Fluorescently-labeled Proteins Research Articles

Autocorrelation analysis of a phenotypic screen reveals hidden drug activity

Phenotype based screening is a powerful tool to evaluate cellular drug response. Through high content fluorescence imaging of simple fluorescent labels and complex image analysis phenotypic measurements can identify subtle compound-induced cellular changes unique to compound mechanisms of action (MoA). Recently, a screen of 1008 compounds in three cell lines was reported where analysis detected changes in cellular phenotypes and accurately identified compound MoA for roughly half the compounds. However, we were surprised that DNA alkylating agents and other compounds known to induce or impact the DNA damage response produced no measured activity in cells with fluorescently labeled 53BP1—a canonical DNA damage marker. We hypothesized that phenotype analysis is not sensitive enough to detect small changes in 53BP1 distribution and analyzed the screen images with autocorrelation image analysis. We found that autocorrelation analysis, which quantifies fluorescently-labeled protein clustering, identified higher compound activity for compounds and MoAs known to impact the DNA damage response, suggesting altered 53BP1 recruitment to damaged DNA sites. We then performed experiments under more ideal imaging settings and found autocorrelation analysis to be a robust measure of changes to 53BP1 clustering in the DNA damage response. These results demonstrate the capacity of autocorrelation to detect otherwise undetectable compound activity and suggest that autocorrelation analysis of specific proteins could serve as a powerful screening tool.

Computational tools for quantifying actin filament numbers, lengths, and bundling.

The actin cytoskeleton is a dynamic filamentous network that assembles into specialized structures to enable cells to perform essential processes. Direct visualization of fluorescently-labeled cytoskeletal proteins has provided numerous insights into the dynamic processes that govern the assembly of actin-based structures. However, accurate analysis of these experiments is often complicated by the interdependent and kinetic natures of the reactions involved. It is often challenging to disentangle these processes to accurately track their evolution over time. Here, we describe two programs written in the MATLAB programming language that facilitate counting, length measurements, and quantification of bundling of actin filaments visualized in fluorescence micrographs. To demonstrate the usefulness of our programs, we describe their application to the analysis of two representative reactions: (1) a solution of pre-assembled filaments under equilibrium conditions, and (2) a reaction in which actin filaments are crosslinked together over time. We anticipate that these programs can be applied to extract equilibrium and kinetic information from a broad range of actin-based reactions, and that their usefulness can be expanded further to investigate the assembly of other biopolymers.

KCK-tag-induced quantitative and qualitative property changes of DNA-binding protein ParB.

Lysine-cysteine-lysine (KCK) tags are commonly employed to label proteins with maleimide-fluorescent dye conjugates to enhance dye-labeling efficiency. In our study, we systematically quantified the actions of a DNA-binding CTPase ParB protein and its KCK-tagged versions on flow-stretched lambda DNA using an in vitro single-molecule DNA flow-stretching assay. We measured DNA compaction rates in a series of experimental conditions, including the presence of the specific binding sequence (parS), CTP, or its non-hydrolyzable analog CTPγS. We found that KCK tag (either at N- or C-terminus) artificially increased the compaction rates of flow-stretched DNAs and even reversed ParB proteins’ response to these experimental conditions. DNA flow-stretching experiments with fluorescently-labeled ParB protein showed that the appended KCK-tag enhances protein loading onto a DNA. Furthermore, DNA compaction by ParB protein with a negatively-charged glutamate-cysteine-glutamate (ECE) tag was inefficient compared with its non-tagged ParB protein. These results suggest that the quantitative and qualitative changes of the KCK-tagged proteins were, at least in part, derived from electrostatic interactions between positively-charged KCK-tag and negatively-charged DNA backbone. Interestingly, we found that the KCK-tagged ParB proteins do not lead to any noticeable changes from in vivo fluorescence imaging and chromatin immunoprecipitation (ChIP) assays. It implies that a lack of perturbation in in vivo assays does not necessarily guarantee intact in vitro results. Despite the small size of KCK-tag, when it is used for fluorescent-dye labeling on a protein, rigorous test must be accompanied to ensure absence of any in vitro artifacts.

Spatial resolution of virus replication: RSV and cytoplasmic inclusion bodies.

Respiratory Syncytial Virus (RSV) is a major cause of respiratory illness in young children, elderly and immunocompromised individuals worldwide representing a severe burden for health systems. The urgent development of vaccines or specific antivirals against RSV is impaired by the lack of knowledge regarding its replication mechanisms. RSV is a negative-sense single-stranded RNA (ssRNA) virus belonging to the Mononegavirales order (MNV) which includes other viruses pathogenic to humans as Rabies (RabV), Ebola (EBOV), or measles (MeV) viruses. Transcription and replication of viral genomes occur within cytoplasmatic virus-induced spherical inclusions, commonly referred as inclusion bodies (IBs). Recently IBs were shown to exhibit properties of membrane-less organelles (MLO) arising by liquid-liquid phase separation (LLPS). Compartmentalization of viral RNA synthesis steps in viral-induced MLO is indeed a common feature of MNV. Strikingly these key compartments still remain mysterious. Most of our current knowledge on IBs relies on the use of fluorescence microscopy. The ability to fluorescently label IBs in cells has been key to uncover their dynamics and nature. The generation of recombinant viruses expressing a fluorescently-labeled viral protein and the immunolabeling or the expression of viral fusion proteins known to be recruited in IBs are some of the tools used to visualize IBs in infected cells. In this chapter, microscope techniques and the most relevant studies that have shed light on RSV IBs fundamental aspects, including biogenesis, organization and dynamics are being discussed and brought to light with the investigations carried out on other MNV.

Adsorption of conjugates of lysozyme and fluorescein isothiocyanate in hydrophobic interaction chromatography

Bioconjugates, such as antibody-drug conjugates or fluorescent-labeled proteins, are highly interesting for various applications in medicine and biology. In their production, not only the synthesis is challenging but also the downstream processing, for which hydrophobic interaction chromatography (HIC) is often used. However, in-depth studies of the adsorption of bioconjugates in HIC are still rare. Therefore, in the present work, three different conjugates of lysozyme and fluorescein isothiocyanate (FITC) were synthesized and isolated, and their adsorption on the hydrophobic resin Toyopearl PPG-600 M was systematically studied in batch experiments. The influence of sodium chloride and ammonium sulfate with ionic strengths up to 2000 mM on the adsorption isotherms was investigated at pH 7.0 and 25 °C, and the results were compared to those for pure lysozyme. The conjugation leads to an increase of the adsorption in all studied cases. All studied conjugates contain only a single FITC and differ only in the position of the conjugation on the lysozyme. Despite this, strong differences in the adsorption behavior were observed. Moreover, a mathematical model was developed, which enables the prediction of the adsorption isotherms in the studied systems for varying ionic strengths.

Ribosome selectivity and nascent chain context in modulating the incorporation of fluorescent non-canonical amino acid into proteins

Fluorescence reporter groups are important tools to study the structure and dynamics of proteins. Genetic code reprogramming allows for cotranslational incorporation of non-canonical amino acids at any desired position. However, cotranslational incorporation of bulky fluorescence reporter groups is technically challenging and usually inefficient. Here we analyze the bottlenecks for the cotranslational incorporation of NBD-, BodipyFL- and Atto520-labeled Cys-tRNACys into a model protein using a reconstituted in-vitro translation system. We show that the modified Cys-tRNACys can be rejected during decoding due to the reduced ribosome selectivity for the modified aa-tRNA and the competition with native near-cognate aminoacyl-tRNAs. Accommodation of the modified Cys-tRNACys in the A site of the ribosome is also impaired, but can be rescued by one or several Gly residues at the positions −1 to −4 upstream of the incorporation site. The incorporation yield depends on the steric properties of the downstream residue and decreases with the distance from the protein N-terminus to the incorporation site. In addition to the full-length translation product, we find protein fragments corresponding to the truncated N-terminal peptide and the C-terminal fragment starting with a fluorescence-labeled Cys arising from a StopGo-like event due to a defect in peptide bond formation. The results are important for understanding the reasons for inefficient cotranslational protein labeling with bulky reporter groups and for designing new approaches to improve the yield of fluorescence-labeled protein.

Lung-resident memory CD4 T cells recognizing antigen in the airway enhance the kinetics of antigen presentation and naive T cell activation in draining lymph nodes

Abstract CD4+ tissue-resident memory (TRM) T cells act as sentinels at previous sites of infection and rapidly initiate protective inflammatory responses upon antigen re-encounter. Here, we tested if beyond local impacts in the lung, TRM responses lead to faster egress of antigen-loaded dendritic cells (DC) resulting in their more rapid accumulation in draining lymph nodes (DLN). To do so, we used a mouse model originally developed to examine CD4+ TRM-mediated protection against influenza virus infection. We established a TRM population in otherwise naïve mice by intranasal transfer of memory CD4 T cells specific for ovalbumin (OVA). The mice were then challenged with fluorescently-labelled OVA protein intranasally. Faster migration from the lung to the DLN by OVA+ DC was seen within 48 hours of protein administration in mice receiving TRM compared to controls without TRM transfer. We hypothesized that the enhanced kinetics of OVA presentation in DLN mediated by TRM antigen recognition would result in more rapid systemic activation of OVA-specific CD4 T cells. We thus transferred an indicator population of naïve OVA-specific CD4 T cells intravenously to mice receiving TRM transfer or not prior to OVA protein administration. Faster kinetics of naïve T cell activation were seen in recipients of TRM within 3 days as judged by expression of activation markers and proliferation. Most importantly, by day 4, responding naïve cells reached the lung in greater numbers and were more functional in mice receiving TRM. Our results reveal that CD4+ TRM activation can enhance the kinetics of antigen presentation in DLNs and suggest this mechanism can be harnessed by vaccines to fully integrate protective local and systemic CD4 T cell responses. Supported by grants from NIH (R21 R21AI148882)

Development of Myotropic Extracellular Vesicles for Targeted Delivery of Therapeutics to Skeletal Muscle

Muscular pathologies comprise a family of diseases and disorders resulting in the degeneration of skeletal muscle tissue. Current therapeutics are non‐specific to skeletal muscle, which often reduces efficacy and causes harmful off‐target effects in patients. Thus, a myotropic (muscle‐targeted) drug delivery system is needed to shuttle therapeutics to muscle cells to improve efficacy and limit off‐target effects. The objective of this investigation was to develop biological lipid nanoparticles, extracellular vesicles (EVs), with enhanced myotropism. We hypothesized that displaying myotropic transmembrane proteins at the surface of EVs would improve delivery of molecular cargo into muscle cells. To this end, we displayed the myotropic transmembrane proteins MyoMaker (MYMK), MyoMixer (MYMX) and M‐Cadherin (M‐CAD) in the membranes of EVs from Human Embryonic Kidney (HEK293) cells. Using flow cytometry, we measured the incorporation of each protein candidate into the EVs via endogenous green fluorescent protein (GFP) tags included on the cytosolic c‐terminus of each protein. We then measured the delivery of fluorescently‐labeled protein by each myotropic EV candidate in mouse (C2C12) myotubes. In addition to using EVs as delivery vehicles for exogenously loaded therapeutics, the native EV cargo may also be of therapeutic relevance. Given this, we examined the endogenous protein cargo of non‐engineered HEK293‐EVs via mass spectrometry. MYMK displayed the highest degree of incorporation into EVs relative to control HEK293‐EVs (p = 0.006). The MYMK‐EV formulation displayed the greatest increase in protein cargo delivered into myotubes relative to control HEK293‐EVs (~125%, p = 0.0005). Proteomic analysis revealed the top biological processes and molecular functions of HEK293‐EV protein cargo to be ribonucleic acid binding, processing, and metabolism, potentially altering translation in recipient cells. The in vitro data in the present study establish the feasibility of using MYMK‐EVs as a myotropic delivery system. Future investigations are needed to fully elucidate the cell/tissue tropism and physiological effects of MYMK‐EVs in vivo.

Contribution of stress granules and spliceosomal components to the formation of chemoresistance in ovarian cancer cells

The emergence of chemoresistance in cancer cells is one of the main reasons for the high mortality from oncological diseases. We have shown that signaling molecules from dying cancer cells contribute to a more aggressive therapy‐resistant phenotype. However, the molecular mechanisms underlying this effect remain elusive.Here we aimed to find out which proteins secreted by dying tumor cells get into recipient cells and to determine the molecular cascades triggered in response to incubation with therapy‐induced secretomes. We modified the SILAC (Stable Isotope Labeling by Amino acids in Cell culture) technology. A heavy‐labeled primary culture of ovarian cancer cells was treated or untreated with cisplatin for 48 h. Then using ultrafiltration cartridges, a fraction of extracellular vesicles (EV) were isolated from culture media. The obtained EV were resuspended in a medium containing Medium‐labeled amino acids and added to the recipient chemonaive unlabeled tumor cells for 24 h. During incubation, recipient cells absorbed EV with Heavy‐labeled proteins from donor cells and synthesized new proteins with Medium label. Then, we performed proteomic analysis of recipient cells. In total, we identified 4224 proteins. Interestingly, dying tumor cells secrete and recipient cells absorb a large number of spliceosomal and ribosomal proteins along with translation initiation complex. These proteins are often presented in stress granules. In response to EV from dying tumor cells, recipient cells upregulated the translation of genes responsible for the regulation of the cell cycle and oxidative phosphorylation.To our knowledge, this is the first report about the secretion of stress granules and spliceosomal components into the extracellular space and their internalization in other cells. To confirm these data we performed transfection of donor cells with constructs encoding the fluorescent proteins TIA1 (protein of stress granules) and SRSF4 (protein of the spliceosome). We showed that both proteins are re‐located together from the nucleus into the cytoplasm under the cisplatin treatment. Then, we demostrated the absorption of fluorescent‐labeled proteins TIA1 and SRSF4 by recipient cells after incubation with therapy‐induced EV. Our findings demonstrate that in response to cisplatin treatment tumor cells secrete spliceosomal components into the extracellular space (presumably as a part of stress granules), then these components are absorbed by recipient chemonaive cells and trigger a restructuring of metabolism and cell cycle.

Analyzing the Interaction of Fluorescent-Labeled Proteins with Artificial Phospholipid Microvesicles using Quantitative Flow Cytometry.

In the human body, most of the major physiologic reactions involved in the immune response and blood coagulation proceed on the membranes of cells. An important first step in any membrane-dependent reaction is binding of protein on the phospholipid membrane. An approach to studying protein interaction with lipid membranes has been developed using fluorescently labeled proteins and flow cytometry. This method allows the study of protein-membrane interactions using live cells and natural or artificial phospholipid vesicles. The advantage of this method is the simplicity and availability of reagents and equipment. In this method, proteins are labeled using fluorescent dyes. However, both self-made and commercially available, fluorescently labeled proteins can be used. After conjugation with a fluorescent dye, the proteins are incubated with a source of the phospholipid membrane (microvesicles or cells), and the samples are analyzed by flow cytometry. The obtained data can be used to calculate the kinetic constants and equilibrium Kd. In addition, it is possible to estimate the approximate number of protein binding sites on the phospholipid membrane using special calibration beads.

Analyzing the Interaction of Fluorescent-Labeled Proteins with Artificial Phospholipid Microvesicles using Quantitative Flow Cytometry

Analyzing the Interaction of Fluorescent-Labeled Proteins with Artificial Phospholipid Microvesicles using Quantitative Flow Cytometry

Entrapping gold nanoparticles in membranes for simple-to-use enhanced fluorescence detection of proteins

Quantifying proteins under different physiological and pathological conditions can give important insights into human health and disease, since proteins are the functional components of cells. In order to be able to use protein expression levels to diagnose diseases, sensitive protein quantification techniques are required, because some proteins can be present in low concentrations in biofluids at early stages of a disease. Here, a novel and simple-to-implement signal enhancement method for fluorescence-based protein detection is presented, in which an immunoassay was conducted on a nitrocellulose membrane, and a solution of gold nanoparticles was then pipetted onto the membrane before signal acquisition with a fluorescence microscope. The gold nanoparticles were entrapped and adsorbed into the 3D membrane matrix upon drying due to the hydrophobic interactions with the membrane. Through optimizing the concentration and size of the nanoparticles and comparison of different membranes, we were able to achieve metal enhanced fluorescence (MEF). This new gold nanoparticle-based MEF method, together with the 3D membrane platform that improves antibody and gold nanoparticle binding capacity, provided higher signal intensity and assay sensitivity for fluorescence-based protein quantification. Fluorescently-labeled IgG protein was spiked in human plasma with different concentrations, and the lower limit of detection was determined to be 29 ng/mL, three orders of magnitude lower than that of conventional 2D assays and one order of magnitude lower than that of 3D membrane assays without applying gold nanoparticles. This method offers a simple way of improving the sensitivity of fluorescence-based immunoassays that can be easily adopted in a biological lab with basic lab set up. Furthermore, it can be potentially extended to the fluorescence detection of other analytes beyond proteins.

Photosensitive Polymeric Janus Micromotor for Enzymatic Activity Protection and Enhanced Substrate Degradation.

Immobilizing enzymes into microcarriers is a strategy to improve their long-term stability and reusability, hindered by (UV) light irradiation. However, in such approaches, enzyme-substrate interaction is mediated by diffusion, often at slow kinetics. In contrast, enzyme-linked self-propelled motors can accelerate this interaction, frequently mediated by the convection mechanism. This work reports on a new photosensitive polymeric Janus micromotor (JM) for UV-light protection of enzymatic activity and efficient degradation of substrates accelerated by the JMs. The JMs were assembled with UV-photosensitive modified chitosan, co-encapsulating fluorescent-labeled proteins and enzymes as models and magnetite and platinum nanoparticles for magnetic and catalytic motion. The JMs absorbed UV light, protecting the enzymatic activity and accelerating the enzyme-substrate degradation by magnetic/catalytic motion. Immobilizing proteins in photosensitive JMs is a promising strategy to improve the enzyme's stability and hasten the kinetics of substrate degradation, thereby enhancing the enzymatic process's efficiency.

Construction of a highly selective and sensitive carbohydrate-detecting biosensor utilizing Computational Identification of Non-disruptive Conjugation sites (CINC) for flexible and streamlined biosensor design

Fluorescently-labeled solute-binding proteins that alter their fluorescence output in response to ligand binding have been utilized as biosensors for a variety of applications. Coupling protein ligand binding to altered fluorescence output often requires trial and error-based testing of both multiple labeling positions and fluorophores to produce a functional biosensor with the desired properties. This approach is laborious and can lead to reduced ligand binding affinity or altered ligand specificity. Here we report the Computational Identification of Non-disruptive Conjugation sites (CINC) for streamlined identification of fluorophore conjugation sites. By exploiting the structural dynamics properties of proteins, CINC identifies positions where conjugation of a fluorophore results in a fluorescence change upon ligand binding without disrupting protein function. We show that a CINC-developed maltooligosaccharide (MOS)-detecting biosensor is capable of rapid (kon = 20 μM−1s−1), sensitive (sub-μM KD) and selective MOS detection. The MOS-detecting biosensor is modular with respect to the spectroscopic properties and demonstrates portability to detecting MOS released via α-amylase-catalyzed depolymerization of starch using both a stopped-flow and a microplate reader assay. Our MOS-detecting biosensor represents a first-in-class probe whose design was guided by changes in localized dynamics of individual amino acid positions, supporting expansion of the CINC pipeline as an indispensable tool for a wide range of protein engineering applications.

Simultaneous Determination and Subcellular Localization of Protein-Protein Interactions in Plant Cells Using Bimolecular Fluorescence Complementation Assay.

The bimolecular fluorescence complementation (BiFC) assay allows the visualization of protein-protein interactions in their native state within living systems. The BiFC assay is based on the in vivo complementation of nonfluorescent component parts of a fluorescent protein through the interaction or proximity target proteins, each fused to a different component of the fluorescent protein. Expansion of the BiFC toolkit with an increasing spectrum of fluorescence markers and catalog of Gateway-compatible vectors for high-throughput screening, has made BiFC an exceedingly powerful tool in discovering new protein interactions or providing backup evidence for known ones. Apart from the validation of protein-protein interactions, BiFC offers the additional benefit of providing information on the subcellular localization of protein interaction complexes. Subcellular localization to a specific subcellular compartment or organelle may be further validated by the coexpression of a fluorescence-labeled protein marker. Here we describe an efficient yet simple protocol for simultaneous determination and subcellular localization of protein-protein interactions in plant cells.

Deep Learning and Computer Vision Strategies for Automated Gene Editing with a Single-Cell Electroporation Platform.

Single-cell delivery platforms like microinjection and nanoprobe electroporation enable unparalleled control over cell manipulation tasks but are generally limited in throughput. Here, we present an automated single-cell electroporation system capable of automatically detecting cells with artificial intelligence (AI) software and delivering exogenous cargoes of different sizes with uniform dosage. We implemented a fully convolutional network (FCN) architecture to precisely locate the nuclei and cytosol of six cell types with various shapes and sizes, using phase contrast microscopy. Nuclear staining or reporter fluorescence was used along with phase contrast images of cells within the same field of view to facilitate the manual annotation process. Furthermore, we leveraged the near-human inference capabilities of the FCN network in detecting stained nuclei to automatically generate ground-truth labels of thousands of cells within seconds, and observed no statistically significant difference in performance compared to training with manual annotations. The average detection sensitivity and precision of the FCN network were 95±1.7% and 90±1.8%, respectively, outperforming a traditional image-processing algorithm (72±7.2% and 72±5.5%) used for comparison. To test the platform, we delivered fluorescent-labeled proteins into adhered cells and measured a delivery efficiency of 90%. As a demonstration, we used the automated single-cell electroporation platform to deliver Cas9-guide RNA (gRNA) complexes into an induced pluripotent stem cell (iPSC) line to knock out a green fluorescent protein-encoding gene in a population of ~200 cells. The results demonstrate that automated single-cell delivery is a useful cell manipulation tool for applications that demand throughput, control, and precision.

Impact of Surface Chemistry and Doping Concentrations on Biofunctionalization of GaN/Ga‒In‒N Quantum Wells.

The development of sensitive biosensors, such as gallium nitride (GaN)-based quantum wells, transistors, etc., often makes it necessary to functionalize GaN surfaces with small molecules or even biomolecules, such as proteins. As a first step in surface functionalization, we have investigated silane adsorption, as well as the formation of very thin silane layers. In the next step, the immobilization of the tetrameric protein streptavidin (as well as the attachment of chemically modified iron transport protein ferritin (ferritin-biotin-rhodamine complex)) was realized on these films. The degree of functionalization of the GaN surfaces was determined by fluorescence measurements with fluorescent-labeled proteins; silane film thickness and surface roughness were estimated, and also other surface sensitive techniques were applied. The formation of a monolayer consisting of adsorbed organosilanes was accomplished on Mg-doped GaN surfaces, and also functionalization with proteins was achieved. We found that very high Mg doping reduced the amount of surface functionalized proteins. Most likely, this finding was a consequence of the lower concentration of ionizable Mg atoms in highly Mg-doped layers as a consequence of self-compensation effects. In summary, we could demonstrate the necessity of Mg doping for achieving reasonable bio-functionalization of GaN surfaces.

Crosstalk Between Pheromone Signaling and NADPH Oxidase Complexes Coordinates Fungal Developmental Processes.

Sexual and asexual development in filamentous ascomycetes is controlled by components of conserved signaling pathways. Here, we investigated the development of mutant strains lacking genes for kinases MAK2, MEK2, and MIK2, as well as the scaffold protein HAM5 of the pheromone response (PR) pathway. All had a defect in fruiting body development and hyphal fusion. Another phenotype was a defect in melanin-dependent ascospore germination. However, this deficiency was observed only in kinase deletion mutants, but not in strains lacking HAM5. Notably, the same developmental phenotypes were previously described for nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 (NOX1) mutants, but the germination defect was only seen in NOX2 mutants. These data suggest a molecular link between the pheromone signaling pathway and both NOX complexes. Using data from yeast two-hybrid (Y2H) analysis, we found that the scaffolding protein HAM5 interacts with NOR1, the regulator of NOX1 and NOX2 complexes. This interaction was further confirmed using differently fluorescent-labeled proteins to demonstrate that NOR1 and HAM5 co-localize at cytoplasmic spots and tips of mature hyphae. This observation was supported by phenotypic characterization of single and double mutants. The oxidative stress response and the initiation of fruiting bodies were similar in Δham5Δnor1 and Δham5, but distinctly reduced in Δnor1, indicating that the double deletion leads to a partial suppression of the Δnor1 phenotype. We conclude that the PR and NOX1 complexes are connected by direct interaction between HAM5 and NOR1. In contrast, PR kinases are linked to the NOX2 complex without participation of HAM5.

Target Protein Identification on Photocatalyst-Functionalized Magnetic Affinity Beads.

Although various affinity chromatography and photoaffinity labeling methods have been developed for target protein identification of bioactive molecules, it is often difficult to detect proteins that bind the ligand with weak transient affinity using these techniques. We have developed single electron transfer-mediated tyrosine labeling using ruthenium photocatalysts. Proximity labeling using 1-methyl-4-aryl-urazole (MAUra) labels proteins in close proximity to the photocatalyst with high efficiency and selectivity. Performing this labeling reaction on affinity beads makes it possible to label proteins that bind the ligand with weak transient affinity. In this article, novel protocols are described for target protein identification using photocatalyst proximity labeling on ruthenium photocatalyst-functionalized magnetic affinity beads. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of ruthenium photocatalyst Basic Protocol 2: Synthesis of azide- or desthiobiotin-conjugated labeling reagents Basic Protocol 3: Preparation of photocatalyst and ligand-functionalized affinity beads Basic Protocol 4: Target protein labeling in cell lysate Basic Protocol 5: Enrichment of labeled proteins with MAUra-DTB for LC-MS/MS analysis Basic Protocol 6: 2D-DIGE analysis of fluorescence-labeled proteins.

Electrochemical Detection of Endotoxins from Gram-Negative Bacteria Using Immobilized TLR4 Immunoproteins

Diagnostic platforms utilizing biorecognition elements, such as antibodies and DNA molecules, have been demonstrated as useful tools for the rapid detection of pathogens. However, these bioelements are extremely specific to a particular microorganism, which for the purpose of detection, requires prior knowledge of the infection to utilize the proper receptor. In order to circumvent this challenge, our group has been developing biosensors using a family of immunoproteins belonging to the non-specific innate immune system, the Toll-Like Receptor (TLR) immunoproteins, which are programmed to recognize chemical markers from a broad source of microorganisms. This makes them ideal for the first line of detection against an unknown pathogen. For example, TLR4 proteins are selective against endotoxins found in the cell walls of Gram-negative bacteria but not in Gram-positive bacteria. Here we show the results obtained from a Gram-negative bacteria sensor based on TLR4 tethered on the surface of a self-assembled monolayer (SAM) of alkyl thiols on a Au electrode.The TLR4 biosensor assembly involves a multi-step process, starting with the co-adsorption of three alkanethiols onto Au. The resulting SAM possesses distinct characteristics derived from each alkanethiol molecule. A hydroxyl-terminated alkanethiol serves as an anti-fouling agent and a spacer, a ferrocene- (Fc-) terminated alkanethiol is the redox active molecule assisting in charge transfer mediation, and an alkanethiol with a nitrilotriacetic acid- (NTA-) motif permits the chelation of Ni2+ cations, which then immobilizes the TLR4 immunoprotein via its His-tagged recombined C-terminus. Additionally, the desired surface orientation of the TLR4 ligand-recognition sites is also achieved using this anchoring method.1 In nature, TLR4 is a transmembrane monomer that undergoes dimerization once bound to lipopolysaccharide (LPS) endotoxin.2 It is believed that the recombinant TLR4 in our sensors behaves similarly and that dimerization results in an increase in charge transfer resistance (RCT ) with the addition of endotoxin.The sensor assembly process was monitored using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques. CVs were recorded in 200 mM phosphate buffer (PB) pH 7 while tracking the Fc redox chemistry. Similarly, EIS was performed in an electrolyte containing 5 mM ferrocyanide (ferroCN) dissolved in the same buffer. The addition of ferroCN as a second redox probe allows us to monitor the charge transfer mediation between the SAM-bound Fc and the solution-phase ferroCN ions.3 During sensor assembly, CVs showed a peak shift to higher potentials, while EIS showed higher RCT values with the transition from unmodified SAM to TLR4 modification. Both techniques indicated successful assembly. Overall, the sensors show a stable RCT signal prior to LPS exposure. During sensor testing, the amount of LPS added to the sensor was monitored using EIS, tracking the RCT value, as described above. The addition of LPS resulted in a linear RCT change with logarithmic changes in the LPS concentration in the range of 0.01-100 ug/mL, while at higher concentrations, the response deviated from linearity. Early results estimated a limit of detection of 1 ug/mL LPS.Finally, the TLR4-endotoxin interaction has been characterized using the fluorescence-based microscale thermophoresis (MST) technique. In an MST experiment, a fluorophore-labeled molecule is subjected to a pulse of IR laser to heat a focused point in a solution inside a glass capillary, thus inducing microscopic temperature gradients and thermophoresis-induced migration away from that point.4 In our MST experiments, LPS concentrations from 1-2000 ug/mL were combined with a fixed 20 nM fluorescently-labelled TLR4 protein in order to determine their binding affinity. The MST binding curves of TLR4 with endotoxin allowed the determination of the dissociation constant, Kd, which was found to be ca. 11 uM.