Piatkevich, K. Extended Stokes shift in fluorescent proteins: chromophore-protein interactions in a near-infrared TagRFP variant.
Improved split fluorescent proteins for endogenous protein labeling | Nature Communications
Ganesan, S. Improved green and blue fluorescent proteins for expression in bacteria and mammalian cells. Biochemistry 46 , — Bogdanov, A. Suhling, K. Imaging the environment of green fluorescent protein. Nakabayashi, T. Sensing of intracellular environments by fluorescence lifetime imaging of exogenous fluorophores. McLoskey, D. Download references. Correspondence to Alexey M. Reprints and Permissions. By submitting a comment you agree to abide by our Terms and Community Guidelines.
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If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. Article metrics. Advanced search. Skip to main content. Subjects Biological fluorescence Biophotonics Fluorescence imaging Scanning probe microscopy. Abstract Fluorescence lifetime imaging microscopy FLIM measures fluorescence decay rate at every pixel of an image.
Introduction Since the cloning of the avGFP gene in the early s, fluorescent proteins FP have become an indispensable instrument in biology 1. Results and Discussion Here we applied semi-rational molecular evolution to generate a bright EGFP variant with subnanosecond lifetime. Full size table. Figure 1. Full size image. Figure 2. Additional information Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. References 1. Shakhov Authors Search for Anastasia V.
Electronic supplementary material Supplementary Information. About this article. Light absorption and fluorescence of green fluorescent protein GFP -like fluorescent proteins FPs are limited to a visible range of optical spectrum. Bacterial photoreceptors have absorbance spectra in the NIR range due to covalently attached heme-derived linear tetrapyrrole compounds and allow engineering NIR FPs 1. Several photoreceptors from a class of bacterial phytochrome photoreceptors BphPs were developed into bright monomeric NIR FPs, which efficiently bind endogenous biliverdin BV tetrapyrrole in mammalian cells 3 , 4 , 5.
Fourth, CBCR GAF domains are also found as components of complex signaling proteins 15 , suggesting that their structural fold is naturally optimized to use in fusion constructs PCB is naturally present in plant and cyanobacteria but not in mammalian cells, which produce BV 3 , 16 , Characterization of the developed NIR FP showed its numerous advantages over NIR FPs developed from other photoreceptors, including monomeric state, substantially smaller size, significantly higher protein stability in vitro and in mammalian cells, and possibility to be inserted inside of tagged proteins.
To facilitate protein production in mammalian cells, we first codon-optimized the CBCR genes for mammalian cell expression. We subjected the NpR GAF domain to several rounds of random mutagenesis, followed by saturating mutagenesis of the identified residues Supplementary Fig. After each round, we tested the best clones in mammalian cells and selected for the next molecular evolution only those which exhibited the high fluorescence brightness in both bacteria and mammalian cells Fig. Molecular engineering of miRFPnano.
The main mutations are indicated. Mean NIR fluorescence intensity was normalized to mean green fluorescence intensity of co-expressed EGFP and to mean fluorescence intensity of mock-transfected cells. Error bars, s. Notably, with fluorescence quantum yield of Characterization of miRFPnano.
The fluorescence of cells treated with paraformaldehyde was normalized to fluorescence of non-fixed cells. Likely, the compact and tight structure enhanced the miRFPnano resistance to denaturating conditions. The cellular a. In mammalian cells miRFPnano exhibited 2. The high photostability, low cytotoxicity, and high effective brightness make miRFPnano a favorable NIR FP for imaging of long-term cellular events.
Similar cellular stability was observed for enhanced green fluorescent protein EGFP. Live-cell imaging allows monitoring of dynamic events, but some studies require cell fixation. Overall, likely due to the compact and robust protein fold CBCR-derived miRFPnano exhibits the high cellular brightness and significantly enhanced protein stability in vitro and in mammalian cells. To reveal the structural basis of the protein stability, brightness, and specificity to BV chromophore, we determined the crystal structure of miRFPnano at 1. Carbon, nitrogen, oxygen, and sulfur atoms are in white, blue, red, and yellow, respectively.
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Its role is likely played by the side chain of D56, which forms H-bonds with pyrrole nitrogens of the rings A, B, and C Of the 18 amino acid substitutions introduced into parental NpRg Supplementary Fig. One of the important substitutions, NH makes a strong H-bond with the ring D, which is absent in NpRg, thus preventing rotation of this ring and non-radiative energy dissipation via photoswitching. F25C makes additional space in the chromophore-binding pocket, possibly enhancing BV accommodation. T57R substitution introduced a flexible positively charged residue near the chromophore binding site, enabling electrostatic attraction of BV and its additional shielding from solvent.
Substitutions Y27F and N99I increased the hydrophobicity of the chromophore environment. Such a favorable chromophore binding pocket within a compact single GAF domain fold should make miRFPnano a robust probe for various applications. To test performance of miRFPnano as fluorescent probe for labeling of intracellular structures, we constructed several miRFPnano N- and C-terminal protein fusions.
In live mammalian cells these fusions exhibited proper localization, including the fusions associated with or forming filaments Fig. Cell images showed homogenous distribution of miRFPnano and absence of intracellular aggregates Fig. Left images are zoom-in of the indicated areas of the right images. In number of cases, placing of a FP tag at the termini of proteins affects their function or leads to incorrect localization Such proteins can be labeled with FP inserted in a middle of the sequence as an internal tag.
Both miRFPnano internal fusions demonstrated perfect membrane localization, co-localizing with mVenus containing a CAAX-motif for membrane targeting.
New Trends in Fluorescence Spectroscopy
In contrast, the internal fusion constructs with two-domain miRFP did not exhibit membrane localization and formed aggregates Fig. Notably, unlike BphPs, GAF domains of CBCRs are often found as modular components of complex signaling proteins 15 , suggesting that miRFPnano has naturally optimized structure for flexible design of fusion constructs. We next evaluated applicability of miRFPnano for imaging of primary cell cultures, such as neurons. Primary rat cortical neurons transfected with miRFPnano exhibited bright homogenous fluorescence without supplying exogenous BV Fig.
PKA is one of the key effectors of cAMP-mediated signaling pathway, while JNK regulates cellular responses to diverse environmental stress signals and inflammatory cytokines Phosphorylation of the substrate peptide by activated kinases leads to a conformation rearrangement of the biosensor and an increase of FRET between donor and acceptor. Important advantage of fully NIR biosensors is their spectral compatibility with GFP-like FPs and common optogenetic tools activatable with blue light.
Similarly to JNK, p38 kinase is activated by stress signals and inflammatory cytokines 30 , Dashed line marks the region used for profile plotting. While the combination of several biosensors enables monitoring of several cell processes, a combination of biosensors with optogenetic tools should allow simultaneous detection and regulation of the processes.
This is a powerful all-optical approach to study cell signaling in native environment. White arrows indicate cells expressing optogenetic regulators. Cells transfected with the optogenetic tools responded to stimuli similarly to the cells expressing the biosensors only Supplementary Fig. However, under blue light cells with the optogenetic constructs exhibited the substantial decrease in response to the stimuli Fig.
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We then estimated the minimal detectable quantity of cells expressing miRFPnano. We next tested miRFPnano in two-color whole-body imaging. Cells transfected with either miRFPnano or miRFP were well spectrally distinguished in mice using two channels with ex. Characterization of miRFPnano in vivo. Mean fluorescence intensity was normalized to mean bioluminescence intensity.
Fluorescence top row and bioluminescence bottom row images of living mice injected with various quantity of HeLa cells expressing miRFPnano. In contrast, two-domain BphP-derived NIR FPs, which have the figure-of-eight knot in their structure and distant termini, are not suitable for internal tagging. The crystal structure of miRFPnano allows to visualize the chromophore and its immediate environment optimized during molecular evolution for BV binding and fluorescence Supplementary Fig. Bound to the conserved Cys86 via its C3 1 atom, BV forms a chromophore, which lacks a double bond between C2 and C3, but has a double bond between C3 1 and C3 2.
The number of conjugated double bonds in this chromophore is the same as in the chromophores of blue-shifted BphP-derived NIR FPs that explains their spectral similarity Fig. Likely, the presence of two protein species in miRFP explains its slightly sigmoidal maturation Fig.
Improved split fluorescent proteins for endogenous protein labeling
JNK is key transducer of exogenous stress signals and is involved in regulation of a number of physiological and pathological processes including apoptosis, proliferation, embryonic development, and inflammation. PKA mediates signals of G protein-coupled receptors and regulates a plethora of downstream effectors involved in key cellular processes. NIR fluorescence makes miRFPnano a useful probe not only for crosstalk-free spectral multiplexing in microscopy but also for deep-tissue imaging. To date, the large number of CBCRs has been cloned.
Molecular Constructions, Polymers and Nanoparticles
The CBCR spectral tuning is mainly associated with the characteristic amino acid motifs 38 , frequently containing Cys residues able to form thioether bonds with different carbon atoms of the PCB chromophore, hence affecting the degree of its electron conjugation. BV synthesis in bacteria was facilitated by co-transformation with a pWA23h plasmid encoding heme oxygenase from Bradyrhizobium ORS hmuO under the rhamnose promoter 17 , LMG host cells Invitrogen were used for protein expression.
For simultaneous site-specific mutagenesis at several positions, an overlap-extension approach was applied. Random mutagenesis was performed with GeneMorph II random mutagenesis kit Agilent Technologies under conditions resulting in a mutation frequency of up to 16 mutations per base pairs. After mutagenesis, a mixture of mutated genes was electroporated into LMG host cells containing the pWA23h plasmid. Typical mutant libraries consisted of more than 10 8 independent clones. To start protein expression 0. Before sorting, bacterial cells were washed with phosphate-buffered saline PBS and diluted with PBS to an optical density of 0.
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