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Small-Molecule Fluorescent Probes for Live-Cell Super-Resolution Microscopy
摘要： Super-resolution fluorescence microscopy is a powerful tool to visualize biomolecules and cellular structures at the nanometer scale. Employing these techniques in living cells has opened up the possibility to study dynamic processes with unprecedented spatial and temporal resolution. Different physical approaches to super-resolution microscopy have been introduced over the last years. A bottleneck to apply these approaches for live-cell imaging has become the availability of appropriate fluorescent probes that can be specifically attached to biomolecules. In this perspective, we discuss the role of small-molecule fluorescent probes for live-cell super-resolution microscopy and the challenges that need to be overcome for their generation. Recent trends in the development of labeling strategies are reviewed together with the required chemical and spectroscopic properties of the probes. Finally, selected examples of the use of small-molecule fluorescent probes in live-cell super-resolution microscopy are given.
关键词： Bioorthogonal chemistry,Super-resolution microscopy,Live-cell imaging,Fluorogenic probes,Protein labeling
更新于2025-09-23 15:19:57
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Anion photoelectron spectroscopy of protein chromophores
摘要： Photoactive proteins that efficiently and selectively transfer light energy into a physical response are ubiquitous in nature. The small molecule chromophores that lie at the heart of these processes often exist as closed-shell anions following deprotonation in proton-transfer reactions. This review highlights the important role that anion photoelectron spectroscopy, combined with computational chemistry calculations, is playing in improving our understanding of the electronic structure and relaxation dynamics of these protein chromophores. We discuss key aspects of anion photoelectron spectroscopy. We then review recent anion photoelectron spectroscopy studies of the deprotonated chromophore anions found in green fluorescent protein (GFP), photoactive yellow protein (PYP) and the deprotonated luciferin anion found in the luciferase enzyme.
关键词： electrospray ionisation,Anion photoelectron spectroscopy,photoactive yellow protein,luciferin,green fluorescent protein,photodetachment
更新于2025-09-19 17:15:36
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Fluorescence resonance energy transfer links membrane ferroportin, hephaestin but not ferroportin, amyloid precursor protein complex with iron efflux
摘要： Iron efflux from mammalian cells is supported by the synergistic actions of the ferrous iron efflux transporter, ferroportin (Fpn) and a multicopper ferroxidase, that is, hephaestin (Heph), ceruloplasmin (Cp) or both. The two proteins stabilize Fpn in the plasma membrane and catalyze extracellular Fe3+ release. The membrane stabilization of Fpn is also stimulated by its interaction with a 22-amino acid synthetic peptide based on a short sequence in the extracellular E2 domain of the amyloid precursor protein (APP). However, whether APP family members interact with Fpn in vivo is unclear. Here, using cyan fluorescent protein (CFP)-tagged Fpn in conjunction with yellow fluorescent protein (YFP)-fusions of Heph and the APP family members APP, APLP1, and APLP2 in HEK293T cells we used fluorescence and surface biotinylation to quantify Fpn membrane occupancy and also measured 59Fe efflux. We demonstrate that Fpn and Heph co-localize, and FRET analysis indicated that the two proteins form an iron-efflux complex. In contrast, none of the full-length, cellular APP proteins exhibited Fpn co-localization or FRET. Moreover, iron supplementation increased surface expression of the iron-efflux complex, and copper depletion knocked down Heph activity and decreased Fpn membrane localization. Whereas cellular APP species had no effects on Fpn and Heph localization, addition of soluble E2 elements derived from APP and APLP2, but not APLP1, increased Fpn membrane occupancy. We conclude that a ferroportin-targeting sequence, K/REWEE, present in APP and APLP2, but not APLP1, helps modulate Fpn-dependent iron efflux in the presence of an active multicopper ferroxidase.
关键词： cell surface protein,hephaestin,iron metabolism,membrane transport,metal transport,Ferroportin,multicopper ferroxidase,APP-like proteins,iron efflux,amyloid precursor protein
更新于2025-09-19 17:15:36
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Near-infrared light remotely up-regulate autophagy with spatiotemporal precision via upconversion optogenetic nanosystem
摘要： In vivo noninvasively manipulating biological functions by the mediation of biosafe near infrared (NIR) light is becoming increasingly popular. For these applications, upconversion rare-earth nanomaterial holds great promise as a novel photonic element, and has been widely adopted in optogenetics. In this article, an upconversion optogenetic nanosystem that was promised to achieve autophagy up-regulation with spatiotemporal precision was designed. The biocompatible system worked via two separated parts: blue light-receptor optogenetics-autophagy upregulation plasmids for import; and upconversion rods-encapsulated flexible capsule for converting tissue-penetrative NIR light into local visible blue light. Results validated that this system could achieve up-regulation of autophagy in vitro (in both HeLa and 293T cell lines) and remotely penetrate tissue (~3.5mm) in vivo. Since autophagy serves at a central position in intracellular signalling pathways, which is correlative with diverse pathologies, we expect that this method could establish an upconversion material-based autophagy up-regulation strategy for fundamental and clinical applications.
关键词： optogenetics,autophagy,upconversion materials,protein-protein interaction (PPI),near-infrared (NIR) light
更新于2025-09-19 17:15:36
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Bimodal detection of proteins by 129Xe NMR and fluorescence spectroscopy
摘要： The full understanding of biological phenomena involves sensitive and non-invasive detection. Here we report the optimization of a probe for intracellular proteins that combines the advantages of fluorescence and hyperpolarized 129Xe NMR detection. The fluorescence detection part is composed of six residues containing a tetracysteine tag (-CCXXCC-) genetically incorporated into the protein of interest and of a small organic molecule, CrAsH. CrAsH becomes fluorescent when it binds to the tetracysteine tag. The part of the biosensor that enables 129Xe NMR detection, linked to the CrAsH moiety by a spacer, is based on a cryptophane core fully suited to reversibly host xenon. We benchmark three different peptides containing the tetracysteine tag and four organic biosensors of different stereochemistry to propose the best couple, fully suited for the in vitro detection of proteins.
关键词： biosensor,tetracysteine-tag,fluorescence,129Xe NMR,cryptophane,fluorescent protein
更新于2025-09-19 17:15:36
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Raman spectra of the GFP-like fluorescent proteins
摘要： The objective of the study was to elucidate optical characteristics of the chromophore structures of fluorescent proteins. Raman spectra of commonly used GFP-like fluorescent proteins (FPs) with diverse emission wavelengths (green, yellow, cyan and red), including the enhanced homogenous FPs EGFP, EYFP, and ECFP (from jellyfish) as well as mNeptune (from sea anemone) were measured. High-quality Raman spectra were obtained and many marker bands for the chromophore of the FPs were identified via assignment of Raman spectra bands. We report the presence of a positive linear correlation between the Raman band shift of C5=C6 and the excitation energy of FPs, demonstrated by plotting absorption maxima (cm-1) against the position of the Raman band C5=C6 in EGFP, ECFP, EYFP, the anionic chromophore and the neutral chromophore. This study revealed new Raman features in the chromophores of the observed FPs, and may contribute to a deeper understanding of the optical properties of FPs.
关键词： Chromophore,Fluorescent protein,Raman spectra
更新于2025-09-19 17:15:36
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Smallest near-infrared fluorescent protein evolved from cyanobacteriochrome as versatile tag for spectral multiplexing
摘要： From a single domain of cyanobacteriochrome (CBCR) we developed a near-infrared (NIR) fluorescent protein (FP), termed miRFP670nano, with excitation at 645 nm and emission at 670 nm. This is the first CBCR-derived NIR FP evolved to efficiently bind endogenous biliverdin chromophore and brightly fluoresce in mammalian cells. miRFP670nano is a monomer with molecular weight of 17 kDa that is 2-fold smaller than bacterial phytochrome (BphP)-based NIR FPs and 1.6-fold smaller than GFP-like FPs. Crystal structure of the CBCR-based NIR FP with biliverdin reveals a molecular basis of its spectral and biochemical properties. Unlike BphP-derived NIR FPs, miRFP670nano is highly stable to denaturation and degradation and can be used as an internal protein tag. miRFP670nano is an effective FRET donor for red-shifted NIR FPs, enabling engineering NIR FRET biosensors spectrally compatible with GFP-like FPs and blue–green optogenetic tools. miRFP670nano unlocks a new source of diverse CBCR templates for NIR FPs.
关键词： near-infrared fluorescent protein,spectral multiplexing,cyanobacteriochrome,FRET biosensors,biliverdin,optogenetics
更新于2025-09-19 17:15:36
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Microtubule network as a potential candidate for targeting by gold nanoparticle-assisted photothermal therapy
摘要： Photothermal therapy is achieving ever-increasing attention as a promising method for killing cancer cells. Although, gold nanoparticles are regarded as one of the most effective photothermal therapy agents, the mechanisms underlying their action have to be addressed. Moreover, studies have showed that gold nanoparticles induce apoptosis in treated cultures. Hence, in this study, we investigated the interaction of folic acid functionalized gold nanoparticles and gold-shelled Fe3O4 nanoparticles with microtubule and microtubule associated protein tau in order to introduce intracellular targets of these nanoparticles and provide a holistic view about the mechanism of action of gold nanoparticles used in photothermal therapy. Various spectroscopic methods were used to find gold nanoparticles interaction with Tubulin and Tau. Our results indicated that these gold nanoparticles interact with both Tau and Tubulin and their affinity increases as temperature rises. Also, the results illustrated that quenching mechanism for gold nanoparticles interaction with Tubulin and Tau was static. The hydrophobic interaction was determined as driving force for gold nanoparticles binding to Tubulin and Tau. Moreover, it was showed that both type of gold nanoparticles stabilize microtubule polymers. These results suggest Tau and Tubulin as intracellular target of gold nanoparticles and propose that microtubule network is at the heart of apoptosis mechanisms initiated by photothermal therapy.
关键词： Gold-shelled Fe3O4 nanoparticles,Tubulin,Gold nanoparticles,Tau protein,Photothermal therapy
更新于2025-09-19 17:15:36
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Azobenzene-based small molecular photoswitches for protein modulation
摘要： Molecular photoswitches are a class of chemical structures that can readily isomerize between distinct geometries upon irradiation with light. Molecular photoswitches are utilized to control protein structure and function with temporal and spatial precision. In this review, we summarize the recent progress in the development of azobenzene-based molecular photoswitches and their applications in the photocontrol of protein structure and function. For clarity of discussion, we divide the known photoswitchable proteins into different categories: protein motifs, ion channels, receptors, and enzymes. Basic approaches and considerations for the structure-guided design of photoswitchable ligands are discussed. The applications and limitations of current photoswitches are also discussed.
关键词： protein modulation,molecular design,azobenzene,photocontrol,photoswitches
更新于2025-09-19 17:15:36
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Rapid monitoring of the target protein expression with a fluorescent signal based on a dicistronic construct in Escherichia coli
摘要： Real?time quantification of recombinant proteins is important in studies on fermentation engineering, cell engineering, etc. Measurement of the expression level of heterologous proteins in bacterial fermentation broth has traditionally relied on time?consuming and labor?intensive procedures, such as polyacrylamide gel electrophoresis, immunoblot analysis, and biological activity assays. We describe a simple, fast, and high sensitive assay for detecting heterologous proteins production in bacteria either at the overall level (fluorescence spectrophotometry) or at the individual level (fluorescence microscopic image) in this study. Based on a dicistronic model, the translation of target gene in the upstream open reading frame (ORF) was coupled with the synthesis of the mCherry reporter in the down?stream ORF in E. coli cells, and subsequently this demonstrated a positive correlation between the expression of target gene and mCherry. Although a time lag exists between the expression of target protein and mCherry reporter, the method described here allows facile monitoring of dynamic changes in target protein expression, relying on indirect determination of the fluorescence intensity of mCherry during fermentation in real?time models. Additionally, the performance of a single bacterial cell factory could be checked under the fluorescence microscope field.
关键词： Dicistron,Recombinant protein expression,Fluorescent signal,Quantification,Real-time
更新于2025-09-19 17:15:36