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The Unique Photophysical Properties of the Peridinin-Chlorophyll-a-Protein
摘要： Peridinin-Chlorophyll-a-Proteins (PCPs) are water-soluble light harvesting complexes from dinoflagellates. They have unique light-harvesting and energy transfer properties which have been studied in details in the last 15 years. This review aims to give an overview on all the main aspects of PCPs photophysics, with an emphasis on some aspects which have not been reviewed in details so far, such as vibrational spectroscopy studies, theoretical calculations, and magnetic resonance studies. A paragraph on the present development of PCPs towards technological applications is also included.
关键词： peridinin-chlorophyll-protein,peridinin,Carotenoids,photoprotection.,light-harvesting
更新于2025-09-11 14:15:04
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Interaction mechanism between TiO <sub/>2</sub> nanostructures and bovine leukemia virus proteins in photoluminescence-based immunosensors
摘要： In this research a mechanism of interaction between a semiconducting TiO2 layer and bovine leukemia virus protein gp51, applied in the design of photoluminescence-based immunosensors, is proposed and discussed. Protein gp51 was adsorbed on the surface of a nanostructured TiO2 thin film, formed on glass substrates (TiO2/glass). A photoluminescence (PL) peak shift from 517 nm to 499 nm was observed after modification of the TiO2/glass by adsorbed gp51 (gp51/TiO2/glass). After incubation of the gp51/TiO2/glass in a solution containing anti-gp51, a new structure (anti-gp51/gp51/TiO2/glass) was formed and the PL peak shifted backwards from 499 nm to 516 nm. The above-mentioned PL shifts are attributed to the variations in the self-trapped exciton energy level, which were induced by the changes of electrostatic interaction between the adsorbed gp51 and the negatively charged TiO2 surface. The strength of the electric field affecting the photoluminescence centers, was determined from variations between the PL-spectra of TiO2/glass, gp51/TiO2/glass and anti-gp51/gp51/TiO2/glass. The principle of how these electric field variations are induced has been predicted. The highlighted origin of the changes in the photoluminescence spectra of TiO2 after its protein modification reveals an understanding of the interaction mechanism between TiO2 and proteins that is the key issue responsible for biosensor performance.
关键词： photoluminescence,protein gp51,electrostatic interaction,immunosensors,TiO2,bovine leukemia virus
更新于2025-09-11 14:15:04
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Detection of soluble expression and in vivo interactions of the inner membrane protein OppC using green fluorescent protein
摘要： In this study, the in vivo interaction system of oligopeptide permease (Opp) proteins was analyzed, and a high expression system of inner membrane protein OppC was constructed by flexible usage of the green fluorescent protein (GFP). The Escherichia coli OppC gene, which encodes a transmembrane component of oligopeptide transporter, was cloned into different vectors. Recombinant plasmids were transformed into different E. coli strains, and the expression conditions were optimized. The effect of plasmids and expression strains on OppC production was evaluated by in-gel and western blot analyses. OppC produced by the pWaldo-GFPe vector, harboring the GFP reporter gene, transformed into E. coli C43(DE3) provided sufficient functional protein for biochemical and biophysical studies. In vivo protein-protein interactions were detected among oligopeptide permease proteins using a GFP fragment reassembly protocol. The substrate binding protein OppA showed no interaction with the other components, while the ATP-binding component OppD did not interact with OppF. OppD and OppF interacted with the transmembrane components OppB and OppC. OppB also showed direct interaction with OppC. In vivo OppC functionality was determined by constructing an OppC gene deletion strain. OppC was shown to be essential for peptide uptake, and non-essential for cell viability. These results could help in elucidating the oligopeptide transport mechanism in bacteria.
关键词： Oligopeptide permease,Protein-protein interaction,Inner membrane protein,Green fluorescent protein
更新于2025-09-10 09:29:36
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Dynamic imaging of small molecule-induced protein-protein interactions in living cells with a fluorophore phase transition-based approach
摘要： Protein-protein interactions (PPIs) mediate signal transduction in cells. Small molecules that regulate PPIs are important tools for biology and biomedicine. Dynamic imaging of small molecule-induced PPIs characterizes and verifies these molecules in living cells. It is thus important to develop cellular assays for dynamic visualization of small molecule-induced protein-protein association and dissociation in living cells. Here we have applied fluorophore phase transition-based principle and designed a PPI assay named SPPIER (separation of phases-based protein interaction reporter). SPPIER utilizes the green fluorescent protein (GFP) and is thus genetically encoded. Upon small molecule-induced PPI, SPPIER rapidly forms highly fluorescent GFP droplets in living cells. SPPIER detects immunomodulatory drugs (IMiDs)-induced PPI between cereblon and the transcription factor Ikaros. It also detects IMiDs analog (e.g. CC-885)-induced PPI between cereblon and GSPT1. SPPIER can be modified to image small molecule-induced protein-protein dissociation, such as nutlin-induced dissociation between HDM2 and p53. The intensive brightness and rapid kinetics of SPPIER enable robust and dynamic visualization of PPIs in living cells.
关键词： GFP,Small molecules,SPPIER,Living cells,Fluorophore phase transition,Protein-protein interactions
更新于2025-09-10 09:29:36
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Chemical cross-linking of a variety of green fluorescent proteins as F?rster resonance energy transfer donors for Yukon orange fluorescent protein: A project-based undergraduate laboratory experience
摘要： F?rster resonance energy transfer (FRET) is the basis for many techniques used in biomedical research. Due to its wide use in molecular sensing, FRET is commonly introduced in many biology, chemistry, and physics courses. While FRET is of great importance in the biophysical sciences, the complexity and dif?culty of constructing FRET experiments has resulted in limited usage in undergraduate laboratory settings. Here, we present a practical undergraduate laboratory experiment for teaching FRET using a diverse set of green-emitting ?uorescent proteins (FPs) as donors for a cross-linked Yukon orange FP. This laboratory experiment enables students to make the connection of basic lab procedures to real world applications and can be applied to molecular biology, biochemistry, physical chemistry, and biophysical laboratory courses.
关键词： Upper-division undergraduate,orange ?uorescent protein,?uorescence lifetimes,proteins,green ?uorescent protein,biochemistry,TCSPC,?uorescence spectroscopy
更新于2025-09-10 09:29:36
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A multicolor large Stokes shift fluorogen-activating RNA aptamer with cationic chromophores
摘要： Large Stokes shift (LSS) fluorescent proteins (FPs) exploit excited state proton transfer pathways to enable fluorescence emission from the phenolate intermediate of 4-hydroxybenzylidene imidazolone (HBI) chromophore. An RNA aptamer named Chili mimics LSS FPs by inducing highly Stokes-shifted emission from several new green and red HBI analogs that are non-fluorescent when free in solution. The ligands are bound by the RNA in their protonated phenol form and feature a cationic aromatic side chain for increased RNA affinity and reduced magnesium dependence. In combination with oxidative functionalization at the C2 position of the imidazolone, this strategy yielded DMHBO+, which binds to the Chili aptamer with a low-nanomolar KD. Because of its highly red-shifted fluorescence emission at 592 nm, the Chili–DMHBO+ complex is an ideal fluorescence donor for F?rster resonance energy transfer (FRET) to the rhodamine dye Atto 590 and will therefore find applications in FRET-based analytical RNA systems.
关键词： RNA aptamer,fluorescence,fluorescence resonance energy transfer,fluorescent protein,large Stokes shift
更新于2025-09-10 09:29:36
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Encyclopedia of Biophysics || Protein Circular Dichroism Analysis
摘要： The far-ultraviolet circular dichroism spectra of proteins contain information about the electrostatic and magnetic environment of the chromophores and may be analyzed to give a close approximation of the content of secondary structure types. Different protein secondary structure types produce characteristic spectral shapes, and, due to the additive nature of the spectra, the shapes contribute to the total spectrum in the proportions that their secondary structures are found within the sample. Typically, radiation of wavelengths 240–190 nm and shorter is used for this type of analysis. More electronic transitions are included by collecting data at shorter wavelengths, so more information is available.
关键词： Deconvolution,Protein Circular Dichroism,Secondary structure analyses
更新于2025-09-10 09:29:36
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A New Device Based on Interferometric Optical Detection Method for Label-Free Screening of C-Reactive Protein
摘要： In previous work, the performance of a compact and cost-effective point-of-care (PoC) device based on the increase relative optical power (IROP) methodology is reported and it is determined the enhancement in comparison with standard high-resolution spectrometry in terms of limit of detection. This paper describes a new label-free IROP-based biomedical device capable of working with low concentration of reagents and low sample volume per measurement in order to be used for screening different steps necessary in immunoassay optimization. This new approach significantly improves the sensing performance in terms of read-out signal (ΔIROP) per nanometer of biofilm in comparison with our previous work. This improvement is achieved due to the implementation of a laser as light source of the optical read-out system and the redesign of Fabry–Perot transducers by optimizing their reflectivity response and reducing their sensing area. For demonstrating the screening capability of this new PoC device in several immunoassays steps and methodologies, a C-reactive protein detection assay was carried out as a potential application and assay model. It is remarkable that only 10 μL of sample was used per measurement. This label-free IROP-based device complies an easy-to-use and cost-effective tool for immunoassays optimization in terms of performance, reagents cost, and measuring time.
关键词： C-reactive protein (CRP),label-free optical screening,immunoassays,point-of-care (PoC) devices,biosensors
更新于2025-09-10 09:29:36
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Expanding the optogenetics toolkit
摘要： Optogenetics has been instrumental for the dissection of functional neural circuits. As this branch of neuroscience flourishes, and as experimental designs become increasingly sophisticated, the need for new opsins—light-activated proteins that can be used to excite or inhibit specific neuronal populations—also grows. Previous efforts have relied on either the discovery of naturally occurring opsin variants with unique features through genome mining, or the engineering of opsins to achieve certain desired properties. Now, researchers at the Janelia Research Campus of the Howard Hughes Medical Institute, led by Joshua Dudman and Alla Karpova, have developed a new protein-engineering approach that could in principle double the number of viable tools for optogenetics. Taking inspiration from evolution, the researchers wondered whether topological inversion—flipping a protein inside-out within the membrane—could generate opsin variants with new properties. They designed a leader sequence containing the signal peptide and the transmembrane domain of neurexin 1B-δ, and they packed in a few more tweaks to further support its desired orientation. They then fused the leader sequence and a well-characterized opsin, ChR2 E123T/T159C. This effectively flipped the opsin within the membrane. Strikingly, the opsin was transformed from an optogenetic activator to a potent inhibitor that functions as a light-activated, nonselective cation pump. They dubbed this new variant ‘full-length inversion of ChR’ (FLInChR). FLInChR suppressed the activity of SNr GABAergic neurons in brain slices. In vivo, FLInChR was able to inhibit SNr projection neurons and modulate animal behaviors. Notably, the extent of inhibition elicited by FLInChR was on par with that observed with ArchT, a commonly used optogenetic inhibitor. It is worth noting that nonselective cation pumping has not been previously achieved through directed molecular engineering. This topological engineering approach could help to create new optogenetic tools for neuroscience research.
关键词： neuroscience,opsins,optogenetics,protein engineering,topological inversion
更新于2025-09-10 09:29:36
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Optical Microrheology of Protein Solutions Using Tailored Nanoparticles
摘要： This work represents a critical re-examination of the application of dynamic light scattering (DLS)–based tracer particle microrheology to measure the zero shear viscosity of aqueous solutions of different proteins up to very high concentrations. It is demonstrated that a combination of surface-functionalized tracer particles, the use of the so-called 3D-DLS technique, and carefully chosen parameters for the scattering experiments is essential for a reliable and artifact-free determination of the viscosity of highly diverse protein solutions, while keeping the amount of protein to a minimum. The major challenges that arise in such microrheology experiments with protein solutions are discussed and used as guiding principles for the synthesis of all-purpose tracer particles with optimal size and an efficient surface functionalization, and the choice of the appropriate amount of tracers in the sample. Potential problems arising from depletion attractions between the tracer particles induced by the proteins are addressed, and compelling evidences for the absence of such effects are presented. The validity of the approach is corroborated by the perfect agreement between the zero shear viscosity obtained from 3D-DLS-based microrheology and literature data from classical rheological measurements for two vastly different protein–solvent systems up to concentrations close to the arrest transition.
关键词： microrheology,tracer particles,protein viscosity,multiple scattering,3D-dynamic light scattering
更新于2025-09-10 09:29:36