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Unimolecular FRET sensors: Simple linker designs and properties
摘要： Protein activation and deactivation is central to a variety of biological mechanisms, including cellular signaling and transport. Unimolecular fluorescent resonance energy transfer (FRET) probes are a class of fusion protein sensors that allow biologists to visualize using an optical microscope whether specific proteins are activated due to the presence nearby of small drug-like signaling molecules, ligands or analytes. Often such probes comprise a donor fluorescent protein attached to a ligand binding domain, a sensor or reporter domain attached to the acceptor fluorescent protein, with these ligand binding and sensor domains connected by a protein linker. Various choices of linker type are possible ranging from highly flexible proteins to hinge-like proteins. It is also possible to select donor and acceptor pairs according to their corresponding F¨oster radius, or even to mutate binding and sensor domains so as to change their binding energy in the activated or inactivated states. The focus of the present work is the exploration through simulation of the impact of such choices on sensor performance.
关键词： FRET Microscopy,Fusion Proteins,Diagnostics,Monte Carlo Simulation,Coarse Graining,Cellular Signaling
更新于2025-09-23 15:21:01
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Fluorescence Microscopy of Single Liposomes with Incorporated Pigment–Proteins
摘要： Reconstitution of transmembrane proteins into liposomes is a widely used method to study their behavior under conditions closely resembling the natural ones. However, this approach does not allow precise control of the liposome size, reconstitution efficiency and the actual protein-to-lipid ratio in the formed proteoliposomes, which might be critical for some applications and/or interpretation of data acquired during the spectroscopic measurements. Here we present a novel strategy employing methods of proteoliposome preparation, fluorescent labelling, purification, and surface immobilization that allow us to quantify these properties using fluorescence microscopy at the single-liposome level and for the first time apply it to study photosynthetic pigment–protein complexes LHCII. We show that LHCII proteoliposome samples, even after purification with a density gradient, always contain a fraction of non-reconstituted protein and are extremely heterogeneous in both protein density and liposome sizes. This strategy enables quantitative analysis of the reconstitution efficiency of different protocols and precise fluorescence spectroscopic study of various transmembrane proteins in a controlled native-like environment.
关键词： Fluorescence Microscopy,Proteoliposomes,Pigment–Proteins,LHCII,Single Liposomes
更新于2025-09-23 15:21:01
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Local Overheating of Biotissue Labeled With Upconversion Nanoparticles Under Yb3+ Resonance Excitation
摘要： Local overheating of biotissue is a critical step for biomedical applications, such as photothermal therapy, enhancement of vascular permeability, remote control of drug release, and so on. Overheating of biological tissue when exposed to light is usually realized by utilizing the materials with a high-absorption cross section (gold, silica, carbon nanoparticles, etc.). Here, we demonstrate core/shell NaYF4:Yb3+, Tm3+/NaYF4 upconversion nanoparticles (UCNPs) commonly used for bioimaging as promising near-infrared (NIR) absorbers for local overheating of biotissue. We assume that achievable temperature of tissue labeled with nanoparticles is high enough because of Yb3+ resonance absorption of NIR radiation, whereas the use of auxiliary light-absorbing materials or shells is optional for photothermal therapy. For this purpose, a computational model of tissue heating based on the energy balance equations was developed and verified with the experimentally obtained thermal-graphic maps of a mouse in response to the 975-nm laser irradiation. Labeling of biotissue with UCNPs was found to increase the local temperature up to 2?C compared to that of the non-labeled area under the laser intensity lower than 1 W/cm2. The cellular response to the UCNP-initiated hyperthermia at subcritical ablation temperatures (lower than 42?C) was demonstrated by measuring the heat shock protein overexpression. This indicates that the absorption cross section of Yb3+ in UCNPs is relatively large, and microscopic temperature of nanoparticles exceeds the integral tissue temperature. In summary, a new approach based on the use of UCNP without any additional NIR absorbers was used to demonstrate a simple approach in the development of photoluminescent probes for simultaneous bioimaging and local hyperthermia.
关键词： near-infrared irradiation,local overheating,photothermal material,bioimaging,heat shock proteins,biotissue laser heating,hyperthermia,upconversion nanoparticles
更新于2025-09-23 15:21:01
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[Methods in Enzymology] || Fluorescence Depolarization Kinetics to Study the Conformational Preference, Structural Plasticity, Binding, and Assembly of Intrinsically Disordered Proteins
摘要： Fluorescence depolarization kinetics measured by the time-resolved fluorescence anisotropy decay serves as a sensitive and powerful methodology to study the conformational dynamics of macromolecules. This methodology allows us to delineate the different modes of biomolecular motional dynamics including the local, segmental, and global rotational dynamics on the timescale ranging from picoseconds to nanoseconds. In this chapter, we describe the principles and applications of this methodology to obtain unique molecular insights into the intrinsically disordered proteins (IDPs). Fluorescence depolarization kinetics, when performed in a site-specific manner, can offer a reliable tool to monitor the intrinsic backbone torsional dynamics of expanded IDPs and is capable of discerning the conformational preference of IDPs. Additionally, the time-resolved fluorescence anisotropy measurements allow us to investigate the mechanism of binding and assembly of a wide range of IDPs that are involved in crucial function and disease.
关键词： Fluorescence depolarization kinetics,conformational dynamics,time-resolved fluorescence anisotropy decay,biomolecular interactions,intrinsically disordered proteins
更新于2025-09-23 15:21:01
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Guided by light: optogenetic control of microtubule gliding assays
摘要： Force generation by molecular motors drives biological processes such as asymmetric cell division and cell migration. Microtubule gliding assays, in which surface-immobilized motor proteins drive microtubule propulsion, are widely used to study basic motor properties as well as the collective behavior of active self-organized systems. Additionally, these assays can be employed for nanotechnological applications such as analyte detection, bio-computation and mechanical sensing. While such assays allow tight control over the experimental conditions, spatiotemporal control of force generation has remained underdeveloped. Here we use light-inducible protein-protein interactions to recruit molecular motors to the surface to control microtubule gliding activity in vitro. We show that using these light-inducible interactions, proteins can be recruited to the surface in patterns, reaching a ~5-fold enrichment within 6 seconds upon illumination. Subsequently, proteins are released with a half-life of 13 seconds when the illumination is stopped. We furthermore demonstrate that light-controlled kinesin recruitment results in reversible activation of microtubule gliding along the surface, enabling efficient control over local microtubule motility. Our approach to locally control force generation offers a way to study the effects of non-uniform pulling forces on different microtubule arrays and also provides novel strategies for local control in nanotechnological applications.
关键词： motor proteins,optogenetics,optical control,Microtubules
更新于2025-09-23 15:21:01
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Sensitive Detection of Dengue Virus Type 2 E-Proteins Signals Using Self-Assembled Monolayers/Reduced Graphene Oxide-PAMAM Dendrimer Thin Film-SPR Optical Sensor
摘要： In this work, sensitive detection of dengue virus type 2 E-proteins (DENV-2 E-proteins) was performed in the range of 0.08 pM to 0.5 pM. The successful DENV detection at very low concentration is a matter of concern for targeting the early detection after the onset of dengue symptoms. Here, we developed a SPR sensor based on self-assembled monolayer/reduced graphene oxide-polyamidoamine dendrimer (SAM/NH2rGO/PAMAM) thin film to detect DENV-2 E-proteins. Surface characterizations involving X-ray diffraction (XRD) and Fourier-transform infrared spectroscopy (FTIR) confirms the incorporation of NH2rGO-PAMAM nanoparticles in the prepared sensor films. The specificity, sensitivity, binding affinity, and selectivity of the SPR sensor were then evaluated. Results indicated that the variation of the sensing layer due to different spin speed, time incubation, and concentration provided a better interaction between the analyte and sensing layer. The linear dependence of the SPR sensor showed good linearity (R2 = 0.92) with the lowest detection of 0.08 pM DENV-2 E-proteins. By using the Langmuir model, the equilibrium association constant was obtained at very high value of 6.6844 TM?1 (R2 = 0.99). High selectivity of the SPR sensor towards DENV-2 E-proteins was achieved in the presence of other competitors.
关键词： optical sensor,E-proteins,SPR sensor,PAMAM dendrimer,reduced graphene oxide,dengue virus,self-assembled monolayer
更新于2025-09-23 15:19:57
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Interactions between CdTe quantum dots and plasma proteins: kinetics, thermodynamics and molecular structure changes
摘要： Environmental particulate matter, especially ultrafine particles (< 100 nm in diameter), can damage the endothelium and favor cardiovascular disease in the general population. With the wide application of nanomaterials, exposure to nanoscale particles (nanoparticles) in the environment is increasing. Systematic study of the interaction of nanoparticles with plasma proteins is critically important for understanding the cardiovascular toxicity of nanomaterials. We combined kinetics and thermodynamics information from surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) and conformational data from fluorescence spectroscopy and circular dichroism (CD) to explore the binding mechanism between cadmium telluride quantum dots (CdTe QDs) and plasma proteins. Special attention was paid to the interaction between CdTe QDs and coagulation-related proteins and the effects of CdTe QDs on protein conformation. The results showed that the binding affinities of CdTe QDs and plasma proteins depend on the nature of the protein and follow the order of fibrinogen (FIB) > plasminogen (PLG) > thrombin (TM) > metallothionein-II (MT-II) > human serum albumin (HSA). The interaction was primarily attributed to hydrophobic forces and the spontaneity of the occurrence of the interaction, and the protein secondary structures of FIB and PLG were changed significantly. The information gained in this study might shed light on the potential toxicity of QDs to the cardiovascular system.
关键词： plasma proteins,circular dichroism,isothermal titration calorimetry,CdTe quantum dots,surface plasmon resonance
更新于2025-09-23 15:19:57
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Single-molecule F?rster Resonance Energy Transfer Measurement Reveals Dynamic Partially Ordered Structure of the Epidermal Growth Factor Receptor C-tail Domain
摘要： Intrinsically disordered proteins (IDPs) or regions (IDRs) are thought to exhibit unique functionalities without forming ordered structures. However, these molecular mechanisms are not easily elucidated, partly because of the difficultly of measuring structural information. In this study, we applied the alternative laser excitation (ALEX) method and circular dichroism (CD) spectroscopy to investigate the structure of the C-terminal tail (CTT) domain of the human epidermal growth factor receptor (EGFR). The single-molecule distributions of F?rster resonance energy transfer (FRET) obtained by ALEX under solution conditions modified by the addition of potassium chloride (KCl), urea or guanidinium chloride (GdmCl) allowed us to separately examine the influences of charge interactions and secondary structure formation. The CD spectrum analyses indicated the types of included secondary structure. The results suggested that the structure of the CTT is influenced by secondary structure formation, which is principally antiparallel β-sheet, rather than by charge interactions, and that phosphorylation of the major Grb2-binding sites partially denature that secondary structure. Our findings suggest that the EGFR CTT might regulate ligand binding kinetics by local β-sheet formation or by the disruption associated with phosphorylation states.
关键词： Circular dichroism,Epidermal Growth Factor Receptor,F?rster resonance energy transfer,Secondary structure,Intrinsically disordered proteins
更新于2025-09-23 15:19:57
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Hybrid quantum dot-based theranostic nanomedicines for tumor-targeted drug delivery and cancer imaging
摘要： Quantum dots (QDs) are considered one of the most efficient tools used in theranostic applications for diagnosis and therapy due to their unique physiochemical characteristics. QDs are semiconductor crystals in the nano-scale range of 2–10 nm which exhibit unique photoluminescence characteristics as well as electronic properties such as tunable emission from visible to near infrared wavelengths and superior light stability. QDs possess strong photoluminescence with high molar extinction coefficient values, which make them the best candidates for cell labeling and detection of cancer biomarkers. QDs are characterized by symmetric narrow-emission spectra and broad-absorption spectra. However, some fears have been raised regarding the toxicity of QDs, especially Cd-containing QDs, due to the release of Cd ions and the generation of reactive oxygen species. Therefore, strategies have been developed to reduce their toxicity and enhance their biocompatibility through hybridization with other moieties such as polymers, proteins, polysaccharides or lipids, offering efficient tumor targeting in addition to inhibiting their release into the systemic circulation. This article discusses QD-based nanohybrids for delivery of anticancer drugs in combination with cancer imaging.
关键词： lipids,proteins,nanohybrids,tumor targeting,inorganic nanoparticles,polymers,cancer theranostics,quantum dots,polysaccharides,drug delivery
更新于2025-09-19 17:15:36
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Rapid and Straightforward Quantification of Recombinant Proteins Using Fluorescence Polarization
摘要： Biotechnology enables the production and commercialization of recombinant proteins for diagnostic, therapeutic and research applications. For instance, monoclonal antibodies nowadays represent a fast growing market that accounts for almost half of the biopharmaceutical compounds business with $75 billion turnover in 2013. Quantification of proteins is an important task in order to optimize cell culture conditions and ensure that every step of the production process is in line with the specifications. For therapeutics, precise and reliable dosage is mandatory to avoid adverse effects for the patients. Current analytical techniques such as ELISA or HPLC are time-consuming and often require purification prior to analysis. In this context, there is a strong demand for a technology that would be to protein quantification what a balance is to weight measurement: rapid, precise, accurate, robust, and straightforward. To this effect, we have developed an assay for the rapid quantification of recombinant proteins. It is based on fluorescence polarization and requires little or no pretreatment of samples. Fluorescence anisotropy of a small specific fluorescent ligand increased upon binding to its recombinant target protein, e.g. a monoclonal antibody, enabling quantification. The assay did not require prior separation of biotechnological cultures making it as straightforward as weighing using a balance. Monitoring of the production of a representative His-tagged protein during E.coli fermentation was obtained by regular sampling and assaying using the fluorescence polarization assay.
关键词： Fluorescence,Recombinant proteins,Assays,Biotechnology,Antibodies
更新于2025-09-19 17:15:36