Ionic Covalent Organic Framework as a Dual Functional Sensor for Temperature and Humidity.

Visual sensing of humidity and temperature by solids plays an important role in the everyday life and in industrial processes. Due to their hydrophobic nature, most covalent organic framework (COF) sensors often exhibit poor optical response when exposed to moisture. To overcome this challenge, the optical response is set out to improve, to moisture by incorporating H-bonding ionic functionalities into the COF network. A highly sensitive COF, consisting of guanidinium and diformylpyridine linkers (TG-DFP), capable of detecting changes in temperature and moisture content is fabricated. The hydrophilic nature of the framework enables enhanced water uptake, allowing the trapped water molecules to form a large number of hydrogen bonds. Despite the presence of non-emissive building blocks, the H-bonds restrict internal bond rotation within the COF, leading to reversible fluorescence and solid-state optical hydrochromism in response to relative humidity and temperature.

Water/Vapor Assisted Fabrication of Large-Area Superprotonic Conductive Covalent Organic Framework Membranes.

Fabrication of large-area ionic covalent organic framework membranes (iCOMs) remains a grand challenge. Herein, the authors report the liquid water and water vapor-assisted fabrication of large-area superprotonic conductive iCOMs. A mixed monomer solution containing 1,3,5-triformylphloroglucinol (TFP) in 1,4-dioxane and p-diaminobenzenesulfonic acid (DABA) in water is first polymerized to obtain a pristine membrane which subsequently underwent crystallization process in mixed vapors containing water vapor. During the polymerization stage, water played a role of a diluting agent, weakening the Coulombic repulsion between sulfonic acid groups. During the crystallization stage, water vapor played a role of a structure-directing agent to facilitate the formation of highly crystalline, large-area iCOMs. The resulting membranes achieved a proton conductivity value of 0.76 S cm-1 at 90 °C under 100% relative humidity, which is among the highest ever reported. Using liquid water and water vapor as versatile additives open a novel avenue to the fabrication of large-area membranes from covalent organic frameworks and other kinds of crystalline organic framework materials.

Reversible Photooxygenation of Anthracene Carboxyimide for Singlet Oxygen Formation: Mechanistic Study and Efficient Nitrite Detection.

Polycyclic aromatic endoperoxides are important sources of singlet oxygen (1 O2 ) and their formation from polyacenes is well established. Anthracene carboxyimides are of particular interest as they exhibit excellent antitumor activity and possess unique photochemical properties. However, the photooxygenation of the synthetically versatile anthracene carboxyimide moiety has not been reported due to its competing [4+4] photodimerization reaction. Herein, we describe the reversible photo-oxidation of an anthracene carboxyimide. X-ray crystallographic analysis surprisingly revealed the formation of a racemic mixture of chiral hydroperoxides, rather than the expected endoperoxide. The photoproduct undergoes both photo- and thermolysis to form 1 O2 . Activation parameters were derived for the thermolysis and the mechanisms of photooxygenation and thermolysis are discussed. The anthracene carboxyimide also showed high selectivity and sensitivity for nitrite anions in acidic aqueous media and possessed stimuli-responsive behaviour.

Fluorescent Molecular Rotors Based on Hinged Anthracene Carboxyimides.

Temperature and viscosity are essential parameters in medicine, environmental science, smart materials, and biology. However, few fluorescent sensor publications mention the direct relationship between temperature and viscosity. Three anthracene carboxyimide-based fluorescent molecular rotors, 1DiAC∙Cl, 2DiAC∙Cl, and 9DiAC∙Cl, were designed and synthesized. Their photophysical properties were studied in various solvents, such as N, N-dimethylacetamide, N, N-dimethylformamide, 1-propanol, ethanol, dimethyl sulfoxide, methanol, and water. Solvent polarizability resulted in a solvatochromism effect for all three rotors and their absorption and emission spectra were analyzed via the Lippert-Mataga equation and multilinear analysis using Kamlet-Taft and Catalán parameters. The rotors exhibited red-shifted absorption and emission bands in solution on account of differences in their torsion angle. The three rotors demonstrated strong fluorescence in a high-viscosity environment due to restricted intramolecular rotation. Investigations carried out under varying ratios of water to glycerol were explored to probe the viscosity-based changes in their optical properties. A good linear correlation between the logarithms of fluorescence intensity and solution viscosity for two rotors, namely 2DiAC∙Cl and 9DiAC∙Cl, was observed as the percentage of glycerol increased. Excellent exponential regression between the viscosity-related temperature and emission intensity was observed for all three investigated rotors.

Taming the Topology of Calix[4]arene-Based 2D-Covalent Organic Frameworks: Interpenetrated vs Noninterpenetrated Frameworks and Their Selective Removal of Cationic Dyes.

A bowl-shaped calix[4]arene with its exciting host-guest chemistry is a versatile supramolecular building block for the synthesis of distinct coordination cages or metal-organic frameworks. However, its utility in the synthesis of crystalline covalent organic frameworks (COFs) remains challenging, presumably due to its conformational flexibility. Here, we report the synthesis of a periodic 2D extended organic network of calix[4]arenes joined by a linear benzidine linker via dynamic imine bonds. By tuning the interaction among neighboring calixarene units through varying the concentration in the reaction mixture, we show the selective formation of interpenetrated (CX4-BD-1) and non-interpenetrated (CX4-BD-2) frameworks. The cone-shaped calixarene moiety in the structural backbone allows for the interweaving of two neighboring layers in CX4-BD-1, making it a unique example of interpenetrated 2D layers. Due to the high negative surface charge from calixarene units, both COFs have shown high performance in charge-selective dye removal and an exceptional selectivity for cationic dyes irrespective of their molecular size. The charge distribution of the COFs and the resulting selectivity for the cationic dyes were further investigated using computational methods.

The Unusual Photochromic and Hydrochromic Switching Behavior of Cellulose-Embedded 1,8-Naphthalimide-Viologen Derivatives in the Solid-State.

Stimuli-responsive chromic materials such as photochromics, hydrochromics, thermochromics, and electrochromics have a long history of capturing the attention of scientists due to their potential industrial applications and novelty in popular culture. However, hybrid chromic materials that combine two or more stimuli-triggered color changing properties are not so well known. Herein, we report a design strategy that has led to a series of emissive 1,8-naphthalimide-viologen dyads which exhibit unusual dual photochromic and hydrochromic switching behavior in the solid-state when embedded in a cellulose matrix. This behavior manifests as reversible solid state fluorescence hydrochromism upon changes in atmospheric relative humidity (RH), and reversible solid state photochromism upon generation of a cellulose-stabilized viologen radical cation. In this design strategy, the bipyridinium unit serves as both a water-sensitive receptor for the hydrochromic fluorophore-receptor system, and a photochromic group, capable of eliciting its own visible colorimetric response, generating a fluorescence quenching radical cation with prolonged exposure to ultraviolet (UV) light. These dyes can be inkjet-printed onto cellulose paper or drop-cast as cellulose powder-based films and can be unidirectionally cycled between three different states which can be characteristically visualized under UV light or visible light. The material's photochromism, hydrochromism, and underlying mechanism of action was investigated using computational analysis, dynamic vapor sorption/desorption isotherms, electron paramagnetic resonance spectroscopy, and variable humidity UV-Vis adsorption and fluorescence spectroscopies.

Luteolin alleviates non-alcoholic fatty liver disease in rats via restoration of intestinal mucosal barrier damage and microbiota imbalance involving in gut-liver axis.

Nonalcoholic fatty liver disease (NAFLD) is demonstrated to be closely related to the disorder of gut microbiota and the intestinal mucosal barrier. Luteolin is a natural flavonoid with various activities. We aimed to investigate whether Luteolin can alleviate NAFLD and its possible mechanism involving the gut-liver axis. A rat NAFLD model was established by feeding a high-fat diet (HFD), and Luteolin was administered intragastrically. The effects of Luteolin on liver biochemical parameters, intestinal histopathology and integrity, gut microbiota, lipopolysaccharides (LPS), inflammatory cytokines, and the Toll-like receptor 4 (TLR4) signaling pathway were evaluated. We found that Luteolin restored the expression of the tight junction proteins in the intestine and ameliorated the increase permeability of the intestinal mucosa to Fluorescein isothiocyanate-dextran (FD4) caused by a high-fat diet, thus enhancing the function of the intestinal barrier. In addition, Luteolin inhibited the TLR4 signaling pathway in the liver, thereby reducing the secretion of pro-inflammatory factors and alleviating NAFLD. 16S rRNA gene sequencing revealed that Luteolin intervention significantly altered the composition of the gut microbiota in NAFLD rats and increased the richness of gut microbiota. Luteolin alleviates NAFLD in rats via restoration and repair of the damaged intestinal mucosal barrier and microbiota imbalance.

Solid-Vapor Interface Engineered Covalent Organic Framework Membranes for Molecular Separation.

Covalent organic frameworks (COFs) with intrinsic, tunable, and uniform pores are potent building blocks for separation membranes, yet poor processing ability and long processing time remain grand challenges. Herein, we report an engineered solid-vapor interface to fabricate a highly crystalline two-dimensional COF membrane with a thickness of 120 nm in 9 h, which is 8 times faster than that in the reported literature. Due to the ultrathin nature and ordered pores, the membrane exhibited an ultrahigh permeance (water, ∼411 L m-2 h-1 bar-1 and acetonitrile, ∼583 L m-2 h-1 bar-1) and excellent rejection of dye molecules larger than 1.4 nm (>98%). The membrane exhibited long-term operation which confirmed its outstanding stability. Our solid-vapor interfacial polymerization method may evolve into a generic platform to fabricate COFs and other organic framework membranes.

Classical and non-classical melatonin receptor agonist-directed micellization of bipyridinium-based supramolecular amphiphiles in water.

The addition of molecular recognition units into structures of amphiphiles is a means by which soft matter capable of undergoing template-directed micellization can be obtained. These supramolecular amphiphiles can bind with molecular templates using non-covalent bonding interactions, forming host-guest complexes that hold the amphiphiles together as they undergo micellization. In most cases, such templates are synthesized and designed for a specific molecular recognition motif. It is not clear, however, to what extent these types of amphiphile systems are responsive to members of a biologically derived class of molecular targets, for example, melatonin receptor agonists and their numerous isosteres. Herein, we describe the template-directed micellization and arrangement at the air-water interface of a bipyridinium-based gemini surfactant, driven by the influence of donor-acceptor CT interactions with a series of bioactive classical and non-classical melatonin isosteres. Under the conditions of templation by either 5-methoxytryptophol, N-acetylserotonin, N-acetyltryptamine, or the pharmaceutical agent agomelatine, favorable Gibbs free energies of micellization were observed with decreases in CMC by up to 70%, and concomitant increases of 28% in surface pressure, and decreases of 20% in contact angle versus untemplated solutions. Solid state thermochromic transition temperatures for inkjet-printed patterns of the templated amphiphile solutions were inversely correlated with trends observed for their respective CMCs, and exhibited no correlation to their binding constants. These findings contend for the generalizable use of melatonin receptor agonists as targets and/or templates for chemical systems, which rely on π-stacking donor-acceptor CT interactions in water to facilitate the actions of binding, sequestration, or template-directed self-assembly.

Prediction of chlorine and fluorine crystal structures at high pressure using symmetry driven structure search with geometric constraints.

The high-pressure properties of fluorine and chlorine are not yet well understood because both are highly reactive and volatile elements, which have made conducting diamond anvil cell and x-ray diffraction experiments a challenge. Here, we use ab initio methods to search for stable crystal structures of both elements at megabar pressures. We demonstrate how symmetry and geometric constraints can be combined to efficiently generate crystal structures that are composed of diatomic molecules. Our algorithm extends the symmetry driven structure search method [R. Domingos et al., Phys. Rev. B 98, 174107 (2018)] by adding constraints for the bond length and the number of atoms in a molecule while still maintaining generality. As a method of validation, we have tested our approach for dense hydrogen and reproduced the known molecular structures of Cmca-12 and Cmca-4. We apply our algorithm to study chlorine and fluorine in the pressure range of 10 GPa-4000 GPa while considering crystal structures with up to 40 atoms per unit cell. We predict chlorine to follow the same series of phase transformations as elemental iodine from Cmca to Immm to Fm3¯m, but at substantially higher pressures. We predict fluorine to transition from a C2/c to Cmca structure at 70 GPa, to a novel orthorhombic and metallic structure with P42/mmc symmetry at 2500 GPa, and finally to its cubic analog form with Pm3¯n symmetry at 3000 GPa.

Azobenzene-Equipped Covalent Organic Framework: Light-Operated Reservoir.

Light-operated materials have gained significant attention for their potential technological importance. To achieve molecular motion within extended networks, stimuli-responsive units require free space. The majority of the so far reported 2D-extended organic networks with responsive moieties restrict their freedom of motion on account of their connectivity providing constrained free volume for efficient molecular motion. We report here a light-responsive azobenzene-functionalized covalent organic framework (TTA-AzoDFP) designed in a way that the pendent azobenzene groups are pointing toward the pore channels with sufficient free volume necessary for the unencumbered dynamic motion to occur inside the pores of the covalent organic framework (COF) and undergo a reversible trans-cis photoisomerization upon light irradiation. The resulting hydrophobic COF was used for the storage of rhodamine B and its controlled release in solution by the mechanical motion of the azobenzene units triggered by ultraviolet-light irradiation. The TTA-AzoDFP displayed unprecedented photoregulated fluorescence emission behavior upon UV-light irradiation. Size, emission, and degree of hydrophobicity with respect to trans-cis-trans photoisomerization could be reversibly controlled by alternating UV- and visible-light exposure. The results reported here demonstrate once again the importance of the careful design of the linkers not only to allow the incorporation of molecular switches within the chemical structure of COFs but also to provide the required free space for not hindering their motion. The results demonstrate that responsive COFs could be suitable platforms for delivery systems that can be controlled by external stimuli.

Conformational Selection of a Polyproline Peptide by Ebola Virus VP30.

An adaptive temperature-based replica-exchange simulation of a peptide extracted from the Ebola virus nucleoprotein containing a polyproline sequence motif is reported. The simulation results of applying the CHARMM36m force field with a generalized Born solvent model is presented. Conformational heterogeneity is described by potentials of mean force (PMFs) for a set of reaction coordinates that define the topological fold space. Starting from an extended backbone conformation of the peptide observed in an X-ray crystallographic assembly with the Ebola virus protein VP30, the PMFs report a conformational landscape populated by chain excursions to collapsed states with limited transitions to either an extended fold or a canonical polyproline type II helix. Clustering of the conformations and applying an elastic network interpolation model yield a multistep pathway of conformational selection that minimizes the net transition-state cost from the population hub to the bound state. Related difference between the pathway endpoints taken from the PMFs reveal a significant free-energy penalty in reaching a population shift. To evaluate sequence fitness of the Ebola virus peptide in generating probability distributions, two human sequence variants are modeled and are found to produce profiles that show extensive deviations, thus suggesting either dissimilar binding mechanisms or the lack of recognition by VP30.

Rim-Differentiated C5-Symmetric Tiara-Pillar[5]arenes.

The synthesis of "rim-differentiated" C5-symmetric pillar[5]arenes, whose two rims are decorated with different chemical functionalities, has remained a challenging task. This is due to the inherent statistical nature of the cyclization of 1,4-disubstituted alkoxybenzenes with different substituents, which leads to four constitutional isomers with only 1/16th being rim-differentiated. Herein, we report a "preoriented" synthetic protocol based on FeCl3-catalyzed cyclization of asymmetrically substituted 2,5-dialkoxybenzyl alcohols. This yields an unprecedented 55% selectivity of the C5-symmetric tiara-like pillar[5]arene isomer among four constitutional isomers. Based on this new method, a series of functionalizable tiara-pillar[5]arene derivatives with C5-symmetry was successfully synthesized, isolated, and fully characterized in the solid state.

Bis-Bipyridinium Gemini Surfactant-Based Supramolecular Helical Fibers and Solid State Thermochromism.

The processability and functional performance of stimuli-responsive supramolecular materials are key factors in determining their utility and potential for mass adoption, usage, and profitability. However, it is difficult to predict how structural changes to the molecular components of these systems will impact their operation. Here, a series of π-electron-deficient bis-bipyridinium gemini surfactants were synthesized and evaluated to elucidate the structure-property relationships that govern their ability to form helical-fiber-based donor-acceptor hydrogels, impact hydrogel strength, and influence their solid-state thermochromism. When combined with the π-electron-rich donor melatonin, the helical-fiber- and hydrogel-forming ability of the gemini surfactants was largely influenced by the dimensions of the rigid bridging group that connects the two bis-bipyridinium units. Dynamic viscoelastic rheology and linear sweep voltammetric analysis revealed a positive correlation between the length of the gemini-surfactant bridging group and both the hydrogel strength and the magnitude of the charge-transfer interaction between the donor-acceptor pair. Solid-state thermochromic transition temperatures of processed aerogels, xerogel films, and inkjet-printed patterns were positively correlated with the strength of the charge transfer interaction between the donor-acceptor pair and, thus, also with the length of the gemini surfactant bridging group. The results provide impactful insights that will enable the development of new donor-acceptor-based thermochromes with versatile processability and tunable functionality.

Assembly and Chiral Memory Effects of Dynamic Macroscopic Supramolecular Helices.

Macroscopic enantiomerically pure helical supramolecular fibers are bottom-up assembled in aqueous media from a chiral π-electron donor template and an achiral π-electron acceptor. The helices can be assembled to the sub-millimeter scale with controlled handedness. These dynamic supramolecular architectures allow for a quantitative exchange of the chiral donor template with achiral analogues. During this process, a chiral memory effect was observed, affording enantiomerically pure helices composed entirely of achiral components.

Melatonin-directed micellization: a case for tryptophan metabolites and their classical bioisosteres as templates for the self-assembly of bipyridinium-based supramolecular amphiphiles in water.

The bulk solution properties of amphiphilic formulations are derivative of their self-assembly into higher ordered supramolecular assemblies known as micelles and of their ordering at the air-water interface. Exerting control over the surface-active properties of amphiphiles and their propensity to aggregate in pure water is most often fine-tuned by covalent modification of their molecular structure. Nevertheless structural constraints which limit the performance of amphiphiles do emerge when trying to develop more sophisticated systems which undergo for example, shape-defined controlled assembly and/or respond to external stimuli. In this regard, the template-modulated assembly of the so-called "supramolecular amphiphiles" continues to make progress ordering molecules that otherwise have very little to no driving force to aggregate in a prescribed manner in aqueous solutions. Herein we describe the template-modulated micellization and ordering at the air-water interface of bipyridinium-based supramolecular amphiphiles triggered by host-guest interactions with high specificity for the neurotransmitter melatonin over its biosynthetic synthon l-tryptophan and the thermodynamic parameters governing the template-modulated micellization process. When bound to the bipyridinium units of micellized surfactant molecules, melatonin effectively serves as "molecular glue" capable of lowering the CMC by 52% as compared to untemplated solutions. Analysis of this system suggests that a hallmark of donor-acceptor template-modulated micellization in water is a strong positively correlated temperature dependence of the CMC and the absence of a U-shaped CMC-temperature curve. Our findings make a case for the incorporation of l-tryptophan-based metabolites and their classical synthetic pharmaceutical bioisosteres as potential targets/components of donor-acceptor CT-based supramolecular amphiphile systems/materials operating in water.

Parallel Tempering of Dark Matter from the Ebola Virus Proteome: Comparison of CHARMM36m and CHARMM22 Force Fields with Implicit Solvent.

Intrinsically disordered proteins are characterized by their large manifold of thermally accessible conformations and their related statistical weights, making them an interesting target of simulation studies. To assess the development of a computational framework for modeling this distinct class of proteins, this work examines temperature-based replica-exchange simulations to generate a conformational ensemble of a 28-residue peptide from the Ebola virus protein VP35. Starting from a prefolded helix-ß-turn-helix topology observed in a crystallographic assembly, the simulation strategy tested is the recently refined CHARMM36m force field combined with a generalized Born solvent model. A comparison of two replica-exchange methods is provided, where one is a traditional approach with a fixed set of temperatures and the other is an adaptive scheme in which the thermal windows are allowed to move in temperature space. The assessment is further extended to include a comparison with equivalent CHARMM22 simulation data sets. The analysis finds CHARMM36m to shift the minimum in the potential of mean force (PMF) to a lower fractional helicity compared with CHARMM22, while the latter showed greater conformational plasticity along the helix-forming reaction coordinate. Among the simulation models, only the adaptive tempering method with CHARMM36m found an ensemble of conformational heterogeneity consisting of transitions between α-helix-ß-hairpin folds and unstructured states that produced a PMF of fractional fold propensity in qualitative agreement with circular dichroism experiments reporting a disordered peptide.

Membrane insertion of fusion peptides from Ebola and Marburg viruses studied by replica-exchange molecular dynamics simulations.

This work presents replica-exchange molecular dynamics simulations of inserting a 16-residue Ebola virus fusion peptide into a membrane bilayer. A computational approach is applied for modeling the peptide at the explicit all-atom level and the membrane-aqueous bilayer by a generalized Born continuum model with a smoothed switching function (GBSW). We provide an assessment of the model calculations in terms of three metrics: (1) the ability to reproduce the NMR structure of the peptide determined in the presence of SDS micelles and comparable structural data on other fusion peptides; (2) determination of the effects of the mutation Trp-8 to Ala and sequence discrimination of the homologous Marburg virus; and (3) calculation of potentials of mean force for estimating the partitioning free energy and their comparison to predictions from the Wimley-White interfacial hydrophobicity scale. We found the GBSW implicit membrane model to produce results of limited accuracy in conformational properties of the peptide when compared to the NMR structure, yet the model resolution is sufficient to determine the effect of sequence differentiation on peptide-membrane integration. © 2016 Wiley Periodicals, Inc.

[3+3] Cyclocondensation of Disubstituted Biphenyl Dialdehydes: Access to Inherently Luminescent and Optically Active Hexa-substituted C3-Symmetric and Asymmetric Trianglimine Macrocycles.

A general synthetic route to inherently luminescent and optically active 6-fold substituted C3-symmetric and asymmetric biphenyl-based trianglimines has been developed. The synthesis of these hexa-substituted triangular macrocycles takes advantage of a convenient method for the synthesis of symmetrically and asymmetrically difunctionalized biphenyl dialdehydes through a convergent two-step aromatic nucleophilic substitution-one-pot Suzuki-coupling reaction protocol. A modular [3+3] diamine-dialdehyde cyclocondensation reaction between both the symmetrically and asymmetrically difunctionalized-4,4'-biphenyldialdehydes with enantiomerically pure (1R,2R)-1,2-diaminocyclohexane was employed to construct the hexa-substituted triangular macrocycles. B97-D/6-311G(2d,p) density functional theory determined structures and X-ray crystallographic analysis reveal that the six substituents appended to the biphenyl legs of the trianglimine macrocycles adopt an alternating conformation not unlike the 1,3,5-alternate conformation observed for calix[6]arenes. Reduction of the imine bonds using NaBH4 afforded the corresponding 6-fold substituted trianglamine without the need to alkylate the amine nitrogen atoms which could hinder their later use as metal coordination sites and without having to introduce asymmetric carbons.

Kinetic, Mutational, and Structural Studies of the Venezuelan Equine Encephalitis Virus Nonstructural Protein 2 Cysteine Protease.

The Venezuelan equine encephalitis virus (VEEV) nonstructural protein 2 (nsP2) cysteine protease (EC 3.4.22.-) is essential for viral replication and is involved in the cytopathic effects (CPE) of the virus. The VEEV nsP2 protease is a member of MEROPS Clan CN and characteristically contains a papain-like protease linked to an S-adenosyl-l-methionine-dependent RNA methyltransferase (SAM MTase) domain. The protease contains an alternative active site motif, (475)NVCWAK(480), which differs from papain's (CGS(25)CWAFS), and the enzyme lacks a transition state-stabilizing residue homologous to Gln-19 in papain. To understand the roles of conserved residues in catalysis, we determined the structure of the free enzyme and the first structure of an inhibitor-bound alphaviral protease. The peptide-like E64d inhibitor was found to bind beneath a ß-hairpin at the interface of the SAM MTase and protease domains. His-546 adopted a conformation that differed from that found in the free enzyme; one or both of the conformers may assist in leaving group departure of either the amine or Cys thiolate during the catalytic cycle. Interestingly, E64c (200 µM), the carboxylic acid form of the E64d ester, did not inhibit the nsP2 protease. To identify key residues involved in substrate binding, a number of mutants were analyzed. Mutation of the motif residue, N475A, led to a 24-fold reduction in kcat/Km, and the conformation of this residue did not change after inhibition. N475 forms a hydrogen bond with R662 in the SAM MTase domain, and the R662A and R662K mutations both led to 16-fold decreases in kcat/Km. N475 forms the base of the P1 binding site and likely orients the substrate for nucleophilic attack or plays a role in product release. An Asn homologous to N475 is similarly found in coronaviral papain-like proteases (PLpro) of the Severe Acute Respiratory Syndrome (SARS) virus and Middle East Respiratory Syndrome (MERS) virus. Mutation of another motif residue, K480A, led to a 9-fold decrease in kcat and kcat/Km. K480 likely enhances the nucleophilicity of the Cys. Consistent with our substrate-bound models, the SAM MTase domain K706A mutation increased Km 4.5-fold to 500 µM. Within the ß-hairpin, the N545A mutation slightly but not significantly increased kcat and Km. The structures and identified active site residues may facilitate the discovery of protease inhibitors with antiviral activity.