ABSTRACT
The metabotropic glutamate receptors (mGluRs) are neuromodulatory family C G protein coupled receptors which assemble as dimers and allosterically couple extracellular ligand binding domains (LBDs) to transmembrane domains (TMDs) to drive intracellular signaling. Pharmacologically, mGluRs can be targeted at the LBDs by glutamate and synthetic orthosteric compounds or at the TMDs by allosteric modulators. Despite the potential of allosteric compounds as therapeutics, an understanding of the functional and structural basis of their effects is limited. Here we use multiple approaches to dissect the functional and structural effects of orthosteric versus allosteric ligands. We find, using electrophysiological and live cell imaging assays, that both agonists and positive allosteric modulators (PAMs) can drive activation and internalization of group II and III mGluRs. The effects of PAMs are pleiotropic, boosting the maximal response to orthosteric agonists and serving independently as internalization-biased agonists across mGluR subtypes. Motivated by this and intersubunit FRET analyses, we determine cryo-electron microscopy structures of mGluR3 in the presence of either an agonist or antagonist alone or in combination with a PAM. These structures reveal PAM-driven re-shaping of intra- and inter-subunit conformations and provide evidence for a rolling TMD dimer interface activation pathway that controls G protein and beta-arrestin coupling.
Subject(s)
Cryoelectron Microscopy , Receptors, Metabotropic Glutamate , Receptors, Metabotropic Glutamate/metabolism , Receptors, Metabotropic Glutamate/chemistry , Receptors, Metabotropic Glutamate/agonists , Allosteric Regulation , Humans , HEK293 Cells , Ligands , Animals , Fluorescence Resonance Energy Transfer , Protein DomainsABSTRACT
T-box riboswitches are noncoding RNA elements involved in genetic regulation of most Gram-positive bacteria. They regulate amino acid metabolism by assessing the aminoacylation status of tRNA, subsequently affecting the transcription or translation of downstream amino acid metabolism-related genes. Here we present single-molecule FRET studies of the Mycobacterium tuberculosis IleS T-box riboswitch, a paradigmatic translational T-box. Results support a two-step binding model, where the tRNA anticodon is recognized first, followed by interactions with the NCCA sequence. Furthermore, after anticodon recognition, tRNA can transiently dock into the discriminator domain even in the absence of the tRNA NCCA-discriminator interactions. Establishment of the NCCA-discriminator interactions significantly stabilizes the fully bound state. Collectively, the data suggest high conformational flexibility in translational T-box riboswitches; and supports a conformational selection model for NCCA recognition. These findings provide a kinetic framework to understand how specific RNA elements underpin the binding affinity and specificity required for gene regulation.
Subject(s)
Anticodon , Mycobacterium tuberculosis , Nucleic Acid Conformation , RNA, Bacterial , RNA, Transfer , Riboswitch , Riboswitch/genetics , RNA, Transfer/metabolism , RNA, Transfer/genetics , RNA, Transfer/chemistry , Mycobacterium tuberculosis/metabolism , Mycobacterium tuberculosis/genetics , Anticodon/metabolism , Anticodon/genetics , RNA, Bacterial/metabolism , RNA, Bacterial/genetics , RNA, Bacterial/chemistry , Fluorescence Resonance Energy Transfer , Protein Biosynthesis , Gene Expression Regulation, Bacterial , KineticsABSTRACT
Much attention has been given to studying the translational diffusion of globular proteins, whereas the translational diffusion of intrinsically disordered proteins (IDPs) is less studied. In this study, we investigate the translational diffusion and how it is affected by the self-association of an IDP, κ-casein, using pulsed-field gradient nuclear magnetic resonance and time-resolved Förster resonance energy transfer. Using the analysis of the shape of diffusion attenuation and the concentration dependence of κ-casein diffusion coefficients and intermolecular interactions, we demonstrate that κ-casein exhibits continuous self-association. When the volume fraction of κ-casein is below 0.08, we observe that κ-casein self-association results in a macroscopic phase separation upon storage at 4 °C. At κ-casein volume fractions above 0.08, self-association leads to the formation of labile gel-like networks without subsequent macroscopic phase separation. Unlike α-casein, which shows a strong concentration dependence and extensive gel-like network formation, only one-third of κ-casein molecules participate in the gel network at a time, resulting in a more dynamic and less extensive structure. These findings highlight the unique association properties of κ-casein, contributing to a better understanding of its behavior under various conditions and its potential role in casein micelle formation.
Subject(s)
Caseins , Fluorescence Resonance Energy Transfer , Intrinsically Disordered Proteins , Caseins/chemistry , Caseins/metabolism , Intrinsically Disordered Proteins/chemistry , Intrinsically Disordered Proteins/metabolism , Diffusion , Nuclear Magnetic Resonance, BiomolecularABSTRACT
Fluorogenic glycomonomers have been used for biological evaluations, and water-soluble and Förster resonance energy transfer (FRET)-sensitive glycopolymers have also been reported. A FRET-sensitive polymer was conveniently prepared from a fluorogenic donor monomer and a fluorogenic acceptor monomer by means of simple radical polymerization in high yield. Continuous fluorospectroscopic monitoring of the polymer in the presence of an enzyme was performed, and the results showed the possible application of the FRET-sensitive glycopolymer for practical use. In addition to the use of aqueous solution phase, the water-soluble and FRET-sensitive glycopolymer was completely captured into an interpenetrating polymer network (IPN) by means of radical polymerization with a combination of acrylamide and bis-acrylamide as used for the cross-linking reagent system. The IPN including the FRET-sensitive glycopolymer was allowed to react with amylases in an aqueous buffer solution at 37 °C, and the enzymatic reaction was continuously and conveniently monitored by means of fluorometric spectroscopy.
Subject(s)
Fluorescence Resonance Energy Transfer , Polymers , Fluorescence Resonance Energy Transfer/methods , Polymers/chemistry , Enzyme Assays/methods , Polymerization , Fluorescent Dyes/chemistryABSTRACT
Circulating tumor DNA (ctDNA) is an emerging biomarker of liquid biopsy for cancer. But it remains a challenge to achieve simple, sensitive and specific detection of ctDNA because of low abundance and single-base mutation. In this work, an excitation/emission-enhanced heterostructure photonic crystal (PC) array synergizing with entropy-driven circuit (EDC) was developed for high-resolution and ultrasensitive analysis of ctDNA. The donor donor-acceptor FÖrster resonance energy transfer ("DD-A" FRET) was integrated in EDC based on the introduction of simple auxiliary strand, which exhibited higher sensitivity than that of traditional EDC. The heterostructure PC array was constructed with the bilayer periodic nanostructures of nanospheres. Because the heterostructure PC has the adjustable dual photonic band gaps (PBGs) by changing nanosphere sizes, and the "DD-A" FRET can offer the excitation and emission peak with enough distance, it helps the successful matches between the dual PBGs of heterostructure PC and the excitation/emission peaks of "DD-A" FRET; thus, the fluorescence from EDC can be enhanced effectively from both of excitation and emission processes on heterostructure PC array. Besides, high-resolution of single-base mutation was obtained through the strict recognition of EDC. Benefiting from the specific spectrum-matched and synergetic amplification of heterostructure PC and EDC with "DD-A" FRET, the proposed array obtained ultrasensitive detection of ctDNA with LOD of 12.9 fM, and achieved the analysis of mutation frequency as low as 0.01%. Therefore, the proposed strategy has the advantages of simple operation, mild conditions (enzyme-free and isothermal), high-sensitivity, high-resolution and high-throughput analysis, showing potential in bioassay and clinical application.
Subject(s)
Biosensing Techniques , Circulating Tumor DNA , Fluorescence Resonance Energy Transfer , Fluorescence Resonance Energy Transfer/methods , Biosensing Techniques/instrumentation , Biosensing Techniques/methods , Humans , Circulating Tumor DNA/blood , Circulating Tumor DNA/isolation & purification , Circulating Tumor DNA/genetics , Circulating Tumor DNA/analysis , Photons , Limit of Detection , Entropy , Neoplasms/blood , Biomarkers, Tumor/blood , Nanospheres/chemistryABSTRACT
Plasticity of synaptic transmission underlies learning and memory. It is accompanied by changes in the density and size of synapses, collectively called structural plasticity. Therefore, understanding the mechanism of structural plasticity is critical for understanding the mechanism of synaptic plasticity. In this chapter, we describe the procedures and equipment required to image structural plasticity of a single dendritic spine, which hosts excitatory synapses in the central nervous system, and underlying molecular interactions/biochemical reactions using two-photon fluorescence lifetime microscopy (2P-FLIM) in combination with Förster resonance energy transfer (FRET)-based biosensors.
Subject(s)
Dendritic Spines , Fluorescence Resonance Energy Transfer , Microscopy, Fluorescence, Multiphoton , Neuronal Plasticity , Dendritic Spines/metabolism , Dendritic Spines/ultrastructure , Dendritic Spines/physiology , Neuronal Plasticity/physiology , Animals , Fluorescence Resonance Energy Transfer/methods , Microscopy, Fluorescence, Multiphoton/methods , Synapses/metabolism , Synapses/physiology , Mice , Biosensing Techniques/methodsABSTRACT
Ratiometric biosensors employing Förster Resonance Energy Transfer (FRET) enable the real-time tracking of metabolite dynamics. Here, we introduce an approach for generating a FRET-based biosensor in which changes in apparent FRET efficiency rely on the analyte-controlled fluorogenicity of a rhodamine rather than the commonly used distance change between donor-acceptor fluorophores. Our fluorogenic, rhodamine-based, chemigenetic biosensor (FOCS) relies on a synthetic, protein-tethered FRET probe, in which the rhodamine acting as the FRET acceptor switches in an analyte-dependent manner from a dark to a fluorescent state. This allows ratiometric sensing of the analyte concentration. We use this approach to generate a chemigenetic biosensor for nicotinamide adenine dinucleotide phosphate (NADPH). FOCS-NADPH exhibits a rapid and reversible response toward NAPDH with a good dynamic range, selectivity, and pH insensitivity. FOCS-NADPH allows real-time monitoring of cytosolic NADPH fluctuations in live cells during oxidative stress or after drug exposure. We furthermore used FOCS-NADPH to investigate NADPH homeostasis regulation through the pentose phosphate pathway of glucose metabolism. FOCS-NADPH is a powerful tool for studying NADPH metabolism and serves as a blueprint for the development of future fluorescent biosensors.
Subject(s)
Biosensing Techniques , Fluorescence Resonance Energy Transfer , Fluorescent Dyes , NADP , Rhodamines , Biosensing Techniques/methods , Rhodamines/chemistry , NADP/metabolism , NADP/analysis , Fluorescent Dyes/chemistry , Fluorescence Resonance Energy Transfer/methods , HumansABSTRACT
A cancer-targeted glutathione (GSH)-gated theranostic probe (CGT probe) for intracellular miRNA imaging and combined treatment of self-sufficient starvation therapy (ST) and chemodynamic therapy (CDT) was developed. The CGT probe is constructed using MnO2 nanosheet (MS) as carrier material to adsorb the elaborately designed functional DNAs. It can be internalized by cancer cells via specific recognition between the AS1411 aptamer and nucleolin. After CGT probe entering the cancer cells, the overexpressed GSH, as gate-control, can degrade MS to Mn2+ which can be used for CDT by Fenton-like reaction. Simultaneously, Mn2+-mediated CDT can further cascade with the enzyme-like activities (catalase-like activity and glucose oxidase-like activity) of CGT probe, achieving self-sufficient ST/CDT synergistic therapy. Meanwhile, the anchored DNAs are released, achieving in situ signal amplification via disubstituted-catalytic hairpin assembly (DCHA) and FRET (fluorescence resonance energy transfer) imaging of miR-21. The in vitro and in vivo experiments demonstrated that accurate and sensitive miRNA detection can be achieved using the CGT probe. Overall, the ingenious CGT probe opens a new avenue for the development of early clinical diagnosis and cancer therapy.
Subject(s)
Fluorescence Resonance Energy Transfer , Glutathione , Manganese Compounds , MicroRNAs , Oxides , Humans , Glutathione/chemistry , Glutathione/metabolism , Animals , Manganese Compounds/chemistry , Oxides/chemistry , Aptamers, Nucleotide/chemistry , Mice , Mice, Nude , Theranostic Nanomedicine/methods , Nucleolin , Neoplasms/diagnostic imaging , Nanostructures/chemistry , Oligodeoxyribonucleotides/chemistry , Mice, Inbred BALB C , Fluorescent Dyes/chemistryABSTRACT
Continuous glucose monitors are crucial for diabetes management, but invasive sampling, signal drift and frequent calibrations restrict their widespread usage. Microneedle sensors are emerging as a minimally-invasive platform for real-time monitoring of clinical parameters in interstitial fluid. Herein, a painless and flexible microneedle sensing patch is constructed by a mechanically-strong microneedle base and a thin layer of fluorescent hydrogel sensor for on-site, accurate, and continuous glucose monitoring. The Förster resonance energy transfer (FRET)-based hydrogel sensors are fabricated by facile photopolymerizations of acryloylated FRET pairs and glucose-specific phenylboronic acid. The optimized hydrogel sensor enables quantification of glucose with reversibility, high selectivity, and signal stability against photobleaching. Poly (ethylene glycol diacrylate)-co-polyacrylamide hydrogel is utilized as the microneedle base, facilitating effective skin piercing and biofluid extraction. The integrated microneedle sensor patch displays a sensitivity of 0.029 mM-1 in the (patho)physiological range, a low detection limit of 0.193 mM, and a response time of 7.7 min in human serum. Hypoglycemia, euglycemia and hyperglycemia are continuously monitored over 6 h simulated meal and rest activities in a porcine skin model. This microneedle sensor with high transdermal analytical performance offers a powerful tool for continuous diabetes monitoring at point-of-care settings.
Subject(s)
Biosensing Techniques , Blood Glucose Self-Monitoring , Blood Glucose , Fluorescence Resonance Energy Transfer , Hydrogels , Needles , Wearable Electronic Devices , Humans , Biosensing Techniques/instrumentation , Hydrogels/chemistry , Blood Glucose Self-Monitoring/instrumentation , Blood Glucose/analysis , Animals , Swine , Polyethylene Glycols/chemistry , Limit of Detection , Acrylic Resins/chemistry , Equipment Design , Continuous Glucose Monitoring , Boronic AcidsABSTRACT
Conformational dynamics of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein (S) mediate exposure of the binding site for the cellular receptor, angiotensin-converting enzyme 2 (ACE2). The N-terminal domain (NTD) of S binds terminal sialic acid (SA) moieties on the cell surface, but the functional role of this interaction in virus entry is unknown. Here, we report that NTD-SA interaction enhances both S-mediated virus attachment and ACE2 binding. Through single-molecule Förster resonance energy transfer imaging of individual S trimers, we demonstrate that SA binding to the NTD allosterically shifts the S conformational equilibrium, favoring enhanced exposure of the ACE2-binding site. Antibodies that target the NTD block SA binding, which contributes to their mechanism of neutralization. These findings inform on mechanisms of S activation at the cell surface.
Subject(s)
Angiotensin-Converting Enzyme 2 , N-Acetylneuraminic Acid , Protein Binding , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Spike Glycoprotein, Coronavirus/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Humans , SARS-CoV-2/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Angiotensin-Converting Enzyme 2/chemistry , N-Acetylneuraminic Acid/metabolism , N-Acetylneuraminic Acid/chemistry , Binding Sites , Single Molecule Imaging , COVID-19/virology , COVID-19/metabolism , Allosteric Regulation , Virus Internalization , Fluorescence Resonance Energy Transfer , Protein Domains , Virus AttachmentABSTRACT
The Arabidopsis thaliana FLAGELLIN-SENSITIVE2 (FLS2), a typical receptor kinase, recognizes the conserved 22 amino acid sequence in the N-terminal region of flagellin (flg22) to initiate plant defense pathways, which was intensively studied in the past decades. However, the dynamic regulation of FLS2 phosphorylation at the plasma membrane after flg22 recognition needs further elucidation. Through single-particle tracking, we demonstrated that upon flg22 treatment the phosphorylation of Ser-938 in FLS2 impacts its spatiotemporal dynamics and lifetime. Following Förster resonance energy transfer-fluorescence lifetime imaging microscopy and protein proximity indexes assays revealed that flg22 treatment increased the co-localization of GFP-tagged FLS2/FLS2S938D but not FLS2S938A with AtRem1.3-mCherry, a sterol-rich lipid marker, indicating that the phosphorylation of FLS2S938 affects FLS2 sorting efficiency to AtRem1.3-associated nanodomains. Importantly, we found that the phosphorylation of Ser-938 enhanced flg22-induced FLS2 internalization and immune responses, demonstrating that the phosphorylation may activate flg22-triggered immunity through partitioning FLS2 into functional AtRem1.3-associated nanodomains, which fills the gap between the FLS2S938 phosphorylation and FLS2-mediated immunity.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Flagellin , Protein Kinases , Single Molecule Imaging , Phosphorylation , Arabidopsis Proteins/metabolism , Arabidopsis Proteins/genetics , Arabidopsis/metabolism , Arabidopsis/genetics , Arabidopsis/immunology , Protein Kinases/metabolism , Protein Kinases/genetics , Flagellin/metabolism , Flagellin/pharmacology , Plant Immunity , Fluorescence Resonance Energy Transfer , Cell Membrane/metabolismABSTRACT
Fluorescence-based LB (liquid biopsy) offers a rapid means of detecting cancer non-invasively. However, the widespread issue of sample loss during purification steps will diminish the accuracy of detection results. Therefore, in this study, we introduce a magnetic lanthanide sensor (MLS) designed for sensitive detection of the characteristic protein, epithelial cell adhesion molecule (EpCAM), on epithelial tumor exosomes. By leveraging the inherent multi-peak emission and time-resolved properties of the sole-component lanthanide element, combined with the self-ratiometric strategy, MLS can overcome limitations imposed by manual operation and/or sample complexity, thereby providing more stable and reliable output results. Specifically, terbium-doped NaYF4 nanoparticles (NaYF4:Tb) and deformable aptamers terminated with BHQ1 were sequentially introduced onto superparamagnetic silica-decorated Fe3O4 nanoparticles. Prior to target binding, emission from NaYF4:Tb at 543 nm was partially quenched due to the fluorescence resonance energy transfer (FRET) from NaYF4:Tb to BHQ1. Upon target binding, changes in the secondary structure of aptamers led to the fluorescence intensity increasing since the deconfinement of distance-dependent FRET effect. The characteristic emission of NaYF4:Tb at 543 nm was then utilized as the detection signal (I1), while the less changed emission at 583 nm served as the reference signal (I2), further reporting the self-ratiometric values of I1 and I2 (I1/I2) to illustrate the epithelial cancerous features of exosomes while ignoring possible sample loss. Consequently, over a wide range of exosome concentrations (2.28 × 102-2.28 × 108 particles per mL), the I1/I2 ratio exhibited a linear increase with exosome concentration [Y(I1/I2) = 0.166 lg (Nexosomes) + 3.0269, R2 = 0.9915], achieving a theoretical detection limit as low as 24 particles per mL. Additionally, MLS effectively distinguished epithelial cancer samples from healthy samples, showcasing significant potential for clinical diagnosis.
Subject(s)
Exosomes , Exosomes/chemistry , Exosomes/metabolism , Humans , Lanthanoid Series Elements/chemistry , Fluorescence Resonance Energy Transfer , Terbium/chemistry , Epithelial Cell Adhesion Molecule/metabolism , Luminescence , Magnetite Nanoparticles/chemistry , Particle Size , Yttrium/chemistry , Biosensing Techniques/methods , FluoridesABSTRACT
Understanding the structure of biomolecules is vital for deciphering their roles in biological systems. Single-molecule techniques have emerged as alternatives to conventional ensemble structure analysis methods for uncovering new biology in molecular dynamics and interaction studies, yet only limited structural information could be obtained experimentally. Here, we address this challenge by introducing iMAX FRET, a one-pot method that allows ab initio 3D profiling of individual molecules using two-color FRET measurements. Through the stochastic exchange of fluorescent weak binders, iMAX FRET simultaneously assesses multiple distances on a biomolecule within a few minutes, which can then be used to reconstruct the coordinates of up to four points in each molecule, allowing structure-based inference. We demonstrate the 3D reconstruction of DNA nanostructures, protein quaternary structures, and conformational changes in proteins. With iMAX FRET, we provide a powerful approach to advance the understanding of biomolecular structure by expanding conventional FRET analysis to three dimensions.
Subject(s)
DNA , Fluorescence Resonance Energy Transfer , Fluorescence Resonance Energy Transfer/methods , DNA/chemistry , Single Molecule Imaging/methods , Nanostructures/chemistry , Proteins/chemistry , Molecular Dynamics SimulationABSTRACT
The conformational dynamics of single-stranded nucleic acids are fundamental for nucleic acid folding and function. However, their elementary chain dynamics have been difficult to resolve experimentally. Here we employ a combination of single-molecule Förster resonance energy transfer, nanosecond fluorescence correlation spectroscopy, and nanophotonic enhancement to determine the conformational ensembles and rapid chain dynamics of short single-stranded nucleic acids in solution. To interpret the experimental results in terms of end-to-end distance dynamics, we utilize the hierarchical chain growth approach, simple polymer models, and refinement with Bayesian inference to generate structural ensembles that closely align with the experimental data. The resulting chain reconfiguration times are exceedingly rapid, in the 10-ns range. Solvent viscosity-dependent measurements indicate that these dynamics of single-stranded nucleic acids exhibit negligible internal friction and are thus dominated by solvent friction. Our results provide a detailed view of the conformational distributions and rapid dynamics of single-stranded nucleic acids.
Subject(s)
DNA, Single-Stranded , Fluorescence Resonance Energy Transfer , Nucleic Acid Conformation , DNA, Single-Stranded/chemistry , Bayes Theorem , Spectrometry, Fluorescence , Viscosity , Solvents/chemistry , Models, MolecularABSTRACT
Investigating ligand-protein complexes is essential in the areas of chemical biology and drug discovery. However, detailed information on key reagents such as fluorescent tracers and associated data for the development of widely used bioluminescence resonance energy transfer (BRET) assays including NanoBRET, time-resolved Förster resonance energy transfer (TR-FRET) and fluorescence polarization (FP) assays are not easily accessible to the research community. We created tracerDB, a curated database of validated tracers. This resource provides an open access knowledge base and a unified system for tracer and assay validation. The database is freely available at https://www.tracerdb.org/ .
Subject(s)
Fluorescence Resonance Energy Transfer , Fluorescence Resonance Energy Transfer/methods , Crowdsourcing , Humans , Fluorescent Dyes/chemistry , Drug Discovery/methods , Ligands , Databases, Factual , Bioluminescence Resonance Energy Transfer Techniques/methods , Fluorescence Polarization/methodsABSTRACT
Integral membrane proteins (IMPs) play central roles in cellular physiology and represent the majority of known drug targets. Single-molecule fluorescence and fluorescence resonance energy transfer (FRET) methods have recently emerged as valuable tools for investigating structure-function relationships in IMPs. This review focuses on the practical foundations required for examining polytopic IMP function using single-molecule FRET (smFRET) and provides an overview of the technical and conceptual frameworks emerging from this area of investigation. In this context, we highlight the utility of smFRET methods to reveal transient conformational states critical to IMP function and the use of smFRET data to guide structural and drug mechanism-of-action investigations. We also identify frontiers where progress is likely to be paramount to advancing the field.
Subject(s)
Fluorescence Resonance Energy Transfer , Membrane Proteins , Single Molecule Imaging , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Membrane Proteins/ultrastructure , Fluorescence Resonance Energy Transfer/methods , Single Molecule Imaging/methods , Humans , AnimalsABSTRACT
Nanomaterials acquire a biomolecular corona upon introduction to biological media, leading to biological transformations such as changes in protein function, unmasking of epitopes, and protein fibrilization. Ex vivo studies to investigate the effect of nanoparticles on protein-protein interactions are typically performed in buffer and are rarely measured quantitatively in live cells. Here, we measure the differential effect of silica nanoparticles on protein association in vitro vs. in mammalian cells. BtubA and BtubB are a pair of bacterial tubulin proteins identified in Prosthecobacter strains that self-assemble like eukaryotic tubulin, first into dimers and then into microtubules in vitro or in vivo. Förster resonance energy transfer labeling of each of the Btub monomers with a donor (mEGFP) and acceptor (mRuby3) fluorescent protein provides a quantitative tool to measure their binding interactions in the presence of unfunctionalized silica nanoparticles in buffer and in cells using fluorescence spectroscopy and microscopy. We show that silica nanoparticles enhance BtubAB dimerization in buffer due to protein corona formation. However, these nanoparticles have little effect on bacterial tubulin self-assembly in the complex mammalian cellular environment. Thus, the effect of nanomaterials on protein-protein interactions may not be readily translated from the test tube to the cell in the absence of particle surface functionalization that can enable targeted protein-nanoparticle interactions to withstand competitive binding in the nanoparticle corona from other biomolecules.
Subject(s)
Bacterial Proteins , Nanoparticles , Silicon Dioxide , Tubulin , Tubulin/metabolism , Tubulin/chemistry , Nanoparticles/chemistry , Silicon Dioxide/chemistry , Silicon Dioxide/metabolism , Bacterial Proteins/metabolism , Bacterial Proteins/chemistry , Fluorescence Resonance Energy Transfer , Humans , Microtubules/metabolism , Protein Multimerization , Protein BindingABSTRACT
The ability of cells to sense and respond to mechanical forces is critical in many physiological and pathological processes. However, determining the mechanisms by which forces affect protein function inside cells remains challenging. Motivated by in vitro demonstrations of fluorescent proteins (FPs) undergoing reversible mechanical switching of fluorescence, we investigated whether force-sensitive changes in FP function could be visualized in cells. Guided by a computational model of FP mechanical switching, we develop a formalism for its detection in Förster resonance energy transfer (FRET)-based biosensors and demonstrate its occurrence in cellulo within a synthetic actin crosslinker and the mechanical linker protein vinculin. We find that in cellulo mechanical switching is reversible and altered by manipulation of cell force generation, external stiffness, and force-sensitive bond dynamics of the biosensor. This work describes a framework for assessing FP mechanical stability and provides a means of probing force-sensitive protein function inside cells.
Subject(s)
Biosensing Techniques , Fluorescence Resonance Energy Transfer , Luminescent Proteins , Fluorescence Resonance Energy Transfer/methods , Luminescent Proteins/metabolism , Luminescent Proteins/genetics , Luminescent Proteins/chemistry , Biosensing Techniques/methods , Humans , Vinculin/metabolism , Vinculin/chemistry , Actins/metabolism , Actins/chemistry , Biomechanical PhenomenaABSTRACT
Fluorescence resonance energy transfer (FRET) biosensors have proven to be an indispensable tool in cell biology and, more specifically, in the study of G-protein signalling. The best method of measuring the activation status or FRET state of a biosensor is often fluorescence lifetime imaging microscopy (FLIM), as it does away with many disadvantages inherent to fluorescence intensity-based methods and is easily quantitated. Despite the significant potential, there is a lack of reliable FLIM-FRET biosensors, and the data processing and analysis workflows reported previously face reproducibility challenges. Here, we established a system in live primary mouse pancreatic ductal adenocarcinoma cells, where we can detect the activation of an mNeonGreen-Gαi3-mCherry-Gγ2 biosensor through the lysophosphatidic acid receptor (LPAR) with 2-photon time-correlated single-photon counting (TCSPC) FLIM. This combination gave a superior signal to the commonly used mTurquoise2-mVenus G-protein biosensor. This system has potential as a platform for drug screening, or to answer basic cell biology questions in the field of G-protein signalling.
Subject(s)
Biosensing Techniques , Fluorescence Resonance Energy Transfer , Animals , Fluorescence Resonance Energy Transfer/methods , Mice , Biosensing Techniques/methods , GTP-Binding Proteins/metabolism , Humans , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Cell Line, Tumor , Receptors, Lysophosphatidic Acid/metabolism , Carcinoma, Pancreatic Ductal/metabolism , Carcinoma, Pancreatic Ductal/pathologyABSTRACT
In this study, a self-responsive fluorescence aptasensor was established for the determination of lactoferrin (Lf) in dairy products. Herein, the aptamer itself functions as both a recognition element that specifically binds to Lf and a fluorescent signal reporter in conjunction with fluorescent moiety. In the presence of Lf, the aptamer preferentially binds to Lf due to its specific and high-affinity recognition by folding into a self-assembled and three-dimensional spatial structure. Meanwhile, its reduced spatial distance in the aptamer-Lf complex induces a FRET phenomenon based on the quenching of 6-FAM by amino acids in the Lf protein, resulting in a turn-off of the fluorescence of the system. As a result, the Lf concentration can be determined straightforwardly corresponding to the change in the self-responsive fluorescence signal. Under the optimized conditions, good linearities (R2 > 0.99) were achieved in an Lf concentration range of 2~10 µg/mL for both standard solutions and the spiked matrix, as well as with the desirable detection limits of 0.68 µg/mL and 0.46 µg/mL, respectively. Moreover, the fluorescence aptasensor exhibited reliable recoveries (89.5-104.3%) in terms of detecting Lf in three commercial samples, which is comparable to the accuracy of the HPCE method. The fluorescence aptasensor offers a user-friendly, cost-efficient, and promising sensor platform for point-of-need detection.