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Recent development of spectroscopic techniques based on quantum states of light can precipitate many breakthroughs in observing and controlling light-matter interactions in biological materials on a fundamental quantum level. For this reason, the generation of entangled light in biologically produced fluorescent proteins would be promising because of their biocompatibility. Here we demonstrate the generation of polarization-entangled two-photon state through spontaneous four-wave mixing in enhanced green fluorescent proteins. The reconstructed density matrix indicates that the entangled state is subject to decoherence originating from two-photon absorption. However, the prepared state is less sensitive to environmental decoherence because of the protective β-barrel structure that encapsulates the fluorophore in the protein. We further explore the quantumness, including classical and quantum correlations, of the state in the decoherence environment. Our method for photonic entanglement generation may have potential for developing quantum spectroscopic techniques and quantum-enhanced measurements in biological materials.
Tagging expressed proteins with the green fluorescent protein (GFP) from Aequorea victoria [1] is a highly specific and sensitive technique for studying the intracellular dynamics of proteins and organelles. We have developed, as a probe, a fusion protein of the carboxyl terminus of dynein and GFP (dynein-GFP), which fluorescently labels the astral microtubules of the budding yeast Saccharomyces cerevisiae. This paper describes the modifications to our multimode microscope imaging system [2,3], the acquisition of three-dimensional (3-D) data sets and the computer processing methods we have developed to obtain time-lapse recordings of fluorescent astral microtubule dynamics and nuclear movements over the complete duration of the 90-120 minute yeast cell cycle. This required low excitation light intensity to prevent GFP photobleaching and phototoxicity, efficient light collection by the microscope optics, a cooled charge-coupled device (CCD) camera with high quantum efficiency, and image reconstruction from serial optical sections through the 6 micron-wide yeast cell to see most or all of the astral molecules. Methods are also described for combining fluorescent images of the microtubules labeled with dynein-GFP with high resolution differential interference contrast (DIC) images of nuclear and cellular morphology [4], and fluorescent images of the chromosomes stained with 4,6-diamidino-2-phenylindole (DAPI) [5].
Fluorescent proteins (FPs) are versatile biomarkers that facilitate effective detection and tracking of macromolecules of interest in real time. Engineered FPs such as superfolder green fluorescent protein (sfGFP) and superfolder Cherry (sfCherry) have exceptional refolding capability capable of delivering fluorescent readout in harsh environments where most proteins lose their native functions. Our recent work on the development of a split FP from a species of strawberry anemone, Corynactis californica, delivered pairs of fragments with up to threefold faster complementation than split GFP. We present the biophysical, biochemical, and structural characteristics of five full-length variants derived from these split C. californica GFP (ccGFP). These ccGFP variants are more tolerant under chemical denaturation with up to 8 kcal/mol lower unfolding free energy than that of the sfGFP. It is likely that some of these ccGFP variants could be suitable as biomarkers under more adverse environments where sfGFP fails to survive. A structural analysis suggests explanations of the variations in stabilities among the ccGFP variants.
Tandem fluorescent protein timers (tFTs) report on protein age through time-dependent change in color, which can be exploited to study protein turnover and trafficking. Each tFT, composed of two fluorescent proteins (FPs) that differ in maturation kinetics, is suited to follow protein dynamics within a specific time range determined by the maturation rates of both FPs. So far, tFTs have been constructed by combining slower-maturing red fluorescent proteins (redFPs) with the faster-maturing superfolder green fluorescent protein (sfGFP). Toward a comprehensive characterization of tFTs, we compare here tFTs composed of different faster-maturing green fluorescent proteins (greenFPs) while keeping the slower-maturing redFP constant (mCherry). Our results indicate that the greenFP maturation kinetics influences the time range of a tFT. Moreover, we observe that commonly used greenFPs can partially withstand proteasomal degradation due to the stability of the FP fold, which results in accumulation of tFT fragments in the cell. Depending on the order of FPs in the timer, incomplete proteasomal degradation either shifts the time range of the tFT toward slower time scales or precludes its use for measurements of protein turnover. We identify greenFPs that are efficiently degraded by the proteasome and provide simple guidelines for the design of new tFTs.
Fluorescent proteins (FP) homologous to the green fluorescent protein (GFP) from the jellyfish Aequorea victoria have revolutionized biomedical research due to their usefulness as genetically encoded fluorescent labels. Fluorescent proteins from copepods are particularly promising due to their high brightness and rapid fluorescence development.
Single-molecule localization microscopy with spontaneously blinking fluorescent tags holds a promise of simplified imaging setup for live-cell application. However, robust blinking has been reported for just a few fluorescent proteins. Here we report on a comparison of spontaneous blinking for three bright green fluorescent proteins, mAvicFP1, AausFP1, and mNeonGreen. mAvicFP1 outperforms other fluorescent proteins in this list in a wide range of camera exposure times and illumination intensities. We establish imaging conditions for live-cell nanoscopy and single-particle tracking with mAvicFP1.
Accumulating evidence suggests that E-selectin, which is physiologically involved in leukocyte recruitment during inflammation, plays an important role in the early stages of tumor cell interactions with vessel walls and contributes to the hematogenous spreading of cancer cells. Therapy designed to block this key step may provide an effective anti-inflammatory and anti-metastatic treatment. It is therefore critical to establish a safe, rapid and sensitive E-selectin adhesion assay. In this regard, we propose a simple and highly sensitive adhesion system based on CHO cells permanently co-expressing E-selectin and the enhanced green fluorescent protein EGFP or the red fluorescent protein DsRed2. This is an inverted adhesion assay in which tumor cells are maintained intact while fluorescent cells expressing E-selectin and EGFP (or DsRed2) are added to them. Adherent cells are then quantified by three different fluorescence-based techniques including spectrofluorimetry, ELISA-type cytofluorimetry and fluorescence microscopy coupled to digital image quantification. In this assay, a battery of cell lines can be analysed at once since only one cell line (fluorescent E-selectin-expressing cells) needs to be harvested. We used this approach to analyze a number of E-selectin-specific binding parameters of intestinal cancer cells in comparison with adhesion to activated endothelial cells or to plastic dishes coated with recombinant E-selectin. Besides the possibility of analyzing a battery of cell lines at once, this assay might be suitable for screening anti-metastatic compounds and could provide valuable information on the metastatic potential of human cancers.
A variety of genetically encoded reporters use changes in fluorescence (or Förster) resonance energy transfer (FRET) to report on biochemical processes in living cells. The standard genetically encoded FRET pair consists of CFPs and YFPs, but many CFP-YFP reporters suffer from low FRET dynamic range, phototoxicity from the CFP excitation light and complex photokinetic events such as reversible photobleaching and photoconversion. We engineered two fluorescent proteins, Clover and mRuby2, which are the brightest green and red fluorescent proteins to date and have the highest Förster radius of any ratiometric FRET pair yet described. Replacement of CFP and YFP with these two proteins in reporters of kinase activity, small GTPase activity and transmembrane voltage significantly improves photostability, FRET dynamic range and emission ratio changes. These improvements enhance detection of transient biochemical events such as neuronal action-potential firing and RhoA activation in growth cones.
Engineered light, oxygen, and voltage (LOV)-based proteins are able to fluoresce without oxygen requirement due to the autocatalytic incorporation of exogenous flavin as a chromophore thus allowing for live cell imaging under hypoxic and anaerobic conditions. They were also discovered to have high sensitivity to transition metal ions and physiological flavin derivatives. These properties make flavin-binding fluorescent proteins (FPs) a perspective platform for biosensor development. However, brightness of currently available flavin-binding FPs is limited compared to GFP-like FPs creating a need for their further enhancement and optimization. In this study, we applied a directed molecular evolution approach to develop a pair of flavin-binding FPs, named miniGFP1 and miniGFP2. The miniGFP proteins are characterized by cyan-green fluorescence with excitation/emission maxima at 450/499 nm and a molecular size of ∼13 kDa. We carried out systematic benchmarking of miniGFPs in Escherichia coli and cultured mammalian cells against spectrally similar FPs including GFP-like FP, bilirubin-binding FP, and bright flavin-binding FPs. The miniGFPs proteins exhibited improved photochemical properties compared to other flavin-binding FPs enabling long-term live cell imaging. We demonstrated the utility of miniGFPs for live cell imaging in bacterial culture under anaerobic conditions and in CHO cells under hypoxia. The miniGFPs' fluorescence was highly sensitive to Cu(II) ions in solution with Kd values of 67 and 68 nM for miniGFP1 and miniGFP2, respectively. We also observed fluorescence quenching of miniGFPs by the reduced form of Cu(I) suggesting its potential application as an optical indicator for Cu(I) and Cu(II). In addition, miniGFPs showed the ability to selectively bind exogenous flavin mononucleotide demonstrating a potential for utilization as a selective fluorescent flavin indicator. Altogether, miniGFPs can serve as a multisensing platform for fluorescence biosensor development for in vitro and in-cell applications.
The flexibility and versatility of self-complementing split fluorescent proteins (FPs) have enabled a wide range of applications. In particular, the FP1-10/11 split system contains a small fragment that facilitates efficient generation of endogenous-tagged cell lines and animals as well as signal amplification using tandem FP11 tags. To improve the FP1-10/11 toolbox we previously developed, here we used a combination of directed evolution and rational design approaches, resulting in two mNeonGreen (mNG)-based split FPs (mNG3A1-10/11 and mNG3K1-10/11) and one mClover-based split FP (CloGFP1-10/11). mNG3A1-10/11 and mNG3K1-10/11 not only enhanced the complementation efficiency at low expression levels, but also allowed us to demonstrate signal amplification using tandem mNG211 fragments in mammalian cells.
The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.
Ratiometric genetically encoded calcium indicators (GECIs) record neural activity with high brightness while mitigating motion-induced artifacts. Recently developed ratiometric GECIs primarily employ cyan and yellow-fluorescent fluorescence resonance energy transfer pairs, and thus fall short in some applications that require deep tissue penetration and resistance to photobleaching. We engineered a set of green-red ratiometric calcium sensors that fused two fluorescent proteins and calcium sensing domain within an alternate configuration. The best performing elements of this palette of sensors, Twitch-GR and Twitch-NR, inherited the superior photophysical properties of their constituent fluorescent proteins. These properties enabled our sensors to outperform existing ratiometric calcium sensors in brightness and photobleaching metrics. In turn, the shot-noise limited signal fidelity of our sensors when reporting action potentials in cultured neurons and in the awake behaving mice was higher than the fidelity of existing sensors. Our sensor enabled a regime of imaging that simultaneously captured neural structure and function down to the deep layers of the mouse cortex.
Photochromic fluorescent proteins (FPs) have proved to be indispensable luminous probes for sophisticated and advanced bioimaging techniques. Among them, an interplay between photoswitching and photoconversion has only been observed in a limited subset of Kaede-like FPs that show potential for discovering the key mechanistic steps during green-to-red photoconversion. Various spectroscopic techniques including femtosecond stimulated Raman spectroscopy (FSRS), X-ray crystallography, and femtosecond transient absorption were employed on a set of five related FPs with varying photoconversion and photoswitching efficiencies. A 3-methyl-histidine chromophore derivative, incorporated through amber suppression using orthogonal aminoacyl tRNA synthetase/tRNA pairs, displays more dynamic photoswitching but greatly reduced photoconversion versus the least-evolved ancestor (LEA). Excitation-dependent measurements of the green anionic chromophore reveal that the varying photoswitching efficiencies arise from both the initial transient dynamics of the bright cis state and the final trans-like photoswitched off state, with an exocyclic bridge H-rocking motion playing an active role during the excited-state energy dissipation. This investigation establishes a close-knit feedback loop between spectroscopic characterization and protein engineering, which may be especially beneficial to develop more versatile FPs with targeted mutations and enhanced functionalities, such as photoconvertible FPs that also feature photoswitching properties.
Green Fluorescent Protein (GFP) was originally found in cnidarians, and later in copepods and cephalochordates (amphioxus) (Branchiostoma spp). Here, we looked for GFP-encoding genes in Asymmetron, an early-diverged cephalochordate lineage, and found two such genes closely related to some of the Branchiostoma GFPs. Dim fluorescence was found throughout the body in adults of Asymmetron lucayanum, and, as in Branchiostoma floridae, was especially intense in the ripe ovaries. Spectra of the fluorescence were similar between Asymmetron and Branchiostoma. Lineage-specific expansion of GFP-encoding genes in the genus Branchiostoma was observed, largely driven by tandem duplications. Despite such expansion, purifying selection has strongly shaped the evolution of GFP-encoding genes in cephalochordates, with apparent relaxation for highly duplicated clades. All cephalochordate GFP-encoding genes are quite different from those of copepods and cnidarians. Thus, the ancestral cephalochordates probably had GFP, but since GFP appears to be lacking in more early-diverged deuterostomes (echinoderms, hemichordates), it is uncertain whether the ancestral cephalochordates (i.e. the common ancestor of Asymmetron and Branchiostoma) acquired GFP by horizontal gene transfer (HGT) from copepods or cnidarians or inherited it from the common ancestor of copepods and deuterostomes, i.e. the ancestral bilaterians.
Forisomes are Ca(2+)-driven, ATP-independent contractile protein bodies that reversibly occlude sieve elements in faboid legumes. They apparently consist of at least three proteins; potential candidates have been described previously as 'FOR' proteins. We isolated three genes from Medicago truncatula that correspond to the putative forisome proteins and expressed their green fluorescent protein (GFP) fusion products in Vicia faba and Glycine max using the composite plant methodology. In both species, expression of any of the constructs resulted in homogenously fluorescent forisomes that formed sieve tube plugs upon stimulation; no GFP fluorescence occurred elsewhere. Isolated fluorescent forisomes reacted to Ca(2+) and chelators by contraction and expansion, respectively, and did not lose fluorescence in the process. Wild-type forisomes showed no affinity for free GFP in vitro. The three proteins shared numerous conserved motifs between themselves and with hypothetical proteins derived from the genomes of M. truncatula, Vitis vinifera and Arabidopsis thaliana. However, they showed neither significant similarities to proteins of known function nor canonical metal-binding motifs. We conclude that 'FOR'-like proteins are components of forisomes that are encoded by a well-defined gene family with relatives in taxa that lack forisomes. Since the mnemonic FOR is already registered and in use for unrelated genes, we suggest the acronym SEO (sieve element occlusion) for this family. The absence of binding sites for divalent cations suggests that the Ca(2+) binding responsible for forisome contraction is achieved either by as yet unidentified additional proteins, or by SEO proteins through a novel, uncharacterized mechanism.
We have developed a screening technology for the identification of short-lived proteins. A green fluorescent protein (GFP)-fusion cDNA library was generated for monitoring degradation kinetics. Cells expressing a subset of the GFP-cDNA expression library were screened to recover those in which the fluorescence signal diminished rapidly when protein synthesis was inhibited. Thirty clones that met the screening criteria were characterized individually. Twenty-three (73%) proved to have a half-life of 4 hours or less.
The cephalochordate Amphioxus naturally co-expresses fluorescent proteins (FPs) with different brightness, which thus offers the rare opportunity to identify FP molecular feature/s that are associated with greater/lower intensity of fluorescence. Here, we describe the spectral and structural characteristics of green FP (bfloGFPa1) with perfect (100%) quantum efficiency yielding to unprecedentedly-high brightness, and compare them to those of co-expressed bfloGFPc1 showing extremely-dim brightness due to low (0.1%) quantum efficiency. This direct comparison of structure-function relationship indicated that in the bright bfloGFPa1, a Tyrosine (Tyr159) promotes a ring flipping of a Tryptophan (Trp157) that in turn allows a cis-trans transformation of a Proline (Pro55). Consequently, the FP chromophore is pushed up, which comes with a slight tilt and increased stability. FPs are continuously engineered for improved biochemical and/or photonic properties, and this study provides new insight to the challenge of establishing a clear mechanistic understanding between chromophore structural environment and brightness level.
Sensitivity, dynamic and detection range as well as exclusion of expression and instrumental artifacts are critical for the quantitation of data obtained with fluorescent protein (FP)-based biosensors in vivo. Current biosensors designs are, in general, unable to simultaneously meet all these criteria. Here, we describe a generalizable platform to create dual-FP biosensors with large dynamic ranges by employing a single FP-cassette, named GO-(Green-Orange) Matryoshka. The cassette nests a stable reference FP (large Stokes shift LSSmOrange) within a reporter FP (circularly permuted green FP). GO- Matryoshka yields green and orange fluorescence upon blue excitation. As proof of concept, we converted existing, single-emission biosensors into a series of ratiometric calcium sensors (MatryoshCaMP6s) and ammonium transport activity sensors (AmTryoshka1;3). We additionally identified the internal acid-base equilibrium as a key determinant of the GCaMP dynamic range. Matryoshka technology promises flexibility in the design of a wide spectrum of ratiometric biosensors and expanded in vivo applications.Single fluorescent protein biosensors are susceptible to expression and instrumental artifacts. Here Ast et al. describe a dual fluorescent protein design whereby a reference fluorescent protein is nested within a reporter fluorescent protein to control for such artifacts while preserving sensitivity and dynamic range.
A simple, cell-based, membrane fusion assay system that uses split green fluorescent proteins (spGFPs) as an indicator was developed. The attachment of the pleckstrin homology (PH) domain to the N-termini of each spGFP not only localized the reporter signal to the plasma membrane but also helped the stable expression of the smaller spGFP of seventeen amino acid residues. It was shown that this system allowed real-time monitoring of membrane fusion by HIV-1 envelope protein (Env) without the addition of external substrates. This method can be adapted to the analyses of other viral membrane fusion.
There is a direct correlation between protein levels and disease states in human serum, which makes it an attractive target for sensors and diagnostics. However, this is challenging because serum features more than 20,000 proteins, with an overall protein content greater than 1 mM. Here we report a sensor based on a hybrid synthetic-biomolecule that uses arrays of green fluorescent protein and nanoparticles to detect proteins at biorelevant concentrations in both buffer and human serum. Distinct and reproducible fluorescence-response patterns were obtained from five serum proteins (human serum albumin, immunoglobulin G, transferrin, fibrinogen and a-antitrypsin), both in buffer and when spiked into human serum. Using linear discriminant analysis we identified these proteins with an identification accuracy of 100% in buffer and 97% in human serum. The arrays were also able to discriminate between different concentrations of the same protein, as well as a mixture of different proteins in human serum.
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