Action spectroscopy of the isolated red Kaede fluorescent protein chromophore.

Incorporation of fluorescent proteins into biochemical systems has revolutionized the field of bioimaging. In a bottom-up approach, understanding the photophysics of fluorescent proteins requires detailed investigations of the light-absorbing chromophore, which can be achieved by studying the chromophore in isolation. This paper reports a photodissociation action spectroscopy study on the deprotonated anion of the red Kaede fluorescent protein chromophore, demonstrating that at least three isomers-assigned to deprotomers-are generated in the gas phase. Deprotomer-selected action spectra are recorded over the S1 ← S0 band using an instrument with differential mobility spectrometry coupled with photodissociation spectroscopy. The spectrum for the principal phenoxide deprotomer spans the 480-660 nm range with a maximum response at ≈610 nm. The imidazolate deprotomer has a blue-shifted action spectrum with a maximum response at ≈545 nm. The action spectra are consistent with excited state coupled-cluster calculations of excitation wavelengths for the deprotomers. A third gas-phase species with a distinct action spectrum is tentatively assigned to an imidazole tautomer of the principal phenoxide deprotomer. This study highlights the need for isomer-selective methods when studying the photophysics of biochromophores possessing several deprotonation sites.

LSSmScarlet, dCyRFP2s, dCyOFP2s and CRISPRed2s, Genetically Encoded Red Fluorescent Proteins with a Large Stokes Shift.

Genetically encoded red fluorescent proteins with a large Stokes shift (LSSRFPs) can be efficiently co-excited with common green FPs both under single- and two-photon microscopy, thus enabling dual-color imaging using a single laser. Recent progress in protein development resulted in a great variety of novel LSSRFPs; however, the selection of the right LSSRFP for a given application is hampered by the lack of a side-by-side comparison of the LSSRFPs' performance. In this study, we employed rational design and random mutagenesis to convert conventional bright RFP mScarlet into LSSRFP, called LSSmScarlet, characterized by excitation/emission maxima at 470/598 nm. In addition, we utilized the previously reported LSSRFPs mCyRFP1, CyOFP1, and mCRISPRed as templates for directed molecular evolution to develop their optimized versions, called dCyRFP2s, dCyOFP2s and CRISPRed2s. We performed a quantitative assessment of the developed LSSRFPs and their precursors in vitro on purified proteins and compared their brightness at 488 nm excitation in the mammalian cells. The monomeric LSSmScarlet protein was successfully utilized for the confocal imaging of the structural proteins in live mammalian cells and multicolor confocal imaging in conjugation with other FPs. LSSmScarlet was successfully applied for dual-color two-photon imaging in live mammalian cells. We also solved the X-ray structure of the LSSmScarlet protein at the resolution of 1.4 Å that revealed a hydrogen bond network supporting excited-state proton transfer (ESPT). Quantum mechanics/molecular mechanics molecular dynamic simulations confirmed the ESPT mechanism of a large Stokes shift. Structure-guided mutagenesis revealed the role of R198 residue in ESPT that allowed us to generate a variant with improved pH stability. Finally, we showed that LSSmScarlet protein is not appropriate for STED microscopy as a consequence of LSSRed-to-Red photoconversion with high-power 775 nm depletion light.

Generation of mWasabi fluorescent protein-binding nanobodies.

mWasabi is a bright monomeric green fluorescent protein. It can be used as a fusion tag to monitor various biological events, e.g. protein localization. Here we report the selection of camelid-derived single-domain antibody fragments (nanobodies) against mWasabi. In this work, phage-display approach was employed to select the high affinity mWasabi-specific Nb (nanobodies). These nanobodies were able to recognize mWasabi or in a fused fashion with PD1. The interesting binding characteristics of these two mWasabi-specific nanobodies could be valuable for design new tools for cellular tracing or targeting based on the mWasabi-fusing protein in many different biological research fields.

Tag-Specific Affinity Purification of Recombinant Proteins by Using Molecularly Imprinted Polymers.

Epitope tagging is widely used to fuse a known epitope to proteins for which no affinity receptor is available by using recombinant DNA technology. One example is FLAG epitope (DYKDDDDK), which provides better purity and recoveries than the favorite poly histidine tag. However, purification requires using anti-FLAG antibody resins, the high cost and nonreusability of which restrict widespread use. One cost-effective solution is provided by the use of bioinspired anti-FLAG molecularly imprinted polymers (MIPs). This work describes the development of MIPs, based on the epitope approach, synthesized from the tetrapeptide DYKD as template that affords purification of FLAG-derived recombinant proteins. Polymer was optimized by using a combinatorial approach to select the functional monomer(s) and cross-linker(s), resulting in the best specific affinity toward FLAG and the peptide DYKD. The imprinted resin obtained was used to purify mCherry proteins tagged with either FLAG or DYKD epitopes from crude cell lysates. Both mCherry variants were highly efficiently purified ( R ≥ 95%, RSD ≤ 15%, n = 3) and impurities were removed. Unlike existing antibody-based resins, the proposed tag-imprinting strategy provides a general method for meeting the growing demand for efficient, inexpensive, and versatile materials for tagged proteins purification.

A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein.

Far-red fluorescent proteins (FPs) are desirable for in vivo imaging because with these molecules less light is scattered, absorbed, or re-emitted by endogenous biomolecules compared with cyan, green, yellow, and orange FPs. We developed a new class of FP from an allophycocyanin α-subunit (APCα). Native APC requires a lyase to incorporate phycocyanobilin. The evolved FP, which we named small ultra-red FP (smURFP), covalently attaches a biliverdin (BV) chromophore without a lyase, and has 642/670-nm excitation-emission peaks, a large extinction coefficient (180,000 M(-1)cm(-1)) and quantum yield (18%), and photostability comparable to that of eGFP. smURFP has significantly greater BV incorporation rate and protein stability than the bacteriophytochrome (BPH) FPs. Moreover, BV supply is limited by membrane permeability, and smURFPs (but not BPH FPs) can incorporate a more membrane-permeant BV analog, making smURFP fluorescence comparable to that of FPs from jellyfish or coral. A far-red and near-infrared fluorescent cell cycle indicator was created with smURFP and a BPH FP.

The effects of photodynamic therapy on leukemia cells mediated by KillerRed, a genetically encoded fluorescent protein photosensitizer.

BACKGROUND: Leukemia is a cancer of blood and bone marrow cells, causing about 300,000 deaths worldwide. Photodynamic therapy (PDT) is a promising alternative for the treatment of malignant tumors. KillerRed is a genetically encoded red fluorescent protein photosensitizer (PS). In this study, we aimed to investigate the effects of KillerRed-mediated PDT on chronic myelogenous leukemia K562 cells, acute monocytic leukemia NB4 cells, and acute monocytic leukemia THP1 cells. METHODS: KillerRed was expressed in Escherichia coli cells, purified by Q-Sepharose column, and confirmed by western-blotting. The PDT effect on cell proliferation was evaluated by Cell Counting Kit-8 (CCK-8). Cell apoptosis was determined by PE Annexin V/7-AAD staining and flow cytometry. The distribution of KillerRed in leukemia cells was detected by confocal laser scanning microscopy (CLSM) and western-blotting. The ROS generation was measured by flow cytometry. RESULTS: Pure KillerRed was obtained with a yield of about 37 mg per liter of bacterial cells. KillerRed photodynamic inactivated the leukemia cells in a concentration-dependent manner, but exhibited no obvious dark toxicity. PDT mediated by KillerRed could also induce apoptotic response (mainly early apoptosis) in the three cell lines. The CLSM imaging indicated that KillerRed was distributed within the cytoplasm and nuclei of leukemia cells, causing damages to the cytoplasm and leaving the nuclear envelope intact during light irradiation. KillerRed distributed both in the cytosol and nuclei was confirmed by western blotting, and ROS significantly increased in PDT treated cells compared to the cells treated with KillerRed alone. CONCLUSIONS: Our studies demonstrated that KillerRed-mediated PDT could effectively inactivate K562, NB4, and THP1 leukemia cells and trigger cell apoptosis, and it has potential to be used individually or complementally, in the treatment of leukemia.

Near-Infrared Markers based on Bacterial Phytochromes with Phycocyanobilin as a Chromophore.

Biomarkers engineered on the basis of bacterial phytochromes with biliverdin IXα (BV) cofactor as a chromophore are increasingly used in cell biology and biomedicine, since their absorption and fluorescence spectra lie within the so-called optical "transparency window" of biological tissues. However, the quantum yield of BV fluorescence in these biomarkers does not exceed 0.145. The task of generating biomarkers with a higher fluorescence quantum yield remains relevant. To address the problem, we proposed the use of phycocyanobilin (PCB) as a chromophore of biomarkers derived from bacterial phytochromes. In this work, we characterized the complexes of iRFP713 evolved from RpBphP2 and its mutant variants with different location of cysteine residues capable of covalent tetrapyrrole attachment with the PCB cofactor. All analyzed proteins assembled with PCB were shown to have a higher fluorescence quantum yield than the proteins assembled with BV. The iRFP713/V256C and iRFP713/C15S/V256C assembled with PCB have a particularly high quantum yield of 0.5 and 0.45, which exceeds the quantum yield of all currently available near-infrared biomarkers. Moreover, PCB has 4 times greater affinity for iRFP713/V256C and iRFP713/C15S/V256C proteins compared to BV. These data establish iRFP713/V256C and iRFP713/C15S/V256C assembled with the PCB chromophore as promising biomarkers for application in vivo. The analysis of the spectral properties of the tested biomarkers allowed for suggesting that the high-fluorescence quantum yield of the PCB chromophore can be attributed to the lower mobility of the D-ring of PCB compared to BV.

Ten-Minute Protein Purification and Surface Tethering for Continuous-Flow Biocatalysis.

Nature applies enzymatic assembly lines to synthesize bioactive compounds. Inspired by such capabilities, we have developed a facile method for spatially segregating attached enzymes in a continuous-flow, vortex fluidic device (VFD). Fused Hisn -tags at the protein termini allow rapid bioconjugation and consequent purification through complexation with immobilized metal affinity chromatography (IMAC) resin. Six proteins were purified from complex cell lysates to average homogeneities of 76 %. The most challenging to purify, tobacco epi-aristolochene synthase, was purified in only ten minutes from cell lysate to near homogeneity (>90 %). Furthermore, this "reaction-ready" system demonstrated excellent stability during five days of continuous-flow processing. Towards multi-step transformations in continuous flow, proteins were arrayed as ordered zones on the reactor surface allowing segregation of catalysts. Ordering enzymes into zones opens up new opportunities for continuous-flow biosynthesis.

Detection of Antibodies in Blood Plasma Using Bioluminescent Sensor Proteins and a Smartphone.

Antibody detection is of fundamental importance in many diagnostic and bioanalytical assays, yet current detection techniques tend to be laborious and/or expensive. We present a new sensor platform (LUMABS) based on bioluminescence resonance energy transfer (BRET) that allows detection of antibodies directly in solution using a smartphone as the sole piece of equipment. LUMABS are single-protein sensors that consist of the blue-light emitting luciferase NanoLuc connected via a semiflexible linker to the green fluorescent acceptor protein mNeonGreen, which are kept close together using helper domains. Binding of an antibody to epitope sequences flanking the linker disrupts the interaction between the helper domains, resulting in a large decrease in BRET efficiency. The resulting change in color of the emitted light from green-blue to blue can be detected directly in blood plasma, even at picomolar concentrations of antibody. Moreover, the modular architecture of LUMABS allows changing of target specificity by simple exchange of epitope sequences, as demonstrated here for antibodies against HIV1-p17, hemagglutinin (HA), and dengue virus type I. The combination of sensitive ratiometric bioluminescent detection and the intrinsic modularity of the LUMABS design provides an attractive generic platform for point-of-care antibody detection that avoids the complex liquid handling steps associated with conventional immunoassays.

Development and investigation of recombinant immunotoxin protein 4D5scFv-mCherry-PE(40).

Development of agents for theranostics implies combining the targeting module, the effector module, and the detection module within the same complex or recombinant protein. We have constructed, isolated, and characterized the 4D5scFv-mCherry-PE(40) protein, which exhibits fluorescent properties and specifically binds to cancer cells expressing the HER2 receptor and reduces their viability. The ability of the obtained targeted antitumor agent 4D5scFv-mCherry-PE(40) to selectively stain the HER2-positive cells and its highly selective cytotoxicity against these cells make the obtained targeted recombinant protein 4D5scFv-mCherry-PE(40) a promising theranostic agent for the diagnostics and therapy of HER2-positive human tumors.

Efficient sample preparation from complex biological samples using a sliding lid for immobilized droplet extractions.

Sample preparation is a major bottleneck in many biological processes. Paramagnetic particles (PMPs) are a ubiquitous method for isolating analytes of interest from biological samples and are used for their ability to thoroughly sample a solution and be easily collected with a magnet. There are three main methods by which PMPs are used for sample preparation: (1) removal of fluid from the analyte-bound PMPs, (2) removal of analyte-bound PMPs from the solution, and (3) removal of the substrate (with immobilized analyte-bound PMPs). In this paper, we explore the third and least studied method for PMP-based sample preparation using a platform termed Sliding Lid for Immobilized Droplet Extractions (SLIDE). SLIDE leverages principles of surface tension and patterned hydrophobicity to create a simple-to-operate platform for sample isolation (cells, DNA, RNA, protein) and preparation (cell staining) without the need for time-intensive wash steps, use of immiscible fluids, or precise pinning geometries. Compared to other standard isolation protocols using PMPs, SLIDE is able to perform rapid sample preparation with low (0.6%) carryover of contaminants from the original sample. The natural recirculation occurring within the pinned droplets of SLIDE make possible the performance of multistep cell staining protocols within the SLIDE by simply resting the lid over the various sample droplets. SLIDE demonstrates a simple easy to use platform for sample preparation on a range of complex biological samples.

A cleavable silica-binding affinity tag for rapid and inexpensive protein purification.

We describe a new affinity purification tag called Car9 that confers proteins to which it is fused micromolar affinity for unmodified silica. When appended to the C-terminus of GFPmut2 through a flexible linker, Car9 promotes efficient adsorption to silica gel and the fusion protein can be released from the particles by incubation with L-lysine. Using a silica gel column and the lysine elution approach in fast protein liquid chromatography (FPLC) mode, Car9-tagged versions of GFPmut2, mCherry and maltose binding protein (MBP) can be recovered from clarified lysates with a purity of 80-90%. Capitalizing on silica's ability to handle large pressure drops, we further show that it is possible to go from cell lysates to purified protein in less than 15 min using a fully disposable device. Finally, we demonstrate that the linker-Car9 region is susceptible to proteolysis by E. coli OmpT and take advantage of this observation to excise the C-terminal extension of GFPmut2-Car9 by incubating purified fusion protein with cells that overproduce the outer membrane protease OmpT. The set of strategies described herein, should reduce the cost of affinity purification by at least 10-fold, cut down purification times to minutes, and allow for the production of proteins with native (or nearly native) termini from their C-terminally-tagged versions.

Predicting the loading of virus-like particles with fluorescent proteins.

The virus-like particle (VLP) of the Cowpea Chlorotic Mottle Virus (CCMV) has often been used to encapsulate foreign cargo. Here we show two different rational design approaches, covalent and noncovalent, for loading teal fluorescent proteins (TFP) into the VLP. The covalent loading approach allows us to gain control over capsid loading on a molecular level. The achieved loading control is used to accurately predict the loading of cargo into CCMV VLP. The effects of molecular confinement were compared for the differently loaded VLPs created with the covalent method. We see that the loading of more than 10 fluorescent proteins in the 18 nm internal cavity of the CCMV capsid gives rise to a maximum efficiency of homo-FRET between the loaded proteins, as measured by fluorescence anisotropy. This shows that already at low levels of VLP loading molecular crowding starts to play a role.

Improving the second-order nonlinear optical response of fluorescent proteins: the symmetry argument.

We have successfully designed and expressed a new fluorescent protein with improved second-order nonlinear optical properties. It is the first time that a fluorescent protein has been rationally altered for this particular characteristic. On the basis of the specific noncentrosymmetry requirements for second-order nonlinear optical effects, we had hypothesized that the surprisingly low first hyperpolarizability (ß) of the enhanced yellow fluorescent protein (eYFP) could be explained by centrosymmetric stacking of the chromophoric Tyr66 and the neighboring Tyr203 residue. The inversion center was removed by mutating Tyr203 into Phe203, with minor changes in the linear optical properties and even an improved fluorescence quantum yield. Structure determination by X-ray crystallography as well as linear optical characterization corroborate a correct folding and maturation. Measurement of ß by means of hyper-Rayleigh scattering (HRS) as well as their analysis using quantum chemistry calculations validate our hypothesis. This observation can eventually lead to improved red fluorescent proteins for even better performance. On the basis of the specific function (second-harmonic generation), the color of its emission, and in analogy with the "fruit" names, we propose SHardonnay as the name for this Tyr203Phe mutant of eYFP.

Optically modulatable blue fluorescent proteins.

Blue fluorescent proteins (BFPs) offer visualization of protein location and behavior, but often suffer from high autofluorescent background and poor signal discrimination. Through dual-laser excitation of bright and photoinduced dark states, mutations to the residues surrounding the BFP chromophore enable long-wavelength optical modulation of BFP emission. Such dark state engineering enables violet-excited blue emission to be increased upon lower energy, green coillumination. Turning this green coillumination on and off at a specific frequency dynamically modulates collected blue fluorescence without generating additional background. Interpreted as transient photoconversion between neutral cis and anionic trans chromophoric forms, mutations tune photoisomerization and ground state tautomerizations to enable long-wavelength depopulation of the millisecond-lived, spectrally shifted dark states. Single mutations to the tyrosine-based blue fluorescent protein T203V/S205V exhibit enhanced modulation depth and varied frequency. Importantly, analogous single point mutations in the nonmodulatable BFP, mKalama1, creates a modulatable variant. Building modulatable BFPs offers opportunities for improved BFP signal discrimination vs background, greatly enhancing their utility.

Purification of the photosynthetic pigment C-phycocyanin from heterotrophic Galdieria sulphuraria.

BACKGROUND: The phycobiliprotein C-phycocyanin (C-PC) is used in cosmetics, diagnostics and foods and also as a nutraceutical or biopharmaceutical. It is produced in the cyanobacterium Arthrospira platensis grown phototrophically in open cultures. C-PC may alternatively be produced heterotrophically in the unicellular rhodophyte Galdieria sulphuraria at higher productivities and under improved hygienic standards if it can be purified as efficiently as C-PC from A. platensis. RESULTS: Ammonium sulfate fractionation, aqueous two-phase extraction, tangential flow ultrafiltration and anion exchange chromatography were evaluated with respect to the purification of C-PC from G. sulphuraria extracts. Galdieria sulphuraria C-PC showed similar properties to those described for cyanobacterial C-PC with respect to separation by all methodologies. The presence of micelles in G. sulphuraria extracts influenced the different procedures. Only chromatography was able to separate C-PC from a second phycobiliprotein, allophycocyanin. CONCLUSION: C-PC from heterotrophic G. sulphuraria shows similar properties to cyanobacterial C-PC and can be purified to the same standards, despite initial C-PC concentrations being low and impurity concentrations high in G. sulphuraria extracts.

Solubilization and folding of a fully active recombinant Gaussia luciferase with native disulfide bonds by using a SEP-Tag.

Gaussia luciferase (GLuc) is the smallest known bioluminescent protein and is attracting much attention as a potential reporter protein. However, its 10 disulfide bond forming cysteines have hampered the efficient production of recombinant GLuc and thus limited its use in bio-imaging application. Here, we demonstrate that the addition of a short solubility enhancement peptide tag (SEP-Tag) to the C-terminus of GLuc (GLuc-C9D) significantly increased the fraction of soluble protein at a standard expression temperature. The expression time was much shorter, and the final yield of GLuc-C9D was significantly higher than with our previous pCold expression system. Reversed phase HPLC indicated that the GLuc-C9D variant folded with a single disulfide bond pattern after proper oxidization. Further, the thermal denaturation of GLuc-C9D was completely reversible, and its secondary structure content remained unchanged until 40°C as assessed by CD spectroscopy. The (1)H-NMR spectrum of GLuc indicated sharp well dispersed peaks typical for natively folded proteins. GLuc-C9D bioluminescence activity was strong and fully retained even after incubation at high temperatures. These results suggest that solubilization using SEP-Tags can be useful for producing large quantities of proteins containing multiple disulfide bonds.

A FRET-facilitated photoswitching using an orange fluorescent protein with the fast photoconversion kinetics.

Fluorescent proteins photoswitchable with noncytotoxic light irradiation and spectrally distinct from multiple available photoconvertible green-to-red probes are in high demand. We have developed a monomeric fluorescent protein, called PSmOrange2, which is photoswitchable with blue light from an orange (ex./em. at 546 nm/561 nm) to a far-red (ex./em. at 619 nm/651 nm) form. Compared to another orange-to-far-red photoconvertable variant, PSmOrange2 has blue-shifted photoswitching action spectrum, 9-fold higher photoconversion contrast, and up to 10-fold faster photoswitching kinetics. This results in the 4-fold more PSmOrange2 molecules being photoconverted in mammalian cells. Compared to common orange fluorescent proteins, such as mOrange, the orange form of PSmOrange has substantially higher photostability allowing its use in multicolor imaging applications to track dynamics of multiple populations of intracellular objects. The PSmOrange2 photochemical properties allow its efficient photoswitching with common two-photon lasers and, moreover, via Förster resonance energy transfer (FRET) from green fluorescent donors. We have termed the latter effect a FRET-facilitated photoswitching and demonstrated it using several sets of interacting proteins. The enhanced photoswitching properties of PSmOrange2 make it a superior photoconvertable protein tag for flow cytometry, conventional microscopy, and two-photon imaging of live cells.

Screening of a highly soluble and oxygen-independent blue fluorescent protein from metagenome.

GFP and its derivatives are commonly used as non-invasive in vivo reporters. These fluorescent proteins have been employed to analyze expression level, localization and movement of proteins, as well as protein interaction. However, they cannot be utilized under anaerobic conditions due to the oxygen requirement for the maturation of the fluorophore. Thus, other proteins with a different mechanism of fluorescence emission are needed. We reported here a blue fluorescent protein, named mBFP, that was derived from metagenomic DNA. This protein consisting of 248 amino acids was overexpressed (>35% of the total protein) in a soluble form in Escherichia coli. mBFP showed a distinct fluorescence pattern that was NADPH-dependent and could be used to image live cells under anaerobic conditions. Thus, mBFP holds great promise for use as a reporter in a broad range of applications.

Expression, purification and some properties of fluorescent chimeras of human small heat shock proteins.

Small heat shock proteins (sHsp) are ubiquitously expressed in all human tissues and have an important housekeeping role in preventing the accumulation of aggregates of improperly folded or denatured proteins. They also participate in the regulation of the cytoskeleton, proliferation, apoptosis and many other vital processes. Fluorescent chimeras composed of sHsp and enhanced fluorescent proteins have been used to determine the intracellular locations of small heat shock proteins and to analyse the hetero-oligomeric complexes formed by different sHsp. However, the biochemical properties and chaperone-like activities of these chimeras have not been investigated. To determine the properties of these chimeras, we fused enhanced yellow and cyan fluorescent proteins (EYFP and ECFP) to the N-termini of four ubiquitously expressed human small heat shock proteins: HspB1, HspB5, HspB6, and HspB8. The eight fluorescent chimeras of small heat shock proteins and isolated fluorescent proteins were expressed in Escherichia coli. The chimeric proteins were isolated and purified via ammonium sulphate fractionation, ion exchange and size-exclusion chromatography. This method provided 20-100 mg of fluorescent chimeras from 1L of bacterial culture. The spectral properties of the chimeras were similar to those of the isolated fluorescent proteins. The fusion of fluorescent proteins to HspB6 and HspB8, which typically form dimers, did not affect their quaternary structures. Oligomers of the fluorescent chimeras of HspB1 and HspB5 were less stable and contained fewer subunits than oligomers formed by the wild-type proteins. Fusion with EYFP decreased the chaperone-like activity of HspB5 and HspB6 whereas fusion with ECFP increased chaperone-like activity. All fluorescent chimeras of HspB1 and HspB8 had higher chaperone-like activity than the wild-type proteins. Thus, although fluorescent chimeras are useful for many purposes, the fluorescent proteins used to form these chimeras may affect certain important properties of sHsp.