The transition metal copper (Cu) is essential for all living organisms but 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 a mutant defective the gene encoding SQUAMOSA PROMOTER BINDING PROTEIN-LIKE7 (SPL7), which acts as transcriptional regulator responses. In response to deficiency, FERRIC REDUCTASE OXIDASE5 (FRO5) FRO4 levels increased strongly, an...

We present the design, synthesis, spectroscopy, and biological applications of Mitochondrial Coppersensor-1 (Mito-CS1), a new type targetable fluorescent sensor for imaging exchangeable mitochondrial copper pools in living cells. Mito-CS1 is bifunctional reporter that combines Cu(+)-responsive platform with mitochondrial-targeting triphenylphosphonium moiety localizing probe to this organelle. Molecular establishes chemical tool can detect changes labile Cu(+) model HEK 293T cell line as...

We present the synthesis and properties of Nickelsensor-1 (NS1), a new water-soluble, turn-on fluorescent sensor that is capable selectively responding to Ni2+ in aqueous solution living cells. NS1 combines BODIPY chromophore mixed N/O/S receptor provide good selectivity for over range biologically abundant metal ions solution. In addition these characteristics, confocal microscopy experiments further show can be delivered into cells report changes intracellular levels respiratory cell model.

Dynamic fluxes of s-block metals like potassium, sodium, and calcium are broad importance in cell signaling. In contrast, the concept mobile transition triggered by activation remains insufficiently explored, large part because copper iron typically studied as static cellular nutrients there a lack direct, selective methods for monitoring their distributions living cells. To help meet this need, we now report Coppersensor-3 (CS3), bright small-molecule fluorescent probe that offers unique...

For reasons that remain insufficiently understood, the brain requires among highest levels of metals in body for normal function. The traditional paradigm this organ and others is fluxes alkali alkaline earth are required signaling, but transition maintained static, tightly bound reservoirs metabolism protection against oxidative stress. Here we show copper an endogenous modulator spontaneous activity, a property functional neural circuitry. Using Copper Fluor-3 (CF3), new fluorescent Cu(+)...

Copper is required for the activity of cytochrome c oxidase (COX), terminal electron-accepting complex mitochondrial respiratory chain. The likely source copper used COX biogenesis a labile pool found in matrix. In mammals, proteins that transport across inner membrane remain unknown. We previously reported carrier family protein Pic2 budding yeast importer. closest ortholog mammalian cells phosphate SLC25A3. Here, to investigate whether SLC25A3 also transports copper, we manipulated its...

Two-dimensional covalent organic frameworks (2D-COFs) are a class of crystalline porous polymers that consist covalently linked, two-dimensional sheets can stack together through noncovalent interactions. Here we report the synthesis novel COF, called PyCOFamide, which has an experimentally observed pore size is greater than 6 nm in diameter. This among largest reported to date for 2D-COF. PyCOFamide exhibits permanent porosity and high crystallinity as evidenced by nitrogen adsorption,...

Copper (Cu), an essential redox active cofactor, participates in fundamental biological processes, but it becomes highly cytotoxic when present excess. Therefore, living organisms have established suitable mechanisms to balance cellular and systemic Cu levels. An important strategy maintain homeostasis consists of regulating uptake mobilization via the conserved family CTR/COPT transport proteins. In model plant Arabidopsis thaliana, COPT1 protein mediates root acquisition, whereas COPT5...

Abstract A novel cytochrome P450 enzyme, TxtE, was recently shown to catalyze the direct aromatic nitration of L ‐tryptophan. This unique chemistry inspired us ask whether TxtE could serve as a platform for engineering new biocatalysts replace current harsh synthetic methods. As first step toward this goal, and better understand wild‐type we obtained high‐resolution structures in its substrate‐free substrate‐bound forms. We also screened library substrate analogues spectroscopic indicators...

Abstract Recent work has shown that engineered variants of cytochrome P450 BM3 (CYP102A1) efficiently catalyze non‐natural reactions, including carbene and nitrene transfer reactions. Given the broad substrate range natural enzymes, we set out to explore if this diversity could be leveraged generate a panel new catalysts for olefin cyclopropanation (i.e., transfer). Here, took step towards goal by characterizing activities four wild‐type P450s have different native substrates. All were...

Chloride-sensitive fluorescent proteins generated from laboratory evolution have a characteristic tyrosine residue that interacts with chloride ion and π-stacks the chromophore. However, engineered yellow-green protein mNeonGreen lacks this interaction but still binds chloride, as seen in recently reported crystal structure. Based on its unique coordination sphere, we were curious if could influence optical properties of mNeonGreen. Here, present structure-guided identification spectroscopic...

Beyond its role as the "queen of electrolytes", chloride can also serve an allosteric regulator or even a signaling ion. To illuminate this essential anion across such spectrum biological processes, researchers have relied on fluorescence imaging with genetically encoded sensors. In large part, these been derived from green fluorescent protein found in jellyfish Aequorea victoria. However, standalone sensor turn-on intensiometric response at physiological pH has yet to be reported. Here, we...

Inarguably, the green fluorescent protein (GFP) family is an exemplary model for engineering, accessing a range of unparalleled functions and utility in biology. The first variant to recognize provide optical output chloride living cells was serendipitously uncovered more than 25 years ago. Since then, researchers have actively expanded potential GFP indicators through site-directed combinatorial site-saturation mutagenesis, along with chimeragenesis. However, date, power directed evolution...

The duality of nitrate is nowhere best exemplified than in human physiology - a detrimental pollutant but also protective nutrient and signaling ion particularly as connected to reactive nitrogen oxides. Aside from limited insights into uptake storage, foundational biology has lagged. Genetically encoded fluorescent biosensors can address this gap with real-time imaging. However, imaging technologies for mammalian cell applications remain rare. Here, we set out design engineer two-domain...

To gain insight in the molecular basis of copper homeostasis during meiosis, we have used DNA microarrays to analyze meiotic gene expression model yeast Schizosaccharomyces pombe. Profiling data identified a novel meiosis-specific gene, termed mfc1(+), that encodes putative major facilitator superfamily-type transporter. Although Mfc1 does not exhibit any significant sequence homology with permease Ctr4, it contains four copper-binding motifs are typically found members transporter family...

The synthesis, X-ray structure, solution stability, and photophysical properties of several trivalent lanthanide complexes Yb(III) Nd(III) using both tetradentate octadentate ligand design strategies incorporating the 1-methyl-3-hydroxy-pyridin-2-one (Me-3,2-HOPO) chelate group are reported. Both have emission bands in Near Infra-Red (NIR) region, this luminescence is retained aqueous (ΦtotYb ∼ 0.09−0.22%). Furthermore, demonstrate very high stability (pYb 18.8−21.9) solution, making them...

Fluorescent proteins have been extensively engineered and applied as optical indicators for chloride in a variety of biological contexts. Surprisingly, given the biodiversity fluorescent proteins, naturally occurring sensor has not reported to date. Here, we present identification spectroscopic characterization yellow protein from jellyfish Phialidium sp. (phiYFP), rare example occurring, excitation ratiometric, turn-on chloride. Our results show that binding tunes pKa chromophore Y66 shifts...

The visualization of chloride in living cells with fluorescent sensors is linked to our ability design hosts that can overcome the energetic penalty desolvation bind water. Fluorescent proteins be used as biological supramolecular address this fundamental challenge. Here, we showcase power protein engineering convert proton-pumping rhodopsin GR from Gloeobacter violaceus into GR1, a red-shifted, turn-on sensor for detergent micelles and live Escherichia coli. This non-natural function was...

Natural and laboratory-guided evolution has created a rich diversity of fluorescent protein (FP)-based sensors for chloride (Cl-). To date, such have been limited to the Aequorea victoria green (avGFP) family, fusions with other FPs unlocked ratiometric imaging applications. Recently, we identified yellow from jellyfish Phialidium sp. (phiYFP) as turn-on, self-ratiometric Cl- sensor. elucidate its working mechanism rare example single FP this capability, tracked excited-state dynamics phiYFP...

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 used and adapted an alternative to synthetic hosts. Here, we report how the mutagenesis β-bulge residues (D137 W138) mNeonGreen, bright, monomeric fluorescent protein, unlocks tunes anion preference at physiological pH for sulfate, resulting turn-off sensor SulfOFF-1. This unprecedented sensing arises from...

Saxitoxin is a secondary metabolite produced by several species of dinoflagellates and cyanobacteria which targets voltage-gated sodium potassium channels in higher vertebrates. However, its molecular target planktonic aquatic community members that co-occur with the toxin producers remains unknown. Previous microarray analysis yeast identified copper iron-homeostasis genes as being differentially regulated response to saxitoxin. This study sought identify microbial cells comparing...