that the protofilament cores of fibrils formed from the various proteins all contain a cross--scaffold, with -strands perpendicular and -sheets parallel to the fibre axis. We have determined the threedimensional structure of an amyloid fibril, formed by the SH3 domain of phosphatidylinositol-3'-kinase, using cryo-electron microscopy and image processing at 25 Å resolution. The structure is a double helix of two protofilament pairs wound around a hollow core, with a helical crossover repeat of 600 Å and an axial subunit repeat of 27 Å. The native SH3 domain is too compact to fit into the fibril density, and must unfold to adopt a longer, thinner shape in the amyloid form. The 2040-Å protofilaments can only accommodate one pair of flat -sheets stacked against each other, with very little inter-strand twist. We propose a model for the polypeptide packing as a basis for understanding the structure of amyloid fibrils in general.
Recent advances in single-molecule detection and single-molecule spectroscopy at room temperature by laser-induced fluorescence
offer new tools for the study of individual macromolecules under physiological conditions. These tools relay conformational
states, conformational dynamics, and activity of single biological molecules to physical observables, unmasked by ensemble
averaging. Distributions and time trajectories of these observables can therefore be measured during a reaction without the
impossible need to synchronize all the molecules in the ensemble. The progress in applying these tools to biological studies
with the use of fluorophores that are site-specifically attached to macromolecules is reviewed.
Chromophore-assisted light inactivation (CALI) offers the only method capable of modulating specific protein activities in
localized regions and at particular times. Here, we generalize CALI so that it can be applied to a wider range of tasks. Specifically,
we show that CALI can work with a genetically inserted epitope tag; we investigate the effectiveness of alternative dyes,
especially fluorescein, comparing them with the standard CALI dye, malachite green; and we study the relative efficiencies
of pulsed and continuous-wave illumination. We then use fluorescein-labeled hemagglutinin antibody fragments, together with
relatively low-power continuous-wave illumination to examine the effectiveness of CALI targeted to kinesin. We show that CALI
can destroy kinesin activity in at least two ways: it can either result in the apparent loss of motor activity, or it can
cause irreversible attachment of the kinesin enzyme to its microtubule substrate. Finally, we apply this implementation of
CALI to an in vitro system of motor proteins and microtubules that is capable of self-organized aster formation. In this system, CALI can effectively
perturb local structure formation by blocking or reducing the degree of aster formation in chosen regions of the sample, without
influencing structure formation elsewhere.
The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is a versatile reporter protein for monitoring gene expression and protein localization in a variety of systems. Applications using GFP reporters have expanded greatly due to the availability of mutants with altered spectral properties, including several blue emission variants, all of which contain the single point mutation Tyr-66 to His in the chromophore region of the protein. However, previously described "BFP" reporters have limited utility, primarily due to relatively dim fluorescence and low expression levels attained in higher eukaryotes with such variants. To improve upon these qualities, we have combined a blue emission mutant of GFP containing four point mutations (Phe-64 to Leu, Ser-65 to Thr, Tyr-66 to His, and Tyr-145 to Phe) with a synthetic gene sequence containing codons preferentially found in highly expressed human proteins. These mutations were chosen to optimize expression of properly folded fluorescent protein in mammalian cells cultured at 37 degreesC and to maximize signal intensity. The combination of improved fluorescence and higher expression levels yield an enhanced blue fluorescent protein that provides greater sensitivity and is suitable for dual color detection with green-emitting fluorophores.
We describe the utilization of a red fluorescent protein (DsRed) as an in vivo marker for Saccharomyces cerevisiae. Clones expressing red and/or green fluorescent proteins with both cytoplasmic and nuclear localization were obtained. A
series of vectors are now available which can be used to create amino-terminal (N-terminal) and carboxyl-terminal (C-terminal)
fusions with the DsRed protein.
We have used an in vitro Golgi protein transport assay dependent on high molecular weight (greater than 100 kD) cytosolic and/or peripheral membrane proteins to study the requirements for transport from the cis- to the medial-compartment. Fractionation of this system indicates that, besides the NEM-sensitive fusion protein (NSF) and the soluble NSF attachment protein (SNAP), at least three high molecular weight protein fractions from bovine liver cytosol are required. The activity from one of these fractions was purified using an assay that included the second and third fractions in a crude state. The result is a protein of 115-kD subunit molecular mass, which we term p115. Immunodepletion of the 115-kD protein from a purified preparation with mAbs removes activity. Peptide sequence analysis of tryptic peptides indicates that p115 is a "novel" protein that has not been described previously. Gel filtration and sedimentation analysis indicate that, in its native state, p115 is a nonglobular homo-oligomer. p115 is present on purified Golgi membranes and can be extracted with high salt concentration or alkaline pH, indicating that it is peripherally associated with the membrane. Indirect immunofluorescence indicates that p115 is associated with the Golgi apparatus in situ.
Chromophore-assisted laser inactivation of protein function has been achieved. After a protein binds a specific ligand or antibody conjugated with malachite green (C.I. 42,000), it is selectively inactivated by laser irradiation at a wavelength of light absorbed by the dye but not significantly absorbed by cellular components. Ligand-bound proteins in solution and on the surfaces of cells can be denatured without other proteins in the same samples being affected. Chromophore-assisted laser inactivation can be used to study cell surface phenomena by inactivating the functions of single proteins on living cells, a molecular extension of cellular laser ablation. It has an advantage over genetics and the use of specific inhibitors in that the protein function of a single cell within the organism can be inactivated by focusing the laser beam.
The ultrastructural organization of the fibrous component of amyloid has been analyzed by means of high resolution electron microscopy of negatively stained isolated amyloid fibrils and of positively stained amyloid fibrils in thin tissue sections. It was found that a number of subunits could be resolved according to their dimensions. The following structural organization is proposed. The amyloid fibril, the fibrous component of amyloid as seen in electron microscopy of thin tissue sections, consists of a number of filaments aggregated side-by-side. These amyloid filaments are approximately 75-80 A in diameter and consist of five (or less likely six) subunits (amyloid protofibrils) which are arranged parallel to each other, longitudinal or slightly oblique to the long axis of the filament. The filament has often seemed to disperse into several longitudinal rows. The amyloid protofibril is about 25-35 A wide and appears to consist of two or three subunit strands helically arranged with a 35-50-A repeat (or, less likely, is composed of globular subunits aggregated end-to-end). These amyloid subprotofibrillar strands measure approximately 10-15 A in diameter.
Chromophore-assisted laser inactivation (CALI) is a technique that selectively inactivates proteins of interest to elucidate their in vivo functions. This method has application to a wide array of biological questions and an understanding of its mechanism is required for its judicious application. We report here that CALI is not mediated by photoinduced thermal denaturation but by photogenerated free radicals. Thermal diffusion calculations suggest that the temperature changes resulting from CALI are too small to cause thermal denaturation, and Arrhenius plots of CALI are inconsistent with a photothermal mechanism. CALI shows an energy dose reciprocity above a threshold and can be inhibited by free-radical quenchers, thus demonstrating a photochemical mechanism of protein inactivation. The type of quenchers that are effective in inhibiting CALI indicates that the active species is a hydrogen abstractor which is not derived from molecular oxygen. We suggest that the active free-radical species is the hydroxyl radical and its very short lifetime explains the spatial specificity of CALI such that half-maximal damage is effected within 15 A from the dye moiety and no significant damage occurs at 34 A. The data are consistent with free-radical formation resulting from a sequential two-photon process.
The jellyfish Aequorea victoria possesses in the margin of its umbrella a green fluorescent protein (GFP, 27 kDa) that serves as the ultimate light emitter in the bioluminescence reaction of the animal. The protein is made up of 238 amino acid residues in a single polypeptide chain and produces a greenish fluorescence (lambda max = 508 nm) when irradiated with long ultraviolet light. The fluorescence is due to the presence of a chromophore consisting of an imidazolone ring, formed by a post-translational modification of the tripeptide -Ser65-Tyr66-Gly67-. GFP has been used extensively as a reporter protein for monitoring gene expression in eukaryotic and prokaryotic cells, but relatively little is known about the chemical mechanism by which fluorescence is produced. To obtain a better understanding of this problem, we studied a peptide fragment of GFP bearing the chromophore and a synthetic model compound of the chromophore. The results indicate that the GFP chromophore consists of an imidazolone ring structure and that the light emitter is the singlet excited state of the phenolate anion of the chromophore. Further, the light emission is highly dependent on the microenvironment around the chromophore and that inhibition of isomerization of the exo-methylene double bond of the chromophore accounts for its efficient light emission.
The 2.1-A resolution crystal structure of wild-type green fluorescent protein and comparison of it with the recently determined structure of the Ser-65 --> Thr (S65T) mutant explains the dual wavelength absorption and photoisomerization properties of the wild-type protein. The two absorption maxima are caused by a change in the ionization state of the chromophore. The equilibrium between these states appears to be governed by a hydrogen bond network that permits proton transfer between the chromophore and neighboring side chains. The predominant neutral form of the fluorophore maximally absorbs at 395 nm. It is maintained by the carboxylate of Glu-222 through electrostatic repulsion and hydrogen bonding via a bound water molecule and Ser-205. The ionized form of the fluorophore, absorbing at 475 nm, is present in a minor fraction of the native protein. Glu-222 donates its charge to the fluorophore by proton abstraction through a hydrogen bond network, involving Ser-205 and bound water. Further stabilization of the ionized state of the fluorophore occurs through a rearrangement of the side chains of Thr-203 and His-148. UV irradiation shifts the ratio of the two absorption maxima by pumping a proton relay from the neutral chromophore's excited state to Glu-222. Loss of the Ser-205-Glu-222 hydrogen bond and isomerization of neutral Glu-222 explains the slow return to the equilibrium dark-adapted state of the chromophore. In the S65T structure, steric hindrance by the extra methyl group stabilizes a hydrogen bonding network, which prevents ionization of Glu-222. Therefore the fluorophore is permanently ionized, causing only a 489-nm excitation peak. This new understanding of proton redistribution in green fluorescent protein should enable engineering of environmentally sensitive fluorescent indicators and UV-triggered fluorescent markers of protein diffusion and trafficking in living cells.
The green fluorescent protein (GFP) from the jellyfish Aequorea Victoria forms an intrinsic chromophore through cyclization and oxidation of an internal tripeptide motif [Prasher, D. C., et al. (1992) Gene 111, 229-233; Cody, C. E., et al. (1993) Biochemistry 32, 1212-1218]. We monitored the formation of the chromophore in vitro using the S65T-GFP chromophore mutant. S65T-GFP recovered from inclusion bodies in Escherichia coli lacks the mature chromophore, suggesting that protein destined for inclusion bodies aggregated prior to productive folding. This material was used to follow the steps leading to chromophore formation. The process of chromophore formation in S65T-GFP was determined to be an ordered reaction consisting of three distinct kinetic steps. Protein folding occurs fairly slowly (k(f) = 2.44 x 10(-3) s(-1)) and prior to any chromophore modification. Next, an intermediate step occurs that includes, but is not necessarily limited to, cyclization of the tripeptide chromophore motif (k(c) = 3.8 x 10(-3) s(-1)). The final and slow step (k(ox) = 1.51 x 10(-4) s(-1)) in chromophore formation involves oxidation of the cyclized chromophore. Since the chromophore forms de novo from purified denatured protein and is a first-order process, we conclude that GFP chromophore formation is an autocatalytic process.
Important Ca2+ signals in the cytosol and organelles are often extremely localized and hard to measure. To overcome this problem we have constructed new fluorescent indicators for Ca2+ that are genetically encoded without cofactors and are targetable to specific intracellular locations. We have dubbed these fluorescent indicators 'cameleons'. They consist of tandem fusions of a blue- or cyan-emitting mutant of the green fluorescent protein (GFP), calmodulin, the calmodulin-binding peptide M13, and an enhanced green- or yellow-emitting GFP. Binding of Ca2+ makes calmodulin wrap around the M13 domain, increasing the fluorescence resonance energy transfer (FRET) between the flanking GFPs. Calmodulin mutations can tune the Ca2+ affinities to measure free Ca2+ concentrations in the range 10(-8) to 10(-2) M. We have visualized free Ca2+ dynamics in the cytosol, nucleus and endoplasmic reticulum of single HeLa cells transfected with complementary DNAs encoding chimaeras bearing appropriate localization signals. Ca2+ concentration in the endoplasmic reticulum of individual cells ranged from 60 to 400 microM at rest, and 1 to 50 microM after Ca2+ mobilization. FRET is also an indicator of the reversible intermolecular association of cyan-GFP-labelled calmodulin with yellow-GFP-labelled M13. Thus FRET between GFP mutants can monitor localized Ca2+ signals and protein heterodimerization in individual live cells.
Peptide aptamers provide probes for biological processes and adjuncts for development of novel pharmaceutical molecules. Such
aptamers are analogous to compounds derived from combinatorial chemical libraries which have specific binding or inhibitory
activities. Much as it is generally difficult to determine the composition of combinatorial chemical libraries in a quantitative
manner, determining the quality and characteristics of peptide libraries displayed in vivo is problematical. To help address these issues we have adapted green fluorescent protein (GFP) as a scaffold for display
of conformationally constrained peptides. The GFP-peptide libraries permit analysis of library diversity and expression levels
in cells and allow enrichment of the libraries for sequences with predetermined characteristics, such as high expression of
correctly folded protein, by selection for high fluorescence.
A novel, brilliantly red fluorescent protein, DsRed has become available recently opening up a wide variety of experimental opportunities for double labeling and fluorescence resonance electron transfer experiments in combination with green fluorescent protein (GFP). Unlike in the case of GFP, proteins tagged with DsRed were often found to aggregate within the cell. Here we report a simple method that allows rescuing the function of an oligomeric protein tagged with DsRed. We demonstrate the feasibility of this approach on the subunit proteins of an oligomeric membrane channel, gap junction connexins. Additionally, DsRed fluorescence was easily detected 12–16 h post transfection, much earlier than previously reported, and could readily be differentiated from co-expressed GFP. Thus, this approach can eliminate the major drawbacks of this highly attractive autofluorescent protein.
Gene expression was visualized in single living mammalian cells with β-lactamase as a reporter that hydrolyzes a substrate
loaded intracellularly as a membrane-permeant ester. Each enzyme molecule changed the fluorescence of many substrate molecules
from green to blue by disrupting resonance energy transfer. This wavelength shift was detectable by eye or color film in individual
cells containing less than 100 β-lactamase molecules. The robust change in emission ratio reveals quantitative heterogeneity
in real-time gene expression, enables clonal selection by flow cytometry, and forms a basis for high-throughput screening
of pharmaceutical candidate drugs in living mammalian cells.
Renaturation of green-fluorescent protein (A-GFP) was achieved for the first time following denaturation in guanidine-HCl or acid. Denaturation was accompanied by the concerted loss of visible fluorescence, alteration of absorption characteristics, and large negative deflection of CD signal in the far UV. Dialysis of a guanidine-denatured sample at pH 8 resulted in 64% renaturation (return to native absorption) and neutralization of an acid-denatured sample restored 90% of the native absorption. Renatured GFP is highly fluorescent and indistinguishable from native GFP with respect to the shape of excitation and emission spectra. Both native and denatured proteins exhibit resistance to trypsin hydrolysis and have identically broad pH and heat stability profiles, all of which suggest full renaturation.
We have determined to 2.1 Å resolution the crystal structure of a dark state, kindling fluorescent protein isolated from the
sea anemone, Anemonia sulcata. The chromophore sequence Met63-Tyr64-Gly65 of the A. sulcata chromoprotein was previously proposed to comprise a 6-membered pyrazine-type heterocycle (Martynov, V. I., Savitsky, A. P.,
Martynova, N. Y., Savitsky, P. A., Lukyanov, K. A., and Lukyanov, S. A. (2001) J. Biol. Chem. 276, 21012–21016). However, our crystallographic data revealed the chromophore to comprise a 5-membered p-hydroxybenzylideneimidazolinone moiety that adopts a non-coplanar trans conformation within the interior of the GFP β-can fold. Unexpectedly, fragmentation of the polypeptide was found to occur
within the chromophore moiety, at the bond between Cys62C and Met63N1. Our structural data reveal that fragmentation of the chromophore represents an intrinsic, autocatalytic step toward the formation
of the mature chromophore within the specific GFP-like proteins.
The green fluorescent protein of Aequorea victoria (GFP) is a natural peptide chromophore without substrate or cofactor requirements for fluorescence. In vitro, a recombinant F64L/S65T GFP mutant (GFPmut1) exhibited pH sensitive fluorescence within the physiologic range. When heterologously expressed in BS-C-1 cells or rabbit proximal tubule cells, uniform cytosolic and nuclear fluorescence was observed. Cytosolic fluorescence constituted over 80% of the total. Excitation scanning of transfected cells revealed two GFPmut1-specific regions that were pH-sensitive over the physiologic range, and each region exhibited a unique pH “bias” in fluorescence emission. Excitation at or near the expected maximum of 488 nm (region II) uniformly resulted in fluorescence that was preferentially altered at acidic pH. In contrast, a novel “wild-type” excitation peak at 400 nm (region I) resulted in alkaline-biased fluorescence similar to that described for the wild-type chromophore in vitro, suggesting that wild-type spectral features disrupted in vitro by mutagenesis may be recovered in intact cells. Calibration of intracellular pH (pHi) with in situ fluorescence following excitation in either region revealed a semilogarithmic relationship between fluorescence intensity and pH within the physiologic range. We therefore measured pHi changes attributable to altered Na/HCO3 cotransport (NBC) activity both in GFPmut1-expressing cells and in paired untransfected cells loaded with BCECF. Basal NBC activity was the same in each group, as was the stimulation of activity by 10% CO2, thus validating the utility of GFPmut1 as a fluorescent probe for pHi and establishing a novel, useful, and practical application for GFPmut1 in monitoring pHi in real time.
VMD is a molecular graphics program designed for the display and analysis of molecular assemblies, in particular biopolymers such as proteins and nucleic acids. VMD can simultaneously display any number of structures using a wide variety of rendering styles and coloring methods. Molecules are displayed as one or more "representations," in which each representation embodies a particular rendering method and coloring scheme for a selected subset of atoms. The atoms displayed in each representation are chosen using an extensive atom selection syntax, which includes Boolean operators and regular expressions. VMD provides a complete graphical user interface for program control, as well as a text interface using the Tcl embeddable parser to allow for complex scripts with variable substitution, control loops, and function calls. Full session logging is supported, which produces a VMD command script for later playback. High-resolution raster images of displayed molecules may be produced by generating input scripts for use by a number of photorealistic image-rendering applications. VMD has also been expressly designed with the ability to animate molecular dynamics (MD) simulation trajectories, imported either from files or from a direct connection to a running MD simulation. VMD is the visualization component of MDScope, a set of tools for interactive problem solving in structural biology, which also includes the parallel MD program NAMD, and the MDCOMM software used to connect the visualization and simulation programs. VMD is written in C++, using an object-oriented design; the program, including source code and extensive documentation, is freely available via anonymous ftp and through the World Wide Web.
The calcium-sensitive photoprotein aequorin and the green fluorescent protein were isolated and purified from Aequorea forskålea. Purified aequorin shows electrophoretic microheterogeneity but appears as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino-terminal sequence determination by the automated Edman technique revealed a single NH2-terminal sequence: Val-Lys-Leu-(Thr)-Pro-Asp- Phe-Asn-Asn-Pro-(-)-Trp-Ile-Gly-Arg-His-aequorin exists as a single polypeptide chain. Apparent molecular weights were determined by sedimentation equilibrium, urea-sodium dodecyl sulfate/polyacrylamide gel electrophoresis, gel filtration of native protein, and gel filtration of denatured protein in 6 M guanidine hydrochloride; all methods suggest an apparent molecular weight of 19 500 ± 2000 for aequorin. Under some conditions the protein dimerizes by disulfide bond formation. Aequorin has a sedimentation coefficient of 2.31 S and a Stokes radius of ∼19 Å; the extinction coefficient (E1cm1%) was calculated to be 27.1. Amino acid analysis revealed a slight preponderance of acidic residues; no carbohydrate moieties were found. Aequorin contains at least one free sulfhydryl group, chemical modification of which results in irreversible loss of luminescent activity. The apparent molecular weight of the green fluorescent protein is 30 000 when determined by urea-sodium dodecyl sulfate/polyacrylamide gel electrophoresis and gel filtration of denatured protein in 6 M guanidine hydrochloride. Amino acid analysis revealed an absence of tryptophan.
The chapter describes the understanding of the physics and physical chemistry of sickle cell hemoglobin polymerization in solutions and in red cells. The polymerization of sickle cell hemoglobin has probably become the best understood of all protein self-assembly systems. The structure of the hemoglobin S molecule, the structure of the various aggregated forms of hemoglobin S, and the structural analysis of the polymers are discussed in the chapter. The chapter discusses the thermodynamics of hemoglobin S polymerization, and includes a description of the nonideal behavior of concentrated hemoglobin S solutions and the effects of physiologically relevant variables, especially oxygen, and the presence of non-S hemoglobins on the polymerization process. Understanding the polymerization process is not only important for understanding the pathophysiology of sickle cell disease, but is critical to the major problem of developing a specific therapy that could be used in the treatment of patients. The kinetic and thermodynamic studies have played a major role by providing relevant and sensitive assays for potential therapeutic agents. The results of the thermodynamic and kinetic studies of solutions are used to explain various properties of cells, including morphological and rheological properties.
Bence Jones protein was enzymatically cleaved into its variable and constant segments, and then the variable segment was precipitated at neutral pH. The precipitate stained well with Congo red and showed green birefringence under the polarized light after such staining. High resolution electron microscopy of shadow-casted and negatively stained preparations revealed that they consisted of three types of fibrillar structures, respectively measuring 10 to 20 Å, 25 to 40 Å and 75 to 100 Å in width. The thinner fibrillar structures dominated the specimens from earlier stages of the precipitation procedure, while thicker ones predominated in the later samples. The sequential appearance of these structures and other observations suggested that they represented fibrils in different stages of development. These light and electron microscopic aspects of the precipitate have many points corresponding with those of the amyloid, although the identity between the two substances remains to be established. Moreover, the fibrillar structures could be produced in only 3 of 13 Bence Jones proteins treated in an identical fashion.
The green-fluorescent protein (GFP) that functions as a bioluminescence energy transfer acceptor in the jellyfish Aequorea has been renatured with up to 90% yield following acid, base, or guanidine denaturation. Renaturation, following pH neutralization or simple dilution of guanidine, proceeds with a half-recovery time of less than 5 min as measured by the return of visible fluorescence. Residual unrenatured protein has been quantitatively removed by chromatography on Sephadex G-75. The chromatographed, renatured GFP has corrected fluorescence excitation and emission spectra identical with those of the native protein at pH 7.0 (excitation lambda max = 398 nm; emission lambda max = 508 nm) and also at pH 12.2 (excitation lambda max = 476 nm; emission lambda max = 505 nm). With its peak position red-shifted 78 nm at pH 12.2, the Aequorea GFP excitation spectrum more closely resembles the excitation spectra of Renilla (sea pansy) and Phialidium (hydromedusan) GFPs at neutral pH. Visible absorption spectra of the native and renatured Aequorea green-fluorescent proteins at pH 7.0 are also identical, suggesting that the chromophore binding site has returned to its native state. Small differences in far-UV absorption and circular dichroism spectra, however, indicate that the renatured protein has not fully regained its native secondary structure.
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A molecular understanding of biology requires that we establish the in situ functions of the proteins in cellular processes. To address this, we developed chromophore- assisted laser inactivation (CALI) for probing the in vivo function of proteins. CALI inactivates specific proteins in living cells by using non-blocking antibodies conjugated with malachite green (MG) dye. MG absorbs 620 nm laser light (which is not absorbed by cells) to generate short lived free radicals with limited range of oxidative damage (15 angstroms) around the dye. This inactivates the bound protein without significantly affecting its neighbors. CALI has been applied to 40 proteins and achieved specific inactivation in almost all those tested. We have developed micro-CALI which uses a focused laser beam (10 micrometers ) to acutely inactivate specific proteins within cells. We have used this to address the molecular mechanisms of neuronal growth cone motility and has implicated a diversity of proteins (e.g. molecular motors, cytoskeletal, and signaling molecules) in discrete steps of growth cone motility. We hope that micro-CALI will be a useful research tool for addressing dynamic processes in biology and medicine.
Using random mutagenesis and visual selection of fluorescent clones, we have isolated a T203I and a E222G mutant of the Aequorea green-fluorescent protein. Each mutant has one of the two fluorescence excitation bands of the wild type deleted and retains the other without a wavelength shift. This finding is consistent with each excitation band corresponding to a distinct spectroscopic state of the chromophore. Both mutations are single amino acid exchanges which in the linear sequence are located remotely from the chromophore but in the folded protein may be situated in its vicinity. We conclude that the mutations influence the fluorescence properties by changing the interactions between the chromophore and its protein environment.
The green fluorescent protein (GFP) of the jellyfish Aequorea victoria is an unusual protein with strong visible absorbance and fluorescence from a p-hydroxybenzylidene-imidazolidinone chromophore, which is generated by cyclization and oxidation of the protein's own Ser-Tyr-Gly sequence at positions 65-67. Cloning of the cDNA and heterologous expression of fluorescent protein in a wide variety of organisms indicate that this unique posttranslational modification must be either spontaneous or dependent only on ubiquitous enzymes and reactants. We report that formation of the final fluorophore requires molecular oxygen and proceeds with a time constant (approximately 4 hr at 22 degrees C and atmospheric pO2) independent of dilution, implying that the oxidation does not require enzymes or cofactors. GFP was mutagenized and screened for variants with altered spectra. The most striking mutant fluoresced blue and contained histidine in place of Tyr-66. The availability of two visibly distinct colors should significantly extend the usefulness of GFP in molecular and cell biology by enabling in vivo visualization of differential gene expression and protein localization and measurement of protein association by fluorescence resonance energy transfer.
Computer simulations of the evolution of linear sequences have demonstrated the importance of recombination of blocks of sequence rather than point mutagenesis alone. Repeated cycles of point mutagenesis, recombination, and selection should allow in vitro molecular evolution of complex sequences, such as proteins. A method for the reassembly of genes from their random DNA fragments, resulting in in vitro recombination is reported. A 1-kb gene, after DNase I digestion and purification of 10- to 50-bp random fragments, was reassembled to its original size and function. Similarly, a 2.7-kb plasmid could be efficiently reassembled. Complete recombination was obtained between two markers separated by 75 bp; each marker was located on a separate gene. Oligonucleotides with 3' and 5' ends that are homologous to the gene can be added to the fragment mixture and incorporated into the reassembled gene. Thus, mixtures of synthetic oligonucleotides and PCR fragments can be mixed into a gene at defined positions based on homology. As an example, a library of chimeras of the human and murine genes for interleukin 1 beta has been prepared. Shuffling can also be used for the in vitro equivalent of some standard genetic manipulations, such as a backcross with parental DNA. The advantages of recombination over existing mutagenesis methods are likely to increase with the numbers of cycles of molecular evolution.
Highly purified recombinant Aequorea green fluorescent protein is able to undergo a reversible oxidation-reduction reaction in the presence of molecular oxygen. In the oxidized form in near UV light, the protein is highly fluorescent, but when reduced with sodium dithionite, it becomes completely non-fluorescent. On exposure to molecular oxygen the reduced, non-fluorescent protein reverts to its original fluorescent state.
A complementary DNA for the Aequorea victoria green fluorescent protein (GFP) produces a fluorescent product when expressed in prokaryotic (Escherichia coli) or eukaryotic (Caenorhabditis elegans) cells. Because exogenous substrates and cofactors are not required for this fluorescence, GFP expression can be used to monitor gene expression and protein localization in living organisms.
The green-fluorescent proteins (GFP) are a unique class of proteins involved in bioluminescence of many cnidaria. The GFPs serve as energy-transfer acceptors, receiving energy from either a luciferase-oxyluciferin complex or a Ca(2+)-activated photoprotein, depending on the organism. Upon mechanical stimulation of the organism, GFP emits green light spectrally identical to its fluorescence emission. These highly fluorescent proteins are unique due to the nature of the covalently attached chromophore, which is composed of modified amino acid residues within the polypeptide. This report describes the characterization of the Aequorea victoria GFP chromophore which is released as a hexapeptide upon digestion of the protein with papain. The chromophore is formed upon cyclization of the residues Ser-dehydroTyr-Gly within the polypeptide. The chromophore structure proposed here differs from that described by Shimomura [(1979) FEBS Lett. 104, 220] in a number of ways.
Green fluorescent proteins (GFPs) are presently attracting tremendous interest as the first general method to create strong visible fluorescence by purely molecular biological means. So far, they have been used as reporters of gene expression, tracers of cell lineage, and as fusion tags to monitor protein localization within living cells. However, the GFP originally cloned from the jellyfish Aequorea victoria has several nonoptimal properties including low brightness, a significant delay between protein synthesis and fluorescence development, and complex photoisomerization. Fortunately, the protein can be re-engineered by mutagenesis to ameliorate these deficiencies and shift the excitation and emission wavelengths, creating different colors and new applications.
Recent publications strongly support the hypothesis that conformational changes in amyloidogenic proteins lead to amyloid fibril formation and cause disease. Biophysical studies on several amyloidogenic proteins provide insights into the conformational changes required for fibrilogenesis. In addition, newly available moderate to high resolution structural studies are bringing us closer to understanding the structure of amyloid.
The green fluorescent protein (GFP) from the Pacific Northwest jellyfish Aequorea victoria has generated intense interest as a marker for gene expression and localization of gene products. The chromophore, resulting
from the spontaneous cyclization and oxidation of the sequence -Ser65 (or Thr65)-Tyr66-Gly67-, requires the native protein fold for both formation and fluorescence emission. The structure of Thr65 GFP has been determined at 1.9 angstrom resolution. The protein fold consists of an 11-stranded β barrel with a coaxial helix,
with the chromophore forming from the central helix. Directed mutagenesis of one residue adjacent to the chromophore, Thr203, to Tyr or His results in significantly red-shifted excitation and emission maxima.
We have constructed a library in Escherichia coli of mutant gfp genes (encoding green fluorescent protein, GFP) expressed from a tightly regulated inducible promoter. We introduced random amino acid (aa) substitutions in the twenty aa flanking the chromophore Ser-Tyr-Gly sequence at aa 65-67. We then used fluorescence-activated cell sorting (FACS) to select variants of GFP that fluoresce between 20-and 35-fold more intensely than wild type (wt), when excited at 488 nm. Sequence analysis reveals three classes of aa substitutions in GFP. All three classes of mutant proteins have highly shifted excitation maxima. In addition, when produced in E. coli, the folding of the mutant proteins is more efficient than folding of wt GFP. These two properties contribute to a greatly increased (100-fold) fluorescence intensity, making the mutants useful for a number of applications.
VMD is a molecular graphics program designed for the display and analysis of molecular assemblies, in particular biopolymers such as proteins and nucleic acids. VMD can simultaneously display any number of structures using a wide variety of rendering styles and coloring methods. Molecules are displayed as one or more "representations," in which each representation embodies a particular rendering method and coloring scheme for a selected subset of atoms. The atoms displayed in each representation are chosen using an extensive atom selection syntax, which includes Boolean operators and regular expressions. VMD provides a complete graphical user interface for program control, as well as a text interface using the Tcl embeddable parser to allow for complex scripts with variable substitution, control loops, and function calls. Full session logging is supported, which produces a VMD command script for later playback. High-resolution raster images of displayed molecules may be produced by generating input scripts for use by a number of photorealistic image-rendering applications. VMD has also been expressly designed with the ability to animate molecular dynamics (MD) simulation trajectories, imported either from files or from a direct connection to a running MD simulation. VMD is the visualization component of MDScope, a set of tools for interactive problem solving in structural biology, which also includes the parallel MD program NAMD, and the MDCOMM software used to connect the visualization and simulation programs. VMD is written in C++, using an object-oriented design; the program, including source code and extensive documentation, is freely available via anonymous ftp and through the World Wide Web.
The docking of transport vesicles with their target membrane is thought to be mediated by p115. We show here that GM130, a cis-Golgi matrix protein, interacts specifically with p115 and so could provide a membrane docking site. Deletion analysis showed that the N-terminus binds to p115, whereas the C-terminus binds to Golgi membranes. Mitotic phosphorylation of GM130 or a peptide derived from the N-terminus prevented binding to p115. The peptide also inhibited the NSF- but not the p97-dependent reassembly of Golgi cisternae from mitotic fragments, unless it was mitotically phosphorylated. Together, these data provide a molecular explanation for the COPI-mediated fragmentation of the Golgi apparatus at the onset of mitosis.
Tissue deposition of normally soluble proteins as insoluble amyloid fibrils is associated with serious diseases including the systemic amyloidoses, maturity onset diabetes, Alzheimer's disease and transmissible spongiform encephalopathy. Although the precursor proteins in different diseases do not share sequence homology or related native structure, the morphology and properties of all amyloid fibrils are remarkably similar. Using intense synchrotron sources we observed that six different ex vivo amyloid fibrils and two synthetic fibril preparations all gave similar high-resolution X-ray fibre diffraction patterns, consistent with a helical array of beta-sheets parallel to the fibre long axis, with the strands perpendicular to this axis. This confirms that amyloid fibrils comprise a structural superfamily and share a common protofilament substructure, irrespective of the nature of their precursor proteins.
We have investigated properties relevant to quantitative imaging in living cells of five green fluorescent protein (GFP) variants that have been used extensively or are potentially useful. We measured the extinction coefficients, quantum yields, pH effects, photobleaching effects, and temperature-dependent chromophore formation of wtGFP, alphaGFP (F99S/M153T/V163A), S65T, EGFP (F64L/S65T), and a blue-shifted variant, EBFP (F64L/S65T/Y66H/Y145F). Absorbance and fluorescence spectroscopy showed little difference between the extinction coefficients and quantum yields of wtGFP and alphaGFP. In contrast, S65T and EGFP extinction coefficients made them both approximately 6-fold brighter than wtGFP when excited at 488 nm, and EBFP absorbed more strongly than the wtGFP when excited in the near-UV wavelength region, although it had a much lower quantum efficiency. When excited at 488 nm, the GFPs were all more resistant to photobleaching than fluorescein. However, the wtGFP and alphaGFP photobleaching patterns showed initial increases in fluorescence emission caused by photoconversion of the protein chromophore. The wtGFP fluorescence decreased more quickly when excited at 395 nm than 488 nm, but it was still more photostable than the EBFP when excited at this wavelength. The wtGFP and alphaGFP were quite stable over a broad pH range, but fluorescence of the other variants decreased rapidly below pH 7. When expressed in bacteria, chromophore formation in wtGFP and S65T was found to be less efficient at 37 degrees C than at 28 degrees C, but the other three variants showed little differences between 37 degrees C and 28 degrees C. In conclusion, no single GFP variant is ideal for every application, but each one offers advantages and disadvantages for quantitative imaging in living cells.
The conformational change hypothesis postulates that tertiary structural changes under partially denaturing conditions convert one of 17 normally soluble and functional human proteins into an alternative conformation that subsequently undergoes self-assembly into an amyloid fibril, the putative causative agent in amyloid disease. This hypothesis is consistent with Anfinsen's view that the tertiary structure of a protein is determined both by its sequence and the aqueous environment; the latter does not always favor the normally folded state. Unlike sickle cell hemoglobin assembly, where owing to a surface mutation, hemoglobin polymerizes in its normally folded conformation, amyloid proteins self-assemble as a result of the formation of an alternative tertiary structure-a conformational intermediate formed under partially denaturing conditions. The pathway by which an amyloidogenic protein assembles into amyloid fibrils appears to involve quaternary structural intermediates that assemble into increasingly complex quaternary structures, including amyloid protofilaments, which ultimately assemble into amyloid fibrils. Several recent studies have discussed the multi-step assembly pathway(s) characterizing amyloid fibril formation.