Development of Near-Infrared Nucleic Acid Mimics of Fluorescent Proteins for In Vivo Imaging of Viral RNA with Turn-On Fluorescence.

GFP-like fluorescent proteins and their molecular mimics have revolutionized bioimaging research, but their emissions are largely limited in the visible to far-red region, hampering the in vivo applications in intact animals. Herein, we structurally modulate GFP-like chromophores using a donor-acceptor-acceptor (D-A-A') molecular configuration to discover a set of novel fluorogenic derivatives with infrared-shifted spectra. These chromophores can be fluorescently elicited by their specific interaction with G-quadruplex (G4), a unique noncanonical nucleic acid secondary structure, via inhibition of the chromophores' twisted-intramolecular charge transfer. This feature allows us to create, for the first time, FP mimics with tunable emission in the near-infrared (NIR) region (Emmax = 664-705 nm), namely, infrared G-quadruplex mimics of FPs (igMFP). Compared with their FP counterparts, igMFPs exhibit remarkably higher quantum yields, larger Stokes shift, and better photostability. In a proof-of-concept application using pathogen-related G4s as the target, we exploited igMFPs to directly visualize native hepatitis C virus (HCV) RNA genome in living cells via their in situ formation by the chromophore-bound viral G4 structure in the HCV core gene. Furthermore, igMFPs are capable of high contrast HCV RNA imaging in living mice bearing a HCV RNA-presenting mini-organ, providing the first application of FP mimics in whole-animal imaging.

Antibody-Independent Quantification of Cytomegalovirus Virion Protein Incorporation Using HiBiT.

Human cytomegalovirus (HCMV) is a large double-stranded DNA virus and member of the ß-herpesvirus family. HCMV is ubiquitous in the human population and causes lifelong infections. HCMV infection is associated with high morbidity and mortality in immunocompromised individuals and the virus is a major cause of virus-mediated congenital disease. There have been a number of HCMV entry receptors identified that use one of two viral receptor binding complexes, including the gH/gL/gO complex and the pentamer made up of gH/gL/UL128/UL130/UL131a. Cytomegaloviruses (CMVs) are typically host-restricted requiring the use of species-specific modeling and culture conditions. We use rat CMV (RCMV) to study CMV-accelerated vascular disease and chronic allograft rejection. RCMV encodes homologous versions of the entry complex proteins but their incorporation and copy number per virion are still unknown. In this methods article, we describe a novel approach of HiBiT tagging viral proteins in order to detect and quantify protein incorporation into particles. This method is independent of protein-specific antibodies and can be standardized using a commercially available HiBiT protein standard. Using bacterial artificial chromosome (BAC) recombineering, we have constructed two individual viruses containing a HiBiT tag fused to the C'-terminus of either the UL128 homolog (R129) or the UL130 homolog (R131). Viruses containing these mutations were rescued, purified and analyzed. Our data demonstrate that R129 and R131 are both incorporated into RCMV virions at equimolar ratios relative to genome copy number, supporting this antibody-free approach for quantifying viral protein incorporation and its application toward the identification of domains required for incorporation.

Design, synthesis and characterization of a fluorescently labeled functional analog of full-length human ghrelin.

Human ghrelin receptor (GHSR) is a recognized prospective target in the diagnosis and therapy of multiple cancer types. To gain a better understanding of this receptor signaling system, we have synthesized a novel full-length ghrelin analog that is fluorescently labeled at the side-chain of a C-terminal cysteine extension. This analog exhibited nanomolar affinity and potency for the ghrelin receptor. It shows comparable efficacy with that of endogenous ghrelin. The fluorescently-labeled ghrelin analog is a valuable tool for in vitro imaging of cell lines that express ghrelin receptor.

Bioluminescent Properties of Semi-Synthetic Obelin and Aequorin Activated by Coelenterazine Analogues with Modifications of C-2, C-6, and C-8 Substituents.

Ca2+-regulated photoproteins responsible for bioluminescence of a variety of marine organisms are single-chain globular proteins within the inner cavity of which the oxygenated coelenterazine, 2-hydroperoxycoelenterazine, is tightly bound. Alongside with native coelenterazine, photoproteins can also use its synthetic analogues as substrates to produce flash-type bioluminescence. However, information on the effect of modifications of various groups of coelenterazine and amino acid environment of the protein active site on the bioluminescent properties of the corresponding semi-synthetic photoproteins is fragmentary and often controversial. In this paper, we investigated the specific bioluminescence activity, light emission spectra, stopped-flow kinetics and sensitivity to calcium of the semi-synthetic aequorins and obelins activated by novel coelenterazine analogues and the recently reported coelenterazine derivatives. Several semi-synthetic photoproteins activated by the studied coelenterazine analogues displayed sufficient bioluminescence activities accompanied by various changes in the spectral and kinetic properties as well as in calcium sensitivity. The poor activity of certain semi-synthetic photoproteins might be attributed to instability of some coelenterazine analogues in solution and low efficiency of 2-hydroperoxy adduct formation. In most cases, semi-synthetic obelins and aequorins displayed different properties upon being activated by the same coelenterazine analogue. The results indicated that the OH-group at the C-6 phenyl ring of coelenterazine is important for the photoprotein bioluminescence and that the hydrogen-bond network around the substituent in position 6 of the imidazopyrazinone core could be the reason of different bioluminescence activities of aequorin and obelin with certain coelenterazine analogues.

Impact of external amino acids on fluorescent protein chromophore biosynthesis revealed by molecular dynamics and mutagenesis studies.

The precise positioning of catalytic amino acids against the substrate in an enzyme active site is a crucial factor in biocatalysis. Biosynthesis of the chromophores of fluorescent proteins (FPs) is an autocatalytic process that must conform to these requirements. Here, we show that, in addition to the internal amino acid residues in the proximity of the chromophore, chromophore biosynthesis is influenced by the remote amino acids exposed on the outer surface of the ß-barrel structure of the FP. It has been shown earlier that chromophore biosynthesis of the red FP from Zoanthus sp. (zoan2RFP) proceeds via an immature green state. At the same time, the green state is the final stage of chromophore biosynthesis of green FP (zoanGFP), which is highly homologous to zoan2RFP. It was also shown that a single N66D substitution in the chromophore-forming sequence of zoanGFP might trigger the synthesis of the red chromophore. However, in this case, the synthesis of the red chromophore is incomplete and occurs only at elevated temperatures. Here, we tried to uncover additional structural determinants that govern the biosynthesis of the red chromophore. A comparison of zoanGFP and zoan2RFP revealed intrabarrel amino acid differences at five positions. Exhaustive substitutions of these five positions in zoanGFP-N66D gave rise to zoanGFPmut with the same intrabarrel amino acid composition as zoan2RFP. zoanGFPmut showed only partial green-to-red chromophore transformation at elevated temperatures. To elucidate the extra factors that can affect red chromophore biosynthesis, we performed comparative molecular dynamics simulations of zoan2RFP and zoanGFPmut. The simulations revealed several external amino acids that might influence the arrangement and flexibility of the chromophore-surrounding amino acid residues in these proteins. Mutagenesis experiments confirmed the crucial role of these residues in red chromophore biosynthesis. The obtained zoanGFPmut2 exhibited complete green-to-red transformation, suggesting that the mutated amino acids exposed on the surface of the ß-barrel contribute to red chromophore biosynthesis.

Atomistic Insights into Photoprotein Formation: Computational Prediction of the Properties of Coelenterazine and Oxygen Binding in Obelin.

Bioluminescence in marine systems is dominated by the use of coelenterazine for light production. The bioluminescent reaction of coelenterazine is an enzyme catalyzed oxidative decarboxylation: coelenterazine reacts with molecular oxygen to form carbon dioxide, coelenteramide, and light. One such class is the Ca2+ -regulated photoproteins. These proteins bind coelenterazine and oxygen, and trap 2-hydroperoxycoelenterazine, an intermediate along the reaction pathway. The reaction is halted until Ca2+ binding triggers the completion of the reaction. There are currently no reported experimental, atomistic descriptions of this ternary Michaelis complex. This study utilized computational techniques to develop an atomistic model of the Michaelis complex. Extensive molecular dynamics simulations were carried out to study the interactions between four tautomeric/protonation states of coelenterazine and wide-type and mutant obelin. Only minor differences in binding modes were observed across all systems. Interestingly, no basic residues were identified in the vicinity of the N7-nitrogen of coelenterazine. This observation was surprising considering that deprotonation at this position is a key mechanistic step in the proposed bioluminescent reaction. This work suggests that coelenterazine binds either as the O10H tautomer, or in the deprotonated form. Implicit ligand sampling simulations were used to identify potential O2 binding and migration pathways within obelin. A key oxygen binding site was identified close to the coelenterazine imidazopyrazinone core. The O2 binding free energy was observed to be dependent on the protonation state of coelenterazine. Taken together, the description of the obelin-coelenterazine-O2 complexes established in this study provides the basis for future computational studies of the bioluminescent mechanism. © 2019 Wiley Periodicals, Inc.

Fluorescent protein nanoparticles: Synthesis and recognition of cellular oxidation damage.

Intracellular reactive oxygen species (ROS) generation are associated with many diseases. Lots of studies focus on the detection of intracellular ROS by small fluorescent molecules. However, ROS recognized by biocompatible nanoparticles are relatively less reported. It is widely known that albumin-based nanomaterials possess unique advantages in biomedical applications because they are biodegradable and biocompatible. Herein, fluorescent protein nanoparticles (PNPs) were prepared using BSA as a starting material without introducing extra fluorescent molecules. The blue fluorescent PNPs were well characterized by FL, FTIR, CD, TEM, DLS, etc. It was revealed that the PNPs exhibited two types of emissive centers through FL spectra and the fluorescence lifetimes. Further mechanism study indicated that the fluorescence of the PNPs was mainly derived from three kinds of aromatic amino acids, namely tryptophan, tyrosine and phenylalanine. Moreover, the fluorescence properties of the PNPs were tightly related to pH. The PNPs displayed excellent stabilities under harsh conditions as well as physiological conditions. In addition, the PNPs (200 µg/mL) were nontoxic to HeLa and GES-1 cell lines, showing good biocompatibility. The cellular uptake of PNPs was occurred only when the cells were stressed with glucose oxidase or H2O2, thereafter the bright blue fluorescence was observed, indicating that it could be utilized for the recognition of cellular oxidation damage. These findings will offer novel clues for the future synthesis of even brighter protein nanoparticles and their biomedical applications.

A genetically encoded photosensitizer protein facilitates the rational design of a miniature photocatalytic CO2-reducing enzyme.

Photosensitizers, which harness light energy to upgrade weak reductants to strong reductants, are pivotal components of the natural and artificial photosynthesis machineries. However, it has proved difficult to enhance and expand their functions through genetic engineering. Here we report a genetically encoded, 27 kDa photosensitizer protein (PSP), which facilitates the rational design of miniature photocatalytic CO2-reducing enzymes. Visible light drives PSP efficiently into a long-lived triplet excited state (PSP*), which reacts rapidly with reduced nicotinamide adenine dinucleotide to generate a super-reducing radical (PSP•), which is strong enough to reduce many CO2-reducing catalysts. We determined the three-dimensional structure of PSP• at 1.8 Šresolution by X-ray crystallography. Genetic engineering enabled the site-specific attachment of a nickel-terpyridine complex and the modular optimization of the photochemical properties of PSP, the chromophore/catalytic centre distance and the catalytic centre microenvironment, which culminated in a miniature photocatalytic CO2-reducing enzyme that has a CO2/CO conversion quantum efficiency of 2.6%.

Rational designing of 8-hydroxyquinolin-imidazolinone-based fluorescent protein mutants with dramatically red shifted emission: A computational study.

Engineering fluorescent proteins to be the customized in vivo labels for monitoring cellular dynamic events is critical in biochemical and biomedical studies. The design and development of novel red fluorescent proteins is one of the most important fronts in this field due to their potential of imaging the entire organism. A recent fluorescent protein mutant eqFP650-67-HqAla with the 8-hydroxyquinolin-imidazolinone (HQI) chromophore has the plausible bathochromic shift of ~30 nm in its emission spectrum wavelength comparing to the parent fluorescent protein eqFP650. However, molecular mechanism of this significant shift remains somewhat obscure. In this study, we carefully benchmarked our computational methods and performed extensive calculations to investigate various structural components' effect on the chromophore's emission energy and decipher the molecular origin of the spectral shift. The influences of conjugation size, substituent group, substituent site as well as the number of substituents have been examined by elaborately designed chromophore derivatives. Accordingly, we proposed several chromophore mutants with dramatic bathochromic shift of up to ~60 nm in their emission spectra. We further evaluated their structural stability in the protein using molecular dynamics simulations. Present theoretical study connects the structural feature of chromophore derivatives in red fluorescent proteins with their splendid performances in shifting the emission frequency and offer the molecular insight. The computational protocol and successive examination procedure to extract the structural effect utilized herein can also be widely applied to other fluorescent proteins in general. © 2018 Wiley Periodicals, Inc.

Synthetic and genetically encoded fluorescent neural activity indicators.

The ultimate goal of neuroscience is to relate the complex activity of cells and cell-networks to behavior and cognition. This requires tools and techniques to visualize neuronal activity. Fluorescence microscopy is an ideal tool to measure activity of cells in the brain due to the high sensitivity of the technique and the growing portfolio of optical hardware and fluorescent sensors. Here, we give a chemist's perspective on the recent progress of fluorescent activity indicators that enable the measurement of cellular events in the living brain. We discuss advances in both chemical and genetically encoded sensors and look forward to hybrid indicators, which incorporate synthetic organic dyes into genetically encoded protein constructs.

Chemoselective Dual Labeling of Native and Recombinant Proteins.

The attachment of two different functionalities in a site-selective fashion represents a great challenge in protein chemistry. We report site specific dual functionalizations of peptides and proteins capitalizing on reactivity differences of cysteines in their free (thiol) and protected, oxidized (disulfide) forms. The dual functionalization of interleukin 2 and EYFP proceeded with no loss of bioactivity in a stepwise fashion applying maleimide and disulfide rebridging allyl-sulfone groups. In order to ensure broader applicability of the functionalization strategy, a novel, short peptide sequence that introduces a disulfide bridge was designed and site-selective dual labeling in the presence of biogenic groups was successfully demonstrated.

A bright cyan-excitable orange fluorescent protein facilitates dual-emission microscopy and enhances bioluminescence imaging in vivo.

Orange-red fluorescent proteins (FPs) are widely used in biomedical research for multiplexed epifluorescence microscopy with GFP-based probes, but their different excitation requirements make multiplexing with new advanced microscopy methods difficult. Separately, orange-red FPs are useful for deep-tissue imaging in mammals owing to the relative tissue transmissibility of orange-red light, but their dependence on illumination limits their sensitivity as reporters in deep tissues. Here we describe CyOFP1, a bright, engineered, orange-red FP that is excitable by cyan light. We show that CyOFP1 enables single-excitation multiplexed imaging with GFP-based probes in single-photon and two-photon microscopy, including time-lapse imaging in light-sheet systems. CyOFP1 also serves as an efficient acceptor for resonance energy transfer from the highly catalytic blue-emitting luciferase NanoLuc. An optimized fusion of CyOFP1 and NanoLuc, called Antares, functions as a highly sensitive bioluminescent reporter in vivo, producing substantially brighter signals from deep tissues than firefly luciferase and other bioluminescent proteins.

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.

Semisynthetic photoprotein reporters for tracking fast Ca(2+) transients.

Changes in the intracellular concentration of free ionized calcium ([Ca(2+)]i) control a host of cellular processes as varied as vision, muscle contraction, neuronal signal transmission, proliferation, apoptosis etc. The disturbance in Ca(2+)-signaling causes many severe diseases. To understand the mechanisms underlying the control by calcium and how disorder of this regulation relates to pathological conditions, it is necessary to measure [Ca(2+)]i. The Ca(2+)-regulated photoproteins which are responsible for bioluminescence of marine coelenterates have been successfully used for this purpose over the years. Here we report the results on comparative characterization of bioluminescence properties of aequorin from Aequorea victoria, obelin from Obelia longissima, and clytin from Clytia gregaria charged by native coelenterazine and coelenterazine analogues f, i, and hcp. The comparison of specific bioluminescence activity, stability, emission spectra, stopped-flow kinetics, sensitivity to calcium, and effect of physiological concentrations of Mg(2+) establishes obelin-hcp as an excellent semisynthetic photoprotein to keep track of fast intracellular Ca(2+) transients. The rate of rise of its light signal on a sudden change of [Ca(2+)] is almost 3- and 11-fold higher than those of obelin and aequorin with native coelenterazine, respectively, and 20 times higher than that of the corresponding aequorin-hcp. In addition, obelin-hcp preserves a high specific bioluminescence activity and displays higher Ca(2+)-sensitivity as compared to obelin charged by native coelenterazine and sensitivity to Ca(2+) comparable with those of aequorin-f and aequorin-hcp.

A FRET-based ratiometric redox probe for detecting oxidative stress by confocal microscopy, FLIM and flow cytometry.

Understanding the role of oxidative stress in disease requires real time monitoring of redox status within a cell. We report a FRET-based, ratiometric redox probe which can be applied to monitor cellular oxidative capacity using three different modalities ­ confocal microscopy, fluorescence lifetime imaging and flow cytometry.

[Frontiers in Live Bone Imaging Researches. Novel fluorescent proteins for in vivo imaging].

Bone tissue is maintained by dynamic equilibrium of various types of cells. Thus, real time intravital imaging of movement and phenotypic changes of cells in the steady state and in pathophysiological state help us to understand molecular mechanism of bone tissue maintenance. Therefore, this review introduces Fucci-Tg mice for cell cycle imaging, caspase-3 indicator SCAT3.1 mice for cell death, and photoconvertible protein Kaede-Tg mice and cell-type specific KikGR mice for visualization of cell movement within intra-organ and between inter-organs.

Polymersomes prepared from thermoresponsive fluorescent protein-polymer bioconjugates: capture of and report on drug and protein payloads.

Polymersomes provide a good platform for targeted drug delivery and the creation of complex (bio)catalytically active systems for research in synthetic biology. To realize these applications requires both spatial control over the encapsulation components in these polymersomes and a means to report where the components are in the polymersomes. To address these twin challenges, we synthesized the protein-polymer bioconjugate PNIPAM-b-amilFP497 composed of thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) and a green-fluorescent protein variant (amilFP497). Above 37 °C, this bioconjugate forms polymersomes that can (co-)encapsulate the fluorescent drug doxorubicin and the fluorescent light-harvesting protein phycoerythrin 545 (PE545). Using fluorescence lifetime imaging microscopy and Förster resonance energy transfer (FLIM-FRET), we can distinguish the co-encapsulated PE545 protein inside the polymersome membrane while doxorubicin is found both in the polymersome core and membrane.

A low-toxic artificial fluorescent glycoprotein can serve as an efficient cytoplasmic labeling in living cell.

To maintain the virtue of good optical property and discard the dross of conventional fluorescent staining dyes, we provide a strategy for designing new fluorescent scaffolds. In this study, a novel fluorescent labeling glycoprotein (chitosan-poly-L-cysteine, CPC) was synthesized through graft copolymerization. CPC gives emission peak at 465-470 nm when excited at 386 nm. The submicro-scale CPC microspheres could be localized and persisted specifically in the cytoplasm of living cells, with strong blue fluorescence. Moreover, CPC was highly resistant to photo bleaching, the fluorescence was remained stable for up to 72 h as the cells grew and developed. The glycoprotein CPC was bio-compatible and in zero grade cytotoxicity as quantified by MTT assay. The fluorescent labeling process with our newly designed glycoprotein CPC is exceptionally efficient.

Single action potentials and subthreshold electrical events imaged in neurons with a fluorescent protein voltage probe.

Monitoring neuronal electrical activity using fluorescent protein-based voltage sensors has been limited by small response magnitudes and slow kinetics of existing probes. Here we report the development of a fluorescent protein voltage sensor, named ArcLight, and derivative probes that exhibit large changes in fluorescence intensity in response to voltage changes. ArcLight consists of the voltage-sensing domain of Ciona intestinalis voltage-sensitive phosphatase and super ecliptic pHluorin that carries the point mutation A227D. The fluorescence intensity of ArcLight A242 decreases by 35% in response to a 100 mV depolarization when measured in HEK293 cells, which is more than five times larger than the signals from previously reported fluorescent protein voltage sensors. We show that the combination of signal size and response speed of these new probes allows the reliable detection of single action potentials and excitatory potentials in individual neurons and dendrites.

Improving a designed photocontrolled DNA-binding protein.

Photocontrolled transcription factors could be powerful tools for probing the roles of transcriptional processes in a variety of settings. Previously, we designed a photocontrolled DNA-binding protein based on a fusion between the bZIP region of GCN4 and photoactive yellow protein from Halorhodospira halophila [Morgan, S. A., et al. (2010) J. Mol. Biol. 399, 94-112]. Here we report a structure-based attempt to improve the degree of photoswitching observed with this chimeric protein. Using computational design tools PoPMuSiC 2.0, Rosetta, Eris, and bCIPA, we identified a series of single- and multiple-point mutations that were expected to stabilize the folded dark state of the protein and thereby enhance the degree of photoswitching. While a number of these mutations, particularly those that introduced a hydrophobic residue at position 143, did significantly enhance dark-state protein stability as judged by urea denaturation studies, dark-state stability did not correlate directly with the degree of photoswitching. Instead, the influence of mutations on the degree of photoswitching was found to be related to their effects on the degree to which DNA binding slowed the pB to pG transition in the PYP photocycle. One mutant, K143F, caused an ∼10-fold slowing of the photocycle and also showed the largest difference in the apparent K(d) for DNA binding, 3.5-fold lower, upon irradiation. This change in the apparent K(d) causes a 12-fold enhancement in the fraction bound DNA upon irradiation due to the cooperativity of DNA binding by this family of proteins. The results highlight the strengths and weaknesses of current approaches to a practical problem in protein design and suggest strategies for further improvement of designed photocontrolled transcription factors.