RESUMO
Microglia and central nervous system (CNS)-associated macrophages (CAMs), such as perivascular and meningeal macrophages, are implicated in virtually all diseases of the CNS. However, little is known about their cell-type-specific roles in the absence of suitable tools that would allow for functional discrimination between the ontogenetically closely related microglia and CAMs. To develop a new microglia gene targeting model, we first applied massively parallel single-cell analyses to compare microglia and CAM signatures during homeostasis and disease and identified hexosaminidase subunit beta (Hexb) as a stably expressed microglia core gene, whereas other microglia core genes were substantially downregulated during pathologies. Next, we generated HexbtdTomato mice to stably monitor microglia behavior in vivo. Finally, the Hexb locus was employed for tamoxifen-inducible Cre-mediated gene manipulation in microglia and for fate mapping of microglia but not CAMs. In sum, we provide valuable new genetic tools to specifically study microglia functions in the CNS.
Assuntos
Encéfalo/patologia , Encefalomielite Autoimune Experimental/patologia , Traumatismos do Nervo Facial/patologia , Microglia/metabolismo , Cadeia beta da beta-Hexosaminidase/metabolismo , Animais , Encéfalo/citologia , Encéfalo/imunologia , Sistemas CRISPR-Cas/genética , Encefalomielite Autoimune Experimental/imunologia , Traumatismos do Nervo Facial/imunologia , Técnicas de Introdução de Genes , Genes Reporter/genética , Loci Gênicos/genética , Humanos , Microscopia Intravital , Substâncias Luminescentes/química , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Microglia/imunologia , Células NIH 3T3 , RNA-Seq , Análise de Célula Única , Transfecção , Cadeia beta da beta-Hexosaminidase/genética , Proteína Vermelha FluorescenteRESUMO
Genetically encoded optical tools have revolutionized modern biology by allowing detection and control of biological processes with exceptional spatiotemporal precision and sensitivity. Natural photoreceptors provide researchers with a vast source of molecular templates for engineering of fluorescent proteins, biosensors, and optogenetic tools. Here, we give a brief overview of natural photoreceptors and their mechanisms of action. We then discuss fluorescent proteins and biosensors developed from light-oxygen-voltage-sensing (LOV) domains and phytochromes, as well as their properties and applications. These fluorescent tools possess unique characteristics not achievable with green fluorescent protein-like probes, including near-infrared fluorescence, independence of oxygen, small size, and photosensitizer activity. We next provide an overview of available optogenetic tools of various origins, such as LOV and BLUF (blue-light-utilizing flavin adenine dinucleotide) domains, cryptochromes, and phytochromes, enabling control of versatile cellular processes. We analyze the principles of their function and practical requirements for use. We focus mainly on optical tools with demonstrated use beyond bacteria, with a specific emphasis on their applications in mammalian cells.
Assuntos
Técnicas Biossensoriais/métodos , Proteínas Luminescentes/química , Optogenética , Arabidopsis/química , Deinococcus/química , Proteínas Luminescentes/genética , Fitocromo/química , Engenharia de Proteínas , Rodopseudomonas/químicaRESUMO
The background light from out-of-focus planes hinders resolution enhancement in structured illumination microscopy when observing volumetric samples. Here we used selective plane illumination and reversibly photoswitchable fluorescent proteins to realize structured illumination within the focal plane and eliminate the out-of-focus background. Theoretical investigation of the imaging properties and experimental demonstrations show that selective plane activation is beneficial for imaging dense microstructures in cells and cell spheroids.
Assuntos
Microscopia de Fluorescência , Microscopia de Fluorescência/métodos , Humanos , Esferoides Celulares , Iluminação/métodos , Proteínas Luminescentes/metabolismo , Proteínas Luminescentes/química , Proteínas de Fluorescência Verde/metabolismoRESUMO
Light-sheet fluorescence microscopy is an invaluable tool for four-dimensional biological imaging of multicellular systems due to the rapid volumetric imaging and minimal illumination dosage. However, it is challenging to retrieve fine subcellular information, especially in living cells, due to the width of the sheet of light (>1 µm). Here, using reversibly switchable fluorescent proteins (RSFPs) and a periodic light pattern for photoswitching, we demonstrate a super-resolution imaging method for rapid volumetric imaging of subcellular structures called multi-sheet RESOLFT. Multiple emission-sheets with a width that is far below the diffraction limit are created in parallel increasing recording speed (1-2 Hz) to provide super-sectioning ability (<100 nm). Our technology is compatible with various RSFPs due to its minimal requirement in the number of switching cycles and can be used to study a plethora of cellular structures. We track cellular processes such as cell division, actin motion and the dynamics of virus-like particles in three dimensions.
Assuntos
Microscopia de Fluorescência , Microscopia de Fluorescência/métodos , Humanos , Proteínas Luminescentes/química , Proteínas Luminescentes/metabolismo , Animais , Actinas/metabolismo , Imageamento Tridimensional/métodos , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/química , Células HeLaRESUMO
Direct investigation of the early cellular changes induced by metastatic cells within the surrounding tissue remains a challenge. Here we present a system in which metastatic cancer cells release a cell-penetrating fluorescent protein, which is taken up by neighbouring cells and enables spatial identification of the local metastatic cellular environment. Using this system, tissue cells with low representation in the metastatic niche can be identified and characterized within the bulk tissue. To highlight its potential, we applied this strategy to study the cellular environment of metastatic breast cancer cells in the lung. We report the presence of cancer-associated parenchymal cells, which exhibit stem-cell-like features, expression of lung progenitor markers, multi-lineage differentiation potential and self-renewal activity. In ex vivo assays, lung epithelial cells acquire a cancer-associated parenchymal-cell-like phenotype when co-cultured with cancer cells and support their growth. These results highlight the potential of this method as a platform for new discoveries.
Assuntos
Linhagem da Célula , Rastreamento de Células/métodos , Metástase Neoplásica/patologia , Células-Tronco Neoplásicas/patologia , Tecido Parenquimatoso/patologia , Coloração e Rotulagem/métodos , Nicho de Células-Tronco , Microambiente Tumoral , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Diferenciação Celular , Técnicas de Cocultura , Células Epiteliais/patologia , Feminino , Humanos , Proteínas Luminescentes/análise , Proteínas Luminescentes/química , Proteínas Luminescentes/metabolismo , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Masculino , Camundongos , Metástase Neoplásica/imunologia , Neutrófilos/patologia , Organoides/patologia , Nicho de Células-Tronco/imunologia , Microambiente Tumoral/imunologia , Proteína Vermelha FluorescenteRESUMO
Genetically encoded sensors enable quantitative imaging of analytes in live cells. Sensors are commonly constructed by combining ligand-binding domains with one or more sensitized fluorescent protein (FP) domains. Sensors based on a single FP can be susceptible to artifacts caused by changes in sensor levels or distribution in vivo. To develop intensiometric sensors with the capacity for ratiometric quantification, dual-FP Matryoshka sensors were generated by using a single cassette with a large Stokes shift (LSS) reference FP nested within the reporter FP (cpEGFP). Here, we present a genetically encoded calcium sensor that employs green apple (GA) Matryoshka technology by incorporating a newly designed red LSSmApple fluorophore. LSSmApple matures faster and provides an optimized excitation spectrum overlap with cpEGFP, allowing for monochromatic coexcitation with blue light. The LSS of LSSmApple results in improved emission spectrum separation from cpEGFP, thereby minimizing fluorophore bleed-through and facilitating imaging using standard dichroic and red FP (RFP) emission filters. We developed an image analysis pipeline for yeast (Saccharomyces cerevisiae) timelapse imaging that utilizes LSSmApple to segment and track cells for high-throughput quantitative analysis. In summary, we engineered a new FP, constructed a genetically encoded calcium indicator (GA-MatryoshCaMP6s), and performed calcium imaging in yeast as a demonstration.
Assuntos
Cálcio , Saccharomyces cerevisiae , Proteínas Luminescentes/química , Cálcio/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteína Vermelha Fluorescente , Corantes FluorescentesRESUMO
Flavin-binding fluorescent proteins are promising genetically encoded tags for microscopy. However, spectral properties of their chromophores (riboflavin, flavin mononucleotide, and flavin adenine dinucleotide) are notoriously similar even between different protein families, which limits applications of flavoproteins in multicolor imaging. Here, we present a palette of 22 finely tuned fluorescent tags based on the thermostable LOV domain from Chloroflexus aggregans. We performed site saturation mutagenesis of three amino acid positions in the flavin-binding pocket, including the photoactive cysteine, to obtain variants with fluorescence emission maxima uniformly covering the wavelength range from 486 to 512 nm. We demonstrate three-color imaging based on spectral separation and two-color fluorescence lifetime imaging of bacteria, as well as two-color imaging of mammalian cells (HEK293T), using the proteins from the palette. These results highlight the possibility of fine spectral tuning of flavoproteins and pave the way for further applications of flavin-binding fluorescent proteins in fluorescence microscopy.
Assuntos
Flavoproteínas , Proteínas Luminescentes , Riboflavina , Humanos , Mononucleotídeo de Flavina/metabolismo , Flavina-Adenina Dinucleotídeo , Flavoproteínas/química , Células HEK293 , Proteínas Luminescentes/químicaRESUMO
Red fluorescent protein (RFP) variants are highly sought after for in vivo imaging since longer wavelengths improve depth and contrast in fluorescence imaging. However, the lower energy emission wavelength usually correlates with a lower fluorescent quantum yield compared to their green emitting counterparts. To guide the rational design of bright variants, we have theoretically assessed two variants (mScarlet and mRouge) which are reported to have very different brightness. Using an α-CASSCF QM/MM framework (chromophore and all protein residues within 6 Å of it in the QM region, for a total of more than 450 QM atoms), we identify key points on the ground and first excited state potential energy surfaces. The brighter variant mScarlet has a rigid scaffold, and the chromophore stays largely planar on the ground state. The dimmer variant mRouge shows more flexibility and can accommodate a pretwisted chromophore conformation which provides easier access to conical intersections. The main difference between the variants lies in the intersection seam regions, which appear largely inaccessible in mScarlet but partially accessible in mRouge. This observation is mainly related with changes in the cavity charge distribution, the hydrogen-bonding network involving the chromophore and a key ARG/THR mutation (which changes both charge and steric hindrance).
Assuntos
Proteínas Luminescentes , Proteína Vermelha Fluorescente , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Teoria Quântica , Modelos Moleculares , Ligação de HidrogênioRESUMO
Autophagy is a widely conserved and multistep cellular catabolic process and maintains cellular homeostasis and normal cellular functions via the degradation of some harmful intracellular components. It was reported that high basal autophagic activity may be closely related to tumorigenesis. So far, the fluorescence imaging technique has been widely used to study autophagic processes, but this method is only suitable for distinguishing autophagosomes and autolysosomes. Simultaneously monitoring multiple autophagic processes remains a significant challenge due to the lack of an efficient detection method. Here, we demonstrated a new method for simultaneously monitoring multiple autophagic processes and assessing autophagic flux in single cells based on in situ fluorescence cross-correlation spectroscopy (FCCS). In this study, microtubule-associated protein 1A/1B-light chain 3B (LC3B) was fused with two tandem fluorescent proteins [mCherry red fluorescent protein (mCherry) and enhanced green fluorescent protein (EGFP)] to achieve the simultaneous labeling and distinguishing of multiple autophagic structures based on the differences in characteristic diffusion time (τD). Furthermore, we proposed a new parameter "delivery efficiency of autophagosome (DEAP)" to assess autophagic flux based on the cross correlation (CC) value. Our results demonstrate that FCCS can efficiently distinguish three autophagic structures, assess the induced autophagic flux, and discriminate different autophagy regulators. Compared with the commonly used fluorescence imaging technique, the resolution of FCCS remains unaffected by Brownian motion and fluorescent monomers in the cytoplasm and is well suitable to distinguishing differently colored autophagic structures and monitoring autophagy.
Assuntos
Autofagia , Análise de Célula Única , Espectrometria de Fluorescência , Humanos , Espectrometria de Fluorescência/métodos , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/análise , Células HeLa , Proteínas Luminescentes/metabolismo , Proteínas Luminescentes/química , Proteína Vermelha Fluorescente , Autofagossomos/metabolismoRESUMO
Flavin-based fluorescent proteins are oxygen-independent reporters that hold great promise for imaging anaerobic and hypoxic biological systems. In this study, we explored the feasibility of applying circular permutation, a valuable method for the creation of fluorescent sensors, to flavin-based fluorescent proteins. We used rational design and structural data to identify a suitable location for circular permutation in iLOV, a flavin-based reporter derived from A. thaliana. However, relocating the N- and C-termini to this position resulted in a significant reduction in fluorescence. This loss of fluorescence was reversible, however, by fusing dimerizing coiled coils at the new N- and C-termini to compensate for the increase in local chain entropy. Additionally, by inserting protease cleavage sites in circularly permuted iLOV, we developed two protease sensors and demonstrated their application in mammalian cells. In summary, our work establishes the first approach to engineer circularly permuted FbFPs optimized for high fluorescence and further showcases the utility of circularly permuted FbFPs to serve as a scaffold for sensor engineering.
Assuntos
Flavinas , Proteínas Luminescentes , Flavinas/química , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Humanos , Engenharia de Proteínas , Arabidopsis/química , Células HEK293RESUMO
Far-red and near-infrared fluorescent proteins have regions of maximum transmission in most tissues and can be widely used as fluorescent biomarkers. We report that fluorescent phycobiliproteins originating from the phycobilisome core subunit ApcF2 can covalently bind biliverdin, named BDFPs. To further improve BDFPs, we conducted a series of studies. Firstly, we mutated K53Q and T144A of BDFPs to increase their effective brightness up to 190 % inâ vivo. Secondly, by homochromatic tandem fusion of high-brightness BDFPs to achieve monomerization, which increases the effective brightness by up to 180 % inâ vivo, and can effectively improve the labeling effect. By combining the above two approaches, the brightness of the tandem BDFPs was much improved compared with that of the previously reported fluorescent proteins in a similar spectral range. The tandem BDFPs were expressed stably while maintaining fluorescence in mammalian cells and Caenorhabditis elegans. They were also photostable and resistant to high temperature, low pH, and chemical denaturation. The tandem BDFPs advantages were proved in applications as biomarkers for imaging in super-resolution microscopy.
Assuntos
Caenorhabditis elegans , Proteínas Luminescentes , Animais , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Caenorhabditis elegans/metabolismo , Humanos , Ficobiliproteínas/química , Ficobiliproteínas/metabolismo , Biliverdina/química , Biliverdina/metabolismo , Corantes Fluorescentes/química , Células HEK293RESUMO
Site-specific installation of non-natural functionality onto proteins has enabled countless applications in biotechnology, chemical biology, and biomaterials science. Though the N-terminus is an attractive derivatization location, prior methodologies targeting this site have suffered from low selectivity, a limited selection of potential chemical modifications, and/or challenges associated with divergent protein purification/modification steps. In this work, we harness the atypically split VidaL intein to simultaneously N-functionalize and purify homogeneous protein populations in a single step. Our methodâreferred to as VidaL-tagged expression and protein ligation (VEPL)âenables modular and scalable production of N-terminally modified proteins with native bioactivity. Demonstrating its flexibility and ease of use, we employ VEPL to combinatorially install 4 distinct (multi)functional handles (e.g., biotin, alkyne, fluorophores) to the N-terminus of 4 proteins that span three different classes: fluorescent (Enhanced Green Fluorescent Protein, mCherry), enzymatic (ß-lactamase), and growth factor (epidermal growth factor). Moving forward, we anticipate that VEPL's ability to rapidly generate and isolate N-modified proteins will prove useful across the growing fields of applied chemical biology.
Assuntos
Inteínas , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/genética , beta-Lactamases/metabolismo , beta-Lactamases/química , Proteínas Luminescentes/química , Fator de Crescimento Epidérmico/metabolismo , Fator de Crescimento Epidérmico/química , Proteína Vermelha Fluorescente , Proteínas/químicaRESUMO
Genetically-encoded redox biosensors have become invaluable tools for monitoring cellular redox processes with high spatiotemporal resolution, coupling the presence of the redox-active analyte with a change in fluorescence signal that can be easily recorded. This review summarizes the available fluorescence recording methods and presents an in-depth classification of the redox biosensors, organized by the analytes they respond to. In addition to the fluorescent protein-based architectures, this review also describes the recent advances on fluorescent, chemigenetic-based redox biosensors and other emerging chemigenetic strategies. This review examines how these biosensors are designed, the biosensors sensing mechanism, and their practical advantages and disadvantages.
Assuntos
Técnicas Biossensoriais , Oxirredução , Técnicas Biossensoriais/métodos , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas Luminescentes/química , AnimaisRESUMO
Protein phase separation plays a very important role in many biological processes and is closely related to the occurrence and development of some serious diseases. So far, the fluorescence imaging method and fluorescence correlation spectroscopy (FCS) have been frequently used to study the phase separation behavior of proteins. Due to the wide size distribution of protein condensates in phase separation from nano-scale to micro-scale in solution and living cells, it is difficult for the fluorescence imaging method and conventional FCS to fully reflect the real state of protein phase separation in the solution due to the low spatio-temporal resolution of the conventional fluorescence imaging method and the limited detection area of FCS. Here, we proposed a novel method for studying the protein phase separation process by objective scanning-based fluorescence cross-correlation spectroscopy (Scan-FCCS). In this study, CRDBP proteins were used as a model and respectively fused with fluorescent proteins (EGFP and mCherry). We first compared conventional FCS and Scan-FCS methods for characterizing the CRDBP protein phase separation behaviors and found that the reproducibility of Scan-FCS is significantly improved by the scanning mode. We studied the self-fusion process of mCherry-CRDBP and EGFP-CRDBP and observed that the phase change concentration of CRDBP was 25 nM and the fusion of mCherry-CRDBP and EGFP-CRDBP at 500 nM was completed within 70 min. We studied the effects of salt concentration and molecular crowding agents on the phase separation of CRDBP and found that salt can prevent the self-fusion of CRDBP and molecular crowding agents can improve the self-fusion of CRDBP. Furthermore, we found the recruitment behavior of CRDBP to ß-catenin proteins and studied their recruitment dynamics. Compared to conventional FCS, Scan-FCCS can significantly improve the reproducibility of measurements due to the dramatic increase of detection zone, and more importantly, this method can provide information about self-fusion and recruitment dynamics in protein phase separation.
Assuntos
Proteínas de Fluorescência Verde , Espectrometria de Fluorescência , Espectrometria de Fluorescência/métodos , Proteínas de Fluorescência Verde/química , Proteínas Luminescentes/química , Proteínas Recombinantes de Fusão/química , Proteína Vermelha Fluorescente , Separação de FasesRESUMO
Antibodies are crucial in various biological applications due to their specific binding to target molecules, altering protein function and structure. The advent of single-chain antibodies such as nanobodies has paved the way for broader applicability in both research and therapies due to their small size and efficient tissue penetration. Recently, several approaches have been reported to optically control the antigen-binding affinity of nanobodies. Here, we show an alternative strategy for creating photo-activatable nanobodies. By fusing the photocleavable protein PhoCl with the N-terminus of the nanobody (named optoNb60), we successfully demonstrated light-dependent restoration of the antigen-binding ability and the following modulation of the activity of a target protein, the beta-2 adrenergic receptor. Moreover, the activation of optoNb60 was monitored by the fluorescence changes upon photoconversion. The compatibility of the uncaging design with the previously reported optogenetic molecules using nanobodies will contribute to the further optimization of the response capabilities of existing optogenetic tools, thereby expanding their applicability.
Assuntos
Receptores Adrenérgicos beta 2 , Anticorpos de Domínio Único , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/imunologia , Humanos , Receptores Adrenérgicos beta 2/metabolismo , Receptores Adrenérgicos beta 2/química , Proteínas Luminescentes/química , Luz , Células HEK293RESUMO
We used site-specific mutagenesis by targeting E179 and F190 on the structure of photoprotein Mnemiopsin 2 (Mn2) from Mnemiopsis leidyi. The tertiary structure of E179S and F190L mutants was made by the MODELLER program. Far-ultraviolet circular dichroism data showed that the overall secondary structural content of photoprotein is not changed upon mutation, however the helicity and stabilizing interactions in helical structure decreases in mutants as compared with the wild-type (WT) photoprotein. Fluorescence spectra data revealed that the tertiary structure of the mutants is more compact than that of WT Mn2. According to the heat-induced denaturation experiments data, the melting temperature (Tm ) for the unfolding of tertiary structure of the F190L variant increases by 3°C compared with that of the WT and E179S mutant. Interestingly, the conformational enthalpy of the F190L mutant (86 kcal mol-1 ) is considerably lower than those in the WT photoprotein (102 kcal mol-1 ) and E179S mutant (106 kcal mol-1 ). The significant difference in the enthalpy of the thermal unfolding process could be explained by considering that the thermally denatured state of the F190L mutant is structurally less expanded than the WT and E179S variants. Bioluminescence activity data showed that the maximum characteristic wavelengths of the mutants undergo blue shift as compared with the WT protein. Initial intensity of the F190L and E179S variants was recorded to be 137.5% and 55.9% of the WT protein, respectively.
Assuntos
Cálcio , Cálcio/química , Mutagênese Sítio-Dirigida , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Dicroísmo Circular , Termodinâmica , Desnaturação ProteicaRESUMO
Förster resonance energy transfer (FRET) spectrometry is a method for determining the quaternary structure of protein oligomers from distributions of FRET efficiencies that are drawn from pixels of fluorescence images of cells expressing the proteins of interest. FRET spectrometry protocols currently rely on obtaining spectrally resolved fluorescence data from intensity-based experiments. Another imaging method, fluorescence lifetime imaging microscopy (FLIM), is a widely used alternative to compute FRET efficiencies for each pixel in an image from the reduction of the fluorescence lifetime of the donors caused by FRET. In FLIM studies of oligomers with different proportions of donors and acceptors, the donor lifetimes may be obtained by fitting the temporally resolved fluorescence decay data with a predetermined number of exponential decay curves. However, this requires knowledge of the number and the relative arrangement of the fluorescent proteins in the sample, which is precisely the goal of FRET spectrometry, thus creating a conundrum that has prevented users of FLIM instruments from performing FRET spectrometry. Here, we describe an attempt to implement FRET spectrometry on temporally resolved fluorescence microscopes by using an integration-based method of computing the FRET efficiency from fluorescence decay curves. This method, which we dubbed time-integrated FRET (or tiFRET), was tested on oligomeric fluorescent protein constructs expressed in the cytoplasm of living cells. The present results show that tiFRET is a promising way of implementing FRET spectrometry and suggest potential instrument adjustments for increasing accuracy and resolution in this kind of study.
Assuntos
Estudos de Viabilidade , Transferência Ressonante de Energia de Fluorescência , Microscopia de Fluorescência , Transferência Ressonante de Energia de Fluorescência/métodos , Microscopia de Fluorescência/métodos , Humanos , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/química , Espectrometria de Fluorescência/métodos , Proteínas Luminescentes/química , Proteínas Luminescentes/metabolismo , FluorescênciaRESUMO
The Keima family comprises large Stokes shift fluorescent proteins that are useful for dual-color fluorescence cross-correlation spectroscopy and multicolor imaging. The tKeima is a tetrameric large Stokes shift fluorescent protein and serves as the ancestor fluorescent protein for both dKeima and mKeima. The spectroscopic properties of tKeima have been previously reported; however, its structural basis and molecular properties have not yet been elucidated. In this study, we present the crystallographic results of the large Stokes shift fluorescent protein tKeima. The purified tKeima protein spontaneously crystallized after purification without further crystallization. The crystal structure of tKeima was determined at 3.0 Å resolution, revealing a ß-barrel fold containing the Gln-Tyr-Gly chromophores mainly with cis-conformation. The tetrameric interfaces of tKeima were stabilized by numerous hydrogen bonds and salt-bridge interactions. These key residues distinguish the substituted residues in dKeima and mKeima. The key structure-based residues involved in the tetramer formation of tKeima provide insights into the generation of a new type of monomeric mKeima. This structural analysis expands our knowledge of the Keima family and provides insights into its protein engineering.
Assuntos
Proteínas Luminescentes , Modelos Moleculares , Proteína Vermelha Fluorescente , Proteínas Luminescentes/química , Cristalografia por Raios X , Conformação Proteica , Sequência de Aminoácidos , Ligação de Hidrogênio , Espectrometria de Fluorescência , Multimerização ProteicaRESUMO
Single-molecule localization microscopy (SMLM) at cryogenic temperature opens new avenues to investigate intact biological samples at the nanoscale and perform cryo-correlative studies. Genetically encoded fluorescent proteins (FPs) are markers of choice for cryo-SMLM, but their reduced conformational flexibility below the glass-transition temperature hampers efficient cryo-photoswitching. We investigated cryo-switching of rsEGFP2, one of the most efficient reversibly switchable fluorescent proteins at ambient temperature due to facile cis-trans isomerization of the chromophore. UV-visible microspectrophotometry and X-ray crystallography revealed a completely different switching mechanism at â¼110 K. At this cryogenic temperature, on-off photoswitching involves the formation of two off-states in cis conformation with blue-shifted absorption relative to that of the trans protonated chromophore populated at ambient temperature. Only one of these off-states can be switched back to the fluorescent on-state by 405 nm light, while both of them are sensitive to UV light at 355 nm. Superior recovery to the fluorescent on-state by 355 nm light was confirmed at the single-molecule level. This suggests, as also shown by simulations, that employing 355 nm light in cryo-SMLM experiments using rsEGFP2 and possibly other FPs could improve the effective labeling efficiency achievable with this technique. The rsEGFP2 photoswitching mechanism discovered in this work adds to the panoply of known switching mechanisms in fluorescent proteins.
Assuntos
Raios Ultravioleta , Temperatura , Proteínas Luminescentes/química , Isomerismo , Conformação ProteicaRESUMO
Fluorescent proteins (FPs) are a powerful tool for examining tissues, cells, and subcellular components inâ vivo and inâ vitro. FusionRed is a particular FP variant mutated from mKate2 that, in addition to lower cytotoxicity and aggregation rates, has shown potential for acting as a tunable photoswitch. This was posited to stem partially from the presence of a bulky side chain at position 158 and a further stabilizing residue at position 157. In this work, we apply computational techniques including classical molecular dynamics (MD) and combined quantum mechanics/molecular mechanics simulations (QM/MM) to explore the effect of mutagenesis at these locations in FusionRed on the chromophore structure, the excited-state surface, and relative positional stability of the chromophore in the protein pocket. We find specific connections between the statistical sampling of the underlying protein structure and the nonradiative decay mechanisms from excited-state dynamics. A single mutation (C158I) that restricts the motion of the chromophore through a favorable hydrophobic interaction corresponds to an increase in fluorescence quantum yield (FQY), while a second rescue mutation (C158I-A157N) partially restores the flexibility of the chromophore and photoswitchability with favorable water interactions on the surface of the protein that counteracts the original interaction. We suggest that applying this understanding of structural features that inhibit or favor rotation on the excited state can be applied for rational design of new, tunable and red photoswitches.