Rational Design of the ß-Bulge Gate in a Green Fluorescent Protein Accelerates the Kinetics of Sulfate Sensing.

Detection of anions in complex aqueous media is a fundamental challenge with practical utility that can be addressed by supramolecular chemistry. Biomolecular hosts such as proteins can be used and adapted as an alternative to synthetic hosts. Here, we report how the mutagenesis of the ß-bulge residues (D137 and W138) in mNeonGreen, a bright, monomeric fluorescent protein, unlocks and tunes the anion preference at physiological pH for sulfate, resulting in the turn-off sensor SulfOFF-1. This unprecedented sensing arises from an enhancement in the kinetics of binding, largely driven by position 138. In line with these data, molecular dynamics (MD) simulations capture how the coordinated entry and gating of sulfate into the ß-barrel is eliminated upon mutagenesis to facilitate binding and fluorescence quenching.

Supramolecular Reinforcement of a Large-Pore 2D Covalent Organic Framework.

Two-dimensional covalent organic frameworks (2D-COFs) are a class of crystalline porous organic polymers that consist of covalently linked, two-dimensional sheets that can stack together through noncovalent interactions. Here we report the synthesis of a novel COF, called PyCOFamide, which has an experimentally observed pore size that is greater than 6 nm in diameter. This is among the largest pore size reported to date for a 2D-COF. PyCOFamide exhibits permanent porosity and high crystallinity as evidenced by the nitrogen adsorption, powder X-ray diffraction, and high-resolution transmission electron microscopy. We show that the pore size of PyCOFamide is large enough to accommodate fluorescent proteins such as Superfolder green fluorescent protein and mNeonGreen. This work demonstrates the utility of noncovalent structural reinforcement in 2D-COFs to produce larger and persistent pore sizes than previously possible.

Discovery and Characterization of a Naturally Occurring, Turn-On Yellow Fluorescent Protein Sensor for Chloride.

Fluorescent proteins have been extensively engineered and applied as optical indicators for chloride in a variety of biological contexts. Surprisingly, given the biodiversity of fluorescent proteins, a naturally occurring chloride sensor has not been reported to date. Here, we present the identification and spectroscopic characterization of the yellow fluorescent protein from the jellyfish Phialidium sp . (phiYFP), a rare example of a naturally occurring, excitation ratiometric, and turn-on fluorescent protein sensor for chloride. Our results show that chloride binding tunes the p Ka of the chromophore Y66 and shifts the equilibrium from the fluorescent phenolate form to the weakly fluorescent phenol form. The latter likely undergoes excited-state proton transfer to generate a turn-on fluorescence response that is pH-dependent. Moreover, anion selectivity and mutagenesis in the chloride binding pocket provide additional evidence for the proposed chloride sensing mechanism. Given these properties, we anticipate that phiYFP, with further engineering, could be a new tool for imaging cellular chloride dynamics.

The mammalian phosphate carrier SLC25A3 is a mitochondrial copper transporter required for cytochrome c oxidase biogenesis.

Copper is required for the activity of cytochrome c oxidase (COX), the terminal electron-accepting complex of the mitochondrial respiratory chain. The likely source of copper used for COX biogenesis is a labile pool found in the mitochondrial matrix. In mammals, the proteins that transport copper across the inner mitochondrial membrane remain unknown. We previously reported that the mitochondrial carrier family protein Pic2 in budding yeast is a copper importer. The closest Pic2 ortholog in mammalian cells is the mitochondrial phosphate carrier SLC25A3. Here, to investigate whether SLC25A3 also transports copper, we manipulated its expression in several murine and human cell lines. SLC25A3 knockdown or deletion consistently resulted in an isolated COX deficiency in these cells, and copper addition to the culture medium suppressed these biochemical defects. Consistent with a conserved role for SLC25A3 in copper transport, its heterologous expression in yeast complemented copper-specific defects observed upon deletion of PIC2 Additionally, assays in Lactococcus lactis and in reconstituted liposomes directly demonstrated that SLC25A3 functions as a copper transporter. Taken together, these data indicate that SLC25A3 can transport copper both in vitro and in vivo.

Identification of mNeonGreen as a pH-Dependent, Turn-On Fluorescent Protein Sensor for Chloride.

Chloride-sensitive fluorescent proteins generated from laboratory evolution have a characteristic tyrosine residue that interacts with a chloride ion and π-stacks with the chromophore. However, the engineered yellow-green fluorescent protein mNeonGreen lacks this interaction but still binds chloride, as seen in a recently reported crystal structure. Based on its unique coordination sphere, we were curious if chloride could influence the optical properties of mNeonGreen. Here, we present the structure-guided identification and spectroscopic characterization of mNeonGreen as a turn-on fluorescent protein sensor for chloride. Our results show that chloride binding lowers the chromophore pKa and shifts the equilibrium away from the weakly fluorescent phenol form to the highly fluorescent phenolate form, resulting in a pH-dependent, turn-on fluorescence response. Moreover, through mutagenesis, we link this sensing mechanism to a non-coordinating residue in the chloride binding pocket. This discovery sets the stage to further engineer mNeonGreen as a new fluorescent protein-based tool for imaging cellular chloride.

Copper is an endogenous modulator of neural circuit spontaneous activity.

For reasons that remain insufficiently understood, the brain requires among the highest levels of metals in the body for normal function. The traditional paradigm for this organ and others is that fluxes of alkali and alkaline earth metals are required for signaling, but transition metals are maintained in static, tightly bound reservoirs for metabolism and protection against oxidative stress. Here we show that copper is an endogenous modulator of spontaneous activity, a property of functional neural circuitry. Using Copper Fluor-3 (CF3), a new fluorescent Cu(+) sensor for one- and two-photon imaging, we show that neurons and neural tissue maintain basal stores of loosely bound copper that can be attenuated by chelation, which define a labile copper pool. Targeted disruption of these labile copper stores by acute chelation or genetic knockdown of the CTR1 (copper transporter 1) copper channel alters the spatiotemporal properties of spontaneous activity in developing hippocampal and retinal circuits. The data identify an essential role for copper neuronal function and suggest broader contributions of this transition metal to cell signaling.

Subcellular metal imaging identifies dynamic sites of Cu accumulation in Chlamydomonas.

We identified a Cu-accumulating structure with a dynamic role in intracellular Cu homeostasis. During Zn limitation, Chlamydomonas reinhardtii hyperaccumulates Cu, a process dependent on the nutritional Cu sensor CRR1, but it is functionally Cu deficient. Visualization of intracellular Cu revealed major Cu accumulation sites coincident with electron-dense structures that stained positive for low pH and polyphosphate, suggesting that they are lysosome-related organelles. Nano-secondary ion MS showed colocalization of Ca and Cu, and X-ray absorption spectroscopy was consistent with Cu(+) accumulation in an ordered structure. Zn resupply restored Cu homeostasis concomitant with reduced abundance of these structures. Cu isotope labeling demonstrated that sequestered Cu(+) became bioavailable for the synthesis of plastocyanin, and transcriptome profiling indicated that mobilized Cu became visible to CRR1. Cu trafficking to intracellular accumulation sites may be a strategy for preventing protein mismetallation during Zn deficiency and enabling efficient cuproprotein metallation or remetallation upon Zn resupply.

Transcriptome sequencing identifies SPL7-regulated copper acquisition genes FRO4/FRO5 and the copper dependence of iron homeostasis in Arabidopsis.

The transition metal copper (Cu) is essential for all living organisms but is toxic when present in excess. To identify Cu deficiency responses comprehensively, we conducted genome-wide sequencing-based transcript profiling of Arabidopsis thaliana wild-type plants and of a mutant defective in the gene encoding SQUAMOSA PROMOTER BINDING PROTEIN-LIKE7 (SPL7), which acts as a transcriptional regulator of Cu deficiency responses. In response to Cu deficiency, FERRIC REDUCTASE OXIDASE5 (FRO5) and FRO4 transcript levels increased strongly, in an SPL7-dependent manner. Biochemical assays and confocal imaging of a Cu-specific fluorophore showed that high-affinity root Cu uptake requires prior FRO5/FRO4-dependent Cu(II)-specific reduction to Cu(I) and SPL7 function. Plant iron (Fe) deficiency markers were activated in Cu-deficient media, in which reduced growth of the spl7 mutant was partially rescued by Fe supplementation. Cultivation in Cu-deficient media caused a defect in root-to-shoot Fe translocation, which was exacerbated in spl7 and associated with a lack of ferroxidase activity. This is consistent with a possible role for a multicopper oxidase in Arabidopsis Fe homeostasis, as previously described in yeast, humans, and green algae. These insights into root Cu uptake and the interaction between Cu and Fe homeostasis will advance plant nutrition, crop breeding, and biogeochemical research.

Non-natural olefin cyclopropanation catalyzed by diverse cytochrome P450s and other hemoproteins.

Recent work has shown that engineered variants of cytochrome P450BM3 (CYP102A1) efficiently catalyze non-natural reactions, including carbene and nitrene transfer reactions. Given the broad substrate range of natural P450 enzymes, we set out to explore if this diversity could be leveraged to generate a broad panel of new catalysts for olefin cyclopropanation (i.e., carbene transfer). Here, we took a step towards this goal by characterizing the carbene transfer activities of four new wild-type P450s that have different native substrates. All four were active and exhibited a range of product selectivities in the model reaction: cyclopropanation of styrene by using ethyl diazoacetate (EDA). Previous work on P450BM3 demonstrated that mutation of the axial coordinating cysteine, universally conserved among P450 enzymes, to a serine residue, increased activity for this non-natural reaction. The equivalent mutation in the selected P450s was found to activate carbene transfer chemistry both in vitro and in vivo. Furthermore, serum albumins complexed with hemin were also found to be efficient in vitro cyclopropanation catalysts.

Structural, functional, and spectroscopic characterization of the substrate scope of the novel nitrating cytochrome P450 TxtE.

A novel cytochrome P450 enzyme, TxtE, was recently shown to catalyze the direct aromatic nitration of L-tryptophan. This unique chemistry inspired us to ask whether TxtE could serve as a platform for engineering new nitration biocatalysts to replace current harsh synthetic methods. As a first step toward this goal, and to better understand the wild-type enzyme, we obtained high-resolution structures of TxtE in its substrate-free and substrate-bound forms. We also screened a library of substrate analogues for spectroscopic indicators of binding and for production of nitrated products. From these results, we found that the wild-type enzyme accepts moderate decoration of the indole ring, but the amino acid moiety is crucial for binding and correct positioning of the substrate and therefore less amenable to modification. A nitrogen atom is essential for catalysis, and a carbonyl must be present to recruit the αB'1 helix of the protein to seal the binding pocket.

Calcium-dependent copper redistributions in neuronal cells revealed by a fluorescent copper sensor and X-ray fluorescence microscopy.

Dynamic fluxes of s-block metals like potassium, sodium, and calcium are of broad importance in cell signaling. In contrast, the concept of mobile transition metals triggered by cell activation remains insufficiently explored, in large part because metals like copper and iron are typically studied as static cellular nutrients and there are a lack of direct, selective methods for monitoring their distributions in living cells. To help meet this need, we now report Coppersensor-3 (CS3), a bright small-molecule fluorescent probe that offers the unique capability to image labile copper pools in living cells at endogenous, basal levels. We use this chemical tool in conjunction with synchotron-based microprobe X-ray fluorescence microscopy (XRFM) to discover that neuronal cells move significant pools of copper from their cell bodies to peripheral processes upon their activation. Moreover, further CS3 and XRFM imaging experiments show that these dynamic copper redistributions are dependent on calcium release, establishing a link between mobile copper and major cell signaling pathways. By providing a small-molecule fluorophore that is selective and sensitive enough to image labile copper pools in living cells under basal conditions, CS3 opens opportunities for discovering and elucidating functions of copper in living systems.

Engineering the ChlorON Series: Turn-On Fluorescent Protein Sensors for Imaging Labile Chloride in Living Cells.

Beyond its role as the "queen of electrolytes", chloride can also serve as an allosteric regulator or even a signaling ion. To illuminate this essential anion across such a spectrum of biological processes, researchers have relied on fluorescence imaging with genetically encoded sensors. In large part, these have been derived from the green fluorescent protein found in the jellyfish Aequorea victoria. However, a standalone sensor with a turn-on intensiometric response at physiological pH has yet to be reported. Here, we address this technology gap by building on our discovery of the anion-sensitive fluorescent protein mNeonGreen (mNG). The targeted engineering of two non-coordinating residues, namely K143 and R195, in the chloride binding pocket of mNG coupled with an anion walking screening and selection strategy resulted in the ChlorON sensors: ChlorON-1 (K143W/R195L), ChlorON-2 (K143R/R195I), and ChlorON-3 (K143R/R195L). In vitro spectroscopy revealed that all three sensors display a robust turn-on fluorescence response to chloride (20- to 45-fold) across a wide range of affinities (Kd ≈ 30-285 mM). We further showcase how this unique sensing mechanism can be exploited to directly image labile chloride transport with spatial and temporal resolution in a cell model overexpressing the cystic fibrosis transmembrane conductance regulator. Building from this initial demonstration, we anticipate that the ChlorON technology will have broad utility, accelerating the path forward for fundamental and translational aspects of chloride biology.

The Arabidopsis COPT6 transport protein functions in copper distribution under copper-deficient conditions.

Copper (Cu), an essential redox active cofactor, participates in fundamental biological processes, but it becomes highly cytotoxic when present in excess. Therefore, living organisms have established suitable mechanisms to balance cellular and systemic Cu levels. An important strategy to maintain Cu homeostasis consists of regulating uptake and mobilization via the conserved family of CTR/COPT Cu transport proteins. In the model plant Arabidopsis thaliana, COPT1 protein mediates root Cu acquisition, whereas COPT5 protein functions in Cu mobilization from intracellular storage organelles. The function of these transporters becomes critical when environmental Cu bioavailability diminishes. However, little is know about the mechanisms that mediate plant Cu distribution. In this report, we present evidence supporting an important role for COPT6 in Arabidopsis Cu distribution. Similarly to COPT1 and COPT2, COPT6 fully complements yeast mutants defective in high-affinity Cu uptake and localizes to the plasma membrane of Arabidopsis cells. Whereas COPT2 mRNA is only up-regulated upon severe Cu deficiency, COPT6 transcript is expressed under Cu excess conditions and displays a more gradual increase in response to decreases in environmental Cu levels. Consistent with COPT6 expression in aerial vascular tissues and reproductive organs, copt6 mutant plants exhibit altered Cu distribution under Cu-deficient conditions, including increased Cu in rosette leaves but reduced Cu levels in seeds. This altered Cu distribution is fully rescued when the wild-type COPT6 gene is reintroduced into the copt6 mutant line. Taken together, these findings highlight the relevance of COPT6 in shoot Cu redistribution when environmental Cu is limited.

Unlocking chloride sensing in the red at physiological pH with a fluorescent rhodopsin-based host.

Chloride is a vital ion for all forms of life. Protein-based fluorescent biosensors can enable researchers to visualize chloride in cells but remain underdeveloped. Here, we demonstrate how a single point mutation in an engineered microbial rhodopsin results in ChloRED-1-CFP. This membrane-bound host is a far-red emitting, ratiometric sensor that provides a reversible readout of chloride in live bacteria at physiological pH, setting the stage to investigate the roles of chloride in diverse biological contexts.

Mfc1 is a novel forespore membrane copper transporter in meiotic and sporulating cells.

To gain insight in the molecular basis of copper homeostasis during meiosis, we have used DNA microarrays to analyze meiotic gene expression in the model yeast Schizosaccharomyces pombe. Profiling data identified a novel meiosis-specific gene, termed mfc1(+), that encodes a putative major facilitator superfamily-type transporter. Although Mfc1 does not exhibit any significant sequence homology with the copper permease Ctr4, it contains four putative copper-binding motifs that are typically found in members of the copper transporter family of copper transporters. Similarly to the ctr4(+) gene, the transcription of mfc1(+) was induced by low concentrations of copper. However, its temporal expression profile during meiosis was distinct to ctr4(+). Whereas Ctr4 was observed at the plasma membrane shortly after induction of meiosis, Mfc1 appeared later in precursor vesicles and, subsequently, at the forespore membrane of ascospores. Using the fluorescent copper-binding tracker Coppersensor-1 (CS1), labile cellular copper was primarily detected in the forespores in an mfc1(+)/mfc1(+) strain, whereas an mfc1Δ/mfc1Δ mutant exhibited an intracellular dispersed punctate distribution of labile copper ions. In addition, the copper amine oxidase Cao1, which localized primarily in the forespores of asci, was fully active in mfc1(+)/mfc1(+) cells, but its activity was drastically reduced in an mfc1Δ/mfc1Δ strain. Furthermore, our data showed that meiotic cells that express the mfc1(+) gene have a distinct developmental advantage over mfc1Δ/mfc1Δ mutant cells when copper is limiting. Taken together, the data reveal that Mfc1 serves to transport copper for accurate and timely meiotic differentiation under copper-limiting conditions.

Inhibition of copper uptake in yeast reveals the copper transporter Ctr1p as a potential molecular target of saxitoxin.

Saxitoxin is a secondary metabolite produced by several species of dinoflagellates and cyanobacteria which targets voltage-gated sodium and potassium channels in higher vertebrates. However, its molecular target in planktonic aquatic community members that co-occur with the toxin producers remains unknown. Previous microarray analysis with yeast identified copper and iron-homeostasis genes as being differentially regulated in response to saxitoxin. This study sought to identify the molecular target in microbial cells by comparing the transcriptional profiles of key copper and iron homeostasis genes (CTR1, FRE1, FET3, CUP1, CRS5) in cells exposed to saxitoxin, excess copper, excess iron, an extracellular Cu(I) chelator, or an intracellular Cu(I) chelator. Protein expression and localization of Ctr1p (copper transporter), Fet3p (multicopper oxidase involved in high-affinity iron uptake), and Aft1p (iron regulator) were also compared among treatments. Combined transcript and protein profiles suggested saxitoxin inhibited copper uptake. This hypothesis was confirmed by intracellular Cu(I) imaging with a selective fluorescent probe for labile copper. On the basis of the combined molecular and physiological results, a model is presented in which the copper transporter Ctr1p serves as a molecular target of saxitoxin and these observations are couched in the context of the eco-evolutionary role this toxin may serve for species that produce it.

Coupling a Live Cell Directed Evolution Assay with Coevolutionary Landscapes to Engineer an Improved Fluorescent Rhodopsin Chloride Sensor.

Our understanding of chloride in biology has been accelerated through the application of fluorescent protein-based sensors in living cells. These sensors can be generated and diversified to have a range of properties using laboratory-guided evolution. Recently, we established that the fluorescent proton-pumping rhodopsin wtGR from Gloeobacter violaceus can be converted into a fluorescent sensor for chloride. To unlock this non-natural function, a single point mutation at the Schiff counterion position (D121V) was introduced into wtGR fused to cyan fluorescent protein (CFP) resulting in GR1-CFP. Here, we have integrated coevolutionary analysis with directed evolution to understand how the rhodopsin sequence space can be explored and engineered to improve this starting point. We first show how evolutionary couplings are predictive of functional sites in the rhodopsin family and how a fitness metric based on a sequence can be used to quantify the known proton-pumping activities of GR-CFP variants. Then, we couple this ability to predict potential functional outcomes with a screening and selection assay in live Escherichia coli to reduce the mutational search space of five residues along the proton-pumping pathway in GR1-CFP. This iterative selection process results in GR2-CFP with four additional mutations: E132K, A84K, T125C, and V245I. Finally, bulk and single fluorescence measurements in live E. coli reveal that GR2-CFP is a reversible, ratiometric fluorescent sensor for extracellular chloride with an improved dynamic range. We anticipate that our framework will be applicable to other systems, providing a more efficient methodology to engineer fluorescent protein-based sensors with desired properties.

Discovery of a monomeric green fluorescent protein sensor for chloride by structure-guided bioinformatics.

Chloride is an essential anion for all forms of life. Beyond electrolyte balance, an increasing body of evidence points to new roles for chloride in normal physiology and disease. Over the last two decades, this understanding has been advanced by chloride-sensitive fluorescent proteins for imaging applications in living cells. To our surprise, these sensors have primarily been engineered from the green fluorescent protein (GFP) found in the jellyfish Aequorea victoria. However, the GFP family has a rich sequence space that could already encode for new sensors with desired properties, thereby minimizing protein engineering efforts and accelerating biological applications. To efficiently sample this space, we present and validate a stepwise bioinformatics strategy focused first on the chloride binding pocket and second on a monomeric oligomerization state. Using this, we identified GFPxm163 from GFPxm found in the jellyfish Aequorea macrodactyla. In vitro characterization shows that the binding of chloride as well as bromide, iodide, and nitrate rapidly tunes the ground state chromophore equilibrium from the phenolate to the phenol state generating a pH-dependent, turn-off fluorescence response. Furthermore, live-cell fluorescence microscopy reveals that GFPxm163 provides a reversible, yet indirect readout of chloride transport via iodide exchange. With this demonstration, we anticipate that the pairing of bioinformatics with protein engineering methods will provide an efficient methodology to discover and design new chloride-sensitive fluorescent proteins for cellular applications.

Biophysical and in silico characterization of NrtA: a protein-based host for aqueous nitrate and nitrite recognition.

Nitrate and nitrite are key components of the global nitrogen cycle. As such, Nature has evolved proteins as biological supramolecular hosts for the recognition, translocation, and transformation of both nitrate and nitrite. To understand the supramolecular principles that govern these anion-protein interactions, here, we employ a hybrid biophysical and in silico approach to characterize the thermodynamic properties and protein dynamics of NrtA from the cyanobacterium Synechocystis sp. PCC 6803 for the recognition of nitrate and nitrite.