Red-shifted Variant Research Articles

Destabilized near-infrared fluorescent nanobodies enable background-free targeting of GFP-based biosensors for imaging and manipulation

Near-infrared (NIR) probes are highly sought after as fluorescent tags for multicolor cellular and in vivo imaging. Here we develop small NIR fluorescent nanobodies, termed NIR-FbLAG16 and NIR-FbLAG30, enabling background-free visualization of various GFP-derived probes and biosensors. We also design a red-shifted variant, NIR-Fb(718), to simultaneously target several antigens within the NIR spectral range. Leveraging the antigen-stabilizing property of the developed NIR-Fbs, we then create two modular systems for precise control of gene expression in GFP-labeled cells. Applying the NIR-Fbs in vivo, we target cells expressing GFP and the calcium biosensor GCaMP6 in the somatosensory cortex of transgenic mice. Simultaneously tracking calcium activity and the reference signal from NIR-FbLAGs bound to GCaMP6 enables ratiometric deep-brain in vivo imaging. Altogether, NIR-FbLAGs present a promising approach for imaging and manipulating various processes in live cells and behaving animals expressing GFP-based probes.

Rational Design of Far-Red Archaerhodopsin-3-Based Fluorescent Genetically Encoded Voltage Indicators: from Elucidation of the Fluorescence Mechanism in Archers to Novel Red-Shifted Variants.

Genetically encoded voltage indicators (GEVIs) have found wide applications as molecular tools for visualization of changes in cell membrane potential. Among others, several classes of archaerhodopsin-3-based GEVIs have been developed and have proved themselves promising in various molecular imaging studies. To expand the application range for this type of GEVIs, new variants with absorption band maxima shifted toward the first biological window and enhanced fluorescence signal are required. Here, we integrate computational and experimental strategies to reveal structural factors that distinguish far-red bright archaerhodopsin-3-based GEVIs, Archers, obtained by directed evolution in a previous study (McIsaac et al., PNAS, 2014) and the wild-type archaerhodopsin-3 with an extremely dim fluorescence signal, aiming to use the obtained information in subsequent rational design. We found that the fluorescence can be enhanced by stabilization of a certain conformation of the protein, which, in turn, can be achieved by tuning the pK a value of two titratable residues. These findings were supported further by introducing mutations into wild-type archeorhodopsin-3 and detecting the enhancement of the fluorescence signal. Finally, we came up with a rational design and proposed previously unknown Archers variants with red-shifted absorption bands (λmax up to 640 nm) and potential-dependent bright fluorescence (quantum yield up to 0.97%).

Engineered red Opto-mGluR6 Opsins, a red-shifted optogenetic excitation tool, an in vitro study.

Degenerative eye diseases cause partial or complete blindness due to photoreceptor degeneration. Optogenetic gene therapy is a revolutionary technique combining genetics and optical methods to control the function of neurons. Due to the inherent risk of photochemical damage, the light intensity necessary to activate Opto-mGluR6 surpasses the safe threshold for retinal illumination. Conversely, red-shifted lights pose a significantly lower risk of inducing such damage compared to blue lights. We designed red-shifted Opto-mGluR6 photopigments with a wide, red-shifted working spectrum compared to Opto-mGluR6 and examined their excitation capability in vitro. ROM19, ROM18 and ROM17, red-shifted variants of Opto-mGluR6, were designed by careful bioinformatics/computational studies. The predicted molecules with the best scores were selected, synthesised and cloned into the pAAV-CMV-IRES-EGFP vector. Expression of constructs was confirmed by functional assessment in engineered HEK-GIRK cells. Spectrophotometry and patch clamp experiments demonstrated that the candidate molecules were sensitive to the desired wavelengths of the light and directly coupled light stimuli to G-protein signalling. Herein, we introduce ROM17, ROM18 and ROM19 as newly generated, red-shifted variants with maximum excitation red-shifted of ~ 40nm, 70 nm and 126 nm compared to Opto-mGluR6.

Structure of the antenna complex expressed during far-red light photoacclimation in Synechococcus sp. PCC 7335

Far-red light photoacclimation, or FaRLiP, is a facultative response exhibited by some cyanobacteria that allows them to absorb and utilize lower energy light (700 to 800 nm) than the wavelengths typically used for oxygenic photosynthesis (400 to 700 nm). During this process, three essential components of the photosynthetic apparatus are altered: photosystem I, photosystem II, and the phycobilisome. In all three cases, at least some of the chromophores found in these pigment-protein complexes are replaced by chromophores that have red-shifted absorbance relative to the analogous complexes produced in visible light. Recent structural and spectroscopic studies have elucidated important features of the two photosystems when altered to absorb and utilize far-red light, but much less is understood about the modified phycobiliproteins made during FaRLiP. We used single-particle, cryo-electron microscopy to determine the molecular structure of a phycobiliprotein core complex comprising allophycocyanin variants that absorb far-red light during FaRLiP in the marine cyanobacterium Synechococcus sp. PCC 7335. The structure reveals the arrangement of the numerous red-shifted allophycocyanin variants and the probable locations of the chromophores that serve as the terminal emitters in this complex. It also suggests how energy is transferred to the photosystem II complexes produced during FaRLiP. The structure additionally allows comparisons with other previously studied allophycocyanins to gain insights into how phycocyanobilin chromophores can be tuned to absorb far-red light. These studies provide new insights into how far-red light is harvested and utilized during FaRLiP, a widespread cyanobacterial photoacclimation mechanism.

Cloning and structural basis of fluorescent protein color variants from identical species of sea anemone, Diadumene lineata

Diadumene lineata is a colorful sea anemone with orange stripe tissue of the body column and plain tentacles with red lines. We subjected Diadumene lineata to expression cloning and obtained genes encoding orange (OFP: DiLiFP561) and red fluorescent proteins (RFPs: DiLiFP570 and DiLiFP571). These proteins formed obligatory tetramers. All three proteins showed bright fluorescence with the brightness of 58.3 mM−1·cm−1 (DiLiFP561), 43.9 mM−1·cm−1 (DiLiFP570), and 31.2 mM−1·cm−1 (DiLiFP571), which were equivalent to that of commonly used red fluorescent proteins. Amplitude-weighted average fluorescence lifetimes of DiLiFP561, DiLiFP570 and DiLiFP571 were determined as 3.7, 3.6 and 3.0 ns. We determined a crystal structure of DiLiFP570 at 1.63 Å resolution. The crystal structure of DiLiFP570 revealed that the chromophore has an extended π-conjugated structure similar to that of DsRed. Most of the amino acid residues surrounding the chromophore were common between DiLiFP570 and DiLiFP561, except M159 of DiLiFP570 (Lysine in DiLiFP561), which is located close to the chromophore hydroxyl group. Interestingly, a similar K-to-M substitution has been reported in a red-shifted variant of DsRed (mRFP1). It is a striking observation that the naturally evolved color-change variants are consistent with the mutation induced via protein engineering processes. The newly cloned proteins are promising as orange and red fluorescent markers for imaging with long fluorescence lifetime.Graphical abstract

Evaluation of Non-invasive Optogenetic Stimulation with Transcranial Functional Ultrasound Imaging.

Optogenetics employs engineered viruses to genetically modify cells to express specific light-sensitive ion channels. The standard method for gene delivery in the brain involves invasive craniotomies that expose the brain and direct injections of viruses that invariably damage neural tissue along the syringe tract. A recently proposed alternative in which non-invasive optogenetics is performed with focused ultrasound (FUS)-mediated blood-brain barrier (BBB) openings has been found to non-invasively facilitate gene delivery for optogenetics in mice. Although gene delivery can be performed non-invasively, validating successful viral transduction and expression of encoded ion channels in target tissue typically involves similar invasive techniques, such as craniotomies in longitudinal studies and/or postmortem histology. Functional ultrasound imaging (fUSi) is an emerging neuroimaging technique that can be used to transcranially detect changes in cerebral blood volume following introduction of a stimulus. In this study, we implemented a fully non-invasive combined FUS-fUSi technique for performing optogenetics in mice. FUS successfully delivered viruses encoding the red-shifted channelrhodopsin variant ChrimsonR in all treated subjects. fUSi successfully identified stimulus-evoked cerebral blood volume changes preferentially in brain regions expressing the light-sensitive ion channels. Improvements in cell-specific targeting of viral vectors and transcranial ultrasound imaging will make the combined technique a useful tool for neuroscience research in small animals.

Engineered Amber-Emitting Nano Luciferase and Its Use for Immunobioluminescence Imaging In Vivo.

The NanoLuc luciferase (NLuc) and its furimazine (FRZ) substrate have revolutionized bioluminescence (BL) assays and imaging. However, the use of the NLuc-FRZ luciferase-luciferin pair for mammalian tissue imaging is hindered by the low tissue penetration of the emitting blue photons. Here, we present the development of an NLuc mutant, QLuc, which catalyzes the oxidation of a synthetic QTZ luciferin for bright and red-shifted emission peaking at ∼585 nm. Compared to other small single-domain NLuc mutants, this amber-light-emitting luciferase exhibited improved performance for imaging deep-tissue targets in live mice. Leveraging this novel bioluminescent reporter, we further pursued in vivo immunobioluminescence imaging (immunoBLI), which used a fusion protein of a single-chain variable antibody fragment (scFv) and QLuc for molecular imaging of tumor-associated antigens in a xenograft mouse model. As one of the most red-shifted NLuc variants, we expect QLuc to find broad applications in noninvasive mammalian imaging. Moreover, the immunoBLI method complements immunofluorescence imaging and immuno-positron emission tomography (immunoPET), serving as a convenient and nonradioactive molecular imaging tool for animal models in basic and preclinical research.

Experimental Characterization of In Silico Red-Shift-Predicted iLOVL470T/Q489K and iLOVV392K/F410V/A426S Mutants.

iLOV is a flavin mononucleotide-binding fluorescent protein used for in vivo cellular imaging similar to the green fluorescent protein. To expand the range of applications of iLOV, spectrally tuned red-shifted variants are desirable to reduce phototoxicity and allow for better tissue penetration. In this report, we experimentally tested two iLOV mutants, iLOVL470T/Q489K and iLOVV392K/F410V/A426S, which were previously computationally proposed by (KhrenovaJ. Phys. Chem. B2017, 121 ( (43), ), pp 10018−1002528992704) to have red-shifted excitation and emission spectra. While iLOVL470T/Q489K is about 20% brighter compared to the WT in vitro, it exhibits a blue shift in contrast to quantum mechanics/molecular mechanics (QM/MM) predictions. Additional optical characterization of an iLOVV392K mutant revealed that V392 is essential for cofactor binding and, accordingly, variants with V392K mutation are unable to bind to FMN. iLOVL470T/Q489K and iLOVV392K/F410V/A426S are expressed at low levels and have no detectable fluorescence in living cells, preventing their utilization in imaging applications.

WormRuler: A software to track body length used to characterize a super red-shifted channelrhodopsin in Caenorhabditis elegans.

Manipulation of neuronal or muscular activity by optogenetics or other stimuli can be directly linked to the analysis of Caenorhabditis elegans ( C. elegans ) body length. Thus, WormRuler was developed as an open-source video analysis toolbox that offers video processing and data analysis in one application. Utilizing this novel tool, the super red-shifted channelrhodopsin variant, ChrimsonSA, was characterized in C. elegans . Expression and activation of ChrimsonSA in GABAergic motor neurons results in their depolarization and therefore elongation of body length, the extent of which providing information about the strength of neuronal transmission.

The molecular basis of spectral tuning in blue- and red-shifted flavin-binding fluorescent proteins

Photoactive biological systems modify the optical properties of their chromophores, known as spectral tuning. Determining the molecular origin of spectral tuning is instrumental for understanding the function and developing applications of these biomolecules. Spectral tuning in flavin-binding fluorescent proteins (FbFPs), an emerging class of fluorescent reporters, is limited by their dependency on protein-bound flavins, whose structure and hence electronic properties cannot be altered by mutation. A blue-shifted variant of the plant-derived improved light, oxygen, voltage FbFP has been created by introducing a lysine within the flavin-binding pocket, but the molecular basis of this shift remains unconfirmed. We here structurally characterize the blue-shifted improved light, oxygen, voltage variant and construct a new blue-shifted CagFbFP protein by introducing an analogous mutation. X-ray structures of both proteins reveal displacement of the lysine away from the chromophore and opening up of the structure as instrumental for the blue shift. Site saturation mutagenesis and high-throughput screening yielded a red-shifted variant, and structural analysis revealed that the lysine side chain of the blue-shifted variant is stabilized close to the flavin by a secondary mutation, accounting for the red shift. Thus, a single additional mutation in a blue-shifted variant is sufficient to generate a red-shifted FbFP. Using spectroscopy, X-ray crystallography, and quantum mechanics molecular mechanics calculations, we provide a firm structural and functional understanding of spectral tuning in FbFPs. We also show that the identified blue- and red-shifted variants allow for two-color microscopy based on spectral separation. In summary, the generated blue- and red-shifted variants represent promising new tools for application in life sciences.

An expanded palette of dopamine sensors for multiplex imaging in vivo.

Genetically encoded dopamine sensors based on green fluorescent protein (GFP) enable high-resolution imaging of dopamine dynamics in behaving animals. However, these GFP-based variants cannot be readily combined with commonly used optical sensors and actuators, due to spectral overlap. We therefore engineered red-shifted variants of dopamine sensors called RdLight1, based on mApple. RdLight1 can be combined with GFP-based sensors with minimal interference, and shows high photostability permitting prolonged continuous imaging. We demonstrate the utility of RdLight1 for receptor-specific pharmacological analysis in cell culture, simultaneous assessment of dopamine release and cell type-specific neuronal activity, and simultaneous subsecond monitoring of multiple neurotransmitters in freely behaving rats. Dual-color photometry revealed that dopamine release in the nucleus accumbens evoked by reward-predictive cues is accompanied by a rapid suppression of glutamate release. By enabling multiplexed imaging of dopamine with other circuit components in vivo, RdLight1 opens avenues for understanding many aspects of dopamine biology.

Synthesis of One Double Bond-Inserted Retinal Analogs and Their Binding Experiments with Opsins: Preparation of Novel Red-Shifted Channelrhodopsin Variants.

In optogenetics, red-shifted channelrhodopsins (ChRs) are eagerly sought. We prepared six kinds of new chromophores with one double bond inserted into the polyene side chain of retinal (A1) or 3,4-didehydroretinal (A2), and examined their binding efficiency with opsins (ReaChR and ChrimsonR). All analogs bound with opsins to afford new ChRs. Among them, A2-10ex (an extra double bond is inserted at the C10-C11 position of A2) showed the greatest red-shift in the absorption spectrum of ChrimsonR, with a maximum absorbance at 654 nm (67 nm red-shifted from that of A1-ChrimsonR). Moreover, a long-wavelength spectral boundary of A2-10ex-ChrimsonR was extended to 756 nm, which reached into the far-red region (710-850 nm).

Target-wide Induction and Synapse Type-Specific Robustness of Presynaptic Homeostasis.

Presynaptic homeostatic plasticity (PHP) is an evolutionarily conserved form of adaptive neuromodulation and is observed at both central and peripheral synapses. In this work, we make several fundamental advances by interrogating the synapse specificity of PHP. We define how PHP remains robust to acute versus long-term neurotransmitter receptor perturbation. We describe a general PHP property that includes global induction and synapse-specific expression mechanisms. Finally, we detail a novel synapse-specific PHP expression mechanism that enables the conversion from short- to long-term PHP expression. If our data can be extended to other systems, including the mammalian central nervous system, they suggest that PHP can be broadly induced and expressed to sustain the function of complex neural circuitry.

MRhubarb: Engineering of monomeric, red-shifted, and brighter variants of iRFP using structure-guided multi-site mutagenesis

Far-red and near-infrared fluorescent proteins (FPs) enable in vivo tissue imaging with greater depth and clarity compared to FPs in the visible spectrum due to reduced light absorbance and scatter by tissues. However current tools are limited by low brightness, limited red-shifting, and a non-ideal dimeric oligomerization state. In this study we developed a monomeric variant of iRFP, termed mRhubarb713, and subsequently used a targeted and expansive multi-site mutagenesis approach to screen for variants with red-shifted spectral activity. Two monomeric variants were discovered, deemed mRhubarb719 and mRhubarb720, with red-shifted spectra and increased quantum yield compared to iRFP. These tools build on previously developed near-IR FPs and should enable improved in vivo imaging studies with a genetically encoded reporter.

Combining retinal-based and chlorophyll-based (oxygenic) photosynthesis: Proteorhodopsin expression increases growth rate and fitness of a ∆PSI strain of Synechocystis sp. PCC6803

To fill the “green absorption gap”, a green absorbing proteorhodopsin was expressed in a PSI-deletion strain (ΔPSI) of Synechocystis sp. PCC6803. Growth-rate measurements, competition experiments and physiological characterization of the proteorhodopsin-expressing strains, relative to the ΔPSI control strain, allow us to conclude that proteorhodopsin can enhance the rate of photoheterotrophic growth of ΔPSI Synechocystis strain. The physiological characterization included measurement of the amount of residual glucose in the spent medium and analysis of oxygen uptake- and production rates. To explore the use of solar radiation beyond the PAR region, a red-shifted variant Proteorhodopsin-D212N/F234S was expressed in a retinal-deficient PSI-deletion strain (ΔPSI/ΔSynACO). Via exogenous addition of retinal analogue an infrared absorbing pigment (maximally at 740 nm) was reconstituted in vivo. However, upon illumination with 746 nm light, it did not significantly stimulate the growth (rate) of this mutant. The inability of the proteorhodopsin-expressing ΔPSI strain to grow photoautotrophically is most likely due to a kinetic rather than a thermodynamic limitation of its NADPH-dehydrogenase in NADP+-reduction.

Mutants of the Flavoprotein iLOV as Prospective Red-Shifted Fluorescent Markers.

We report on novel variants of the flavin-based fluorescent protein iLOV with absorption and emission optical bands shifted to the longer wavelengths relative to their precursor. First attempts (J. Phys. Chem. B 2015, 119, 5176; 2016, 120, 3344) to improve spectral properties of iLOV, a fluorescent marker in living cells, showed that single point mutation Q489K could lead to a promising variant, but its realization was not successful due to unfavorable conformation of the flexible lysine side chain pointing away from the chromophore. The results of molecular simulations presented in this work evidence that location of the charged lysine residue near the chromophore isoalloxazine ring can be fixed by introducing additional mutations in iLOV. Several suggested protein variants are characterized by using classical and QM/MM molecular dynamics simulations followed by optimization of structures in the ground and excited states. Transition energies between the S0 and S1 states are computed by using the advanced quantum chemical methods TD-DFT, SOS-CIS(D), and XMCQDPT2. According to our simulations, the chromophore-containing pockets for the red-shifted iLOV variants containing lysine either at position 392 (V392K/F410V/A426S) or at position 489 (Q489K/L470T) should be stable and exhibit absorption and emission bands red-shifted by 40-50 nm relative to iLOV.

Red-Shifted Aequorin Variants Incorporating Non-Canonical Amino Acids: Applications in In Vivo Imaging.

The increased importance of in vivo diagnostics has posed new demands for imaging technologies. In that regard, there is a need for imaging molecules capable of expanding the applications of current state-of-the-art imaging in vivo diagnostics. To that end, there is a desire for new reporter molecules capable of providing strong signals, are non-toxic, and can be tailored to diagnose or monitor the progression of a number of diseases. Aequorin is a non-toxic photoprotein that can be used as a sensitive marker for bioluminescence in vivo imaging. The sensitivity of aequorin is due to the fact that bioluminescence is a rare phenomenon in nature and, therefore, it does not suffer from autofluorescence, which contributes to background emission. Emission of bioluminescence in the blue-region of the spectrum by aequorin only occurs when calcium, and its luciferin coelenterazine, are bound to the protein and trigger a biochemical reaction that results in light generation. It is this reaction that endows aequorin with unique characteristics, making it ideally suited for a number of applications in bioanalysis and imaging. Herein we report the site-specific incorporation of non-canonical or non-natural amino acids and several coelenterazine analogues, resulting in a catalog of 72 cysteine-free, aequorin variants which expand the potential applications of these photoproteins by providing several red-shifted mutants better suited to use in vivo. In vivo studies in mouse models using the transparent tissue of the eye confirmed the activity of the aequorin variants incorporating L-4-iodophehylalanine and L-4-methoxyphenylalanine after injection into the eye and topical addition of coelenterazine. The signal also remained localized within the eye. This is the first time that aequorin variants incorporating non-canonical amino acids have shown to be active in vivo and useful as reporters in bioluminescence imaging.

A novel screening system based on VanX-mediated autolysis-Application to Gaussia luciferase.

We report a novel bacterial screening protocol based on co-expressing the target protein with VanX, an enzyme which mediates Escherichia coli's autolysis and the release of the target protein into the culture medium, thereby facilitating activity measurement and screening from crude medium. This protocol as assessed with 19 Gaussia luciferase (GLuc) expressing colonies, was able to detect bioluminescence wavelength shift as small as 1.5 nm. We demonstrate the performance and versatility of this protocol by applying it to a semi-rational search for GLuc variants with red-shifted bioluminescence. Six GLuc's sites, F113, I114, W143, L144, A149, and F151, were randomly mutated, and for each site, 50 colonies were cultivated in 3 mL samples, from which bioluminescence was measured without purification. We identified two GLuc single mutation red-shifted variants: W143V and L144A. Their red shifted bioluminescence and biophysical/biochemical properties were confirmed using HPLC purified variants. Biotechnol. Bioeng. 2016;113: 1413-1420. © 2015 Wiley Periodicals, Inc.

Use of genetically-encoded calcium indicators for live cell calcium imaging and localization in virus-infected cells

Calcium signaling is a ubiquitous and versatile process involved in nearly every cellular process, and exploitation of host calcium signals is a common strategy used by viruses to facilitate replication and cause disease. Small molecule fluorescent calcium dyes have been used by many to examine changes in host cell calcium signaling and calcium channel activation during virus infections, but disadvantages of these dyes, including poor loading and poor long-term retention, complicate analysis of calcium imaging in virus-infected cells due to changes in cell physiology and membrane integrity. The recent expansion of genetically-encoded calcium indicators (GECIs), including blue and red-shifted color variants and variants with calcium affinities appropriate for calcium storage organelles like the endoplasmic reticulum (ER), make the use of GECIs an attractive alternative for calcium imaging in the context of virus infections. Here we describe the development and testing of cell lines stably expressing both green cytoplasmic (GCaMP5G and GCaMP6s) and red ER-targeted (RCEPIAer) GECIs. Using three viruses (rotavirus, poliovirus and respiratory syncytial virus) previously shown to disrupt host calcium homeostasis, we show the GECI cell lines can be used to detect simultaneous cytoplasmic and ER calcium signals. Further, we demonstrate the GECI expression has sufficient stability to enable long-term confocal imaging of both cytoplasmic and ER calcium during the course of virus infections.

Reverse-correlation analysis of navigation dynamics in Drosophila larva using optogenetics.

Neural circuits for behavior transform sensory inputs into motor outputs in patterns with strategic value. Determining how neurons along a sensorimotor circuit contribute to this transformation is central to understanding behavior. To do this, a quantitative framework to describe behavioral dynamics is needed. In this study, we built a high-throughput optogenetic system for Drosophila larva to quantify the sensorimotor transformations underlying navigational behavior. We express CsChrimson, a red-shifted variant of channelrhodopsin, in specific chemosensory neurons and expose large numbers of freely moving animals to random optogenetic activation patterns. We quantify their behavioral responses and use reverse-correlation analysis to uncover the linear and static nonlinear components of navigation dynamics as functions of optogenetic activation patterns of specific sensory neurons. We find that linear-nonlinear models accurately predict navigational decision-making for different optogenetic activation waveforms. We use our method to establish the valence and dynamics of navigation driven by optogenetic activation of different combinations of bitter-sensing gustatory neurons. Our method captures the dynamics of optogenetically induced behavior in compact, quantitative transformations that can be used to characterize circuits for sensorimotor processing and their contribution to navigational decision making.