A High-Nuclearity Copper Sulfide Nanocluster [S-Cu50] Featuring a Double-Shell Structure Configuration with Cu(II)/Cu(I) Valences.

This work represents an important step in the quest for creating atomically precise binary semiconductor nanoclusters (BS-NCs). Compared with coinage metal NCs, the preparation of BS-NCs requires strict control of the reaction kinetics to guarantee the formation of an atomically precise single phase under mild conditions, which otherwise could lead to the generation of multiple phases. Herein, we developed an acid-assisted thiolate dissociation approach that employs suitable acid to induce cleavage of the S-C bonds in the Cu-S-R (R = alkyl) precursor, spontaneously fostering the formation of the [Cu-S-Cu] skeleton upon the addition of extra Cu sources. Through this method, a high-nuclearity copper sulfide nanocluster, Cu50S12(SC(CH3)3)20(CF3COO)12 (abbreviated as [S-Cu50] hereafter), has been successfully prepared in high yield, and its atomic structure was accurately modeled through single-crystal X-ray diffraction. It was revealed that [S-Cu50] exhibits a unique double-shell structural configuration of [Cu14S12]@[Cu36S20], and the innermost [Cu14] moiety displays a rhombic dodecahedron geometry, which has never been observed in previously synthesized Cu metal, hydride, or chalcogenide NCs. Importantly, [S-Cu50] represents the first example incorporating mixed Cu(II)/Cu(I) valences in reported atomically precise copper sulfide NCs, which was unambiguously confirmed by XPS, EPR, and XANES. In addition, the electronic structure of [S-Cu50] was established by a variety of optical investigations, including absorption, photoluminescence, and ultrafast transient absorption spectroscopies, as well as theoretical calculations. Moreover, [S-Cu50] is air-stable and demonstrates electrocatalytic activity in ORR with a four-electron pathway.

Single-Pore versus Dual-Pore Bipyridine-Based Covalent-Organic Frameworks: An Insight into the Heterogeneous Catalytic Activity for Selective CH Functionalization.

Exponential growth in the field of covalent-organic frameworks (COFs) is emanating from the direct correlation between designing principles and desired properties. The comparison of catalytic activity between single-pore and dual-pore COFs is of importance to establish structure-function relationship. Herein, the synthesis of imine-linked dual-pore [(BPyDC)]x % -ETTA COFs (x = 0%, 25%, 50%, 75%, 100%) with controllable bipyridine content is fulfilled by three-component condensation of 4,4',4″,4'″-(ethene-1,1,2,2-tetrayl)tetraaniline (ETTA), 4,4'-biphenyldialdehyde, and 2,2'-bipyridyl-5,5'-dialdehyde in different stoichiometric ratio. The strong coordination of bipyridine moieties of [(BPyDC)]x % -ETTA COFs with palladium imparts efficient catalytic active sites for selective functionalization of sp2 CH bond to CX (X = Br, Cl) or CO bonds in good yield. To broaden the scope of regioselective CH functionalization, a wide range of electronically and sterically substituted substrates under optimized catalytic condition are investigated. A comparison of the catalytic activity of palladium decorated dual-pore frameworks with single-pore imine-linked Pd(II) @ Py-2,2'-BPyDC framework  is undertaken. The finding of this work provides a sporadic example of chelation-assisted CH functionalization and disclosed an in-depth comparison of the relationship between superior catalytic activity and core properties of rationally designed imine linked frameworks.

Mono-/Bimetallic Neutral Iridium(III) Complexes Bearing Diketopyrrolopyrrole-Substituted N-Heterocyclic Carbene Ligands: Synthesis and Photophysics.

The synthesis and photophysics (UV-vis absorption, emission, and transient absorption) of four neutral heteroleptic cyclometalated iridium(III) complexes (Ir-1-Ir-4) incorporating thiophene/selenophene-diketopyrrolopyrrole (DPP)-substituted N-heterocyclic carbene (NHC) ancillary ligands are reported. The effects of thiophene versus selenophene substitution on DPP and bis- versus monoiridium(III) complexation on the photophysics of these complexes were systematically investigated via spectroscopic techniques and density functional theory calculations. All complexes exhibited strong vibronically resolved absorption in the regions of 500-700 nm and fluorescence at 600-770 nm, and both are predominantly originated from the DPP-NHC ligand. Complexation induced a pronounced red shift of this low-energy absorption band and the fluorescence band with respect to their corresponding ligands due to the improved planarity and extended π-conjugation in the DPP-NHC ligand. Replacing the thiophene units by selenophenes and/or biscomplexation led to the red-shifted absorption and fluorescence spectra, accompanied by the reduced fluorescence lifetime and quantum yield and enhanced population of the triplet excited states, as reflected by the stronger triplet excited-state absorption and singlet oxygen generation.

How Do Enzymes Orient When Trapped on Metal-Organic Framework (MOF) Surfaces?

Enzyme immobilization in metal-organic frameworks (MOFs) offers retained enzyme integrity and activity, enhanced stability, and reduced leaching. Trapping enzymes on MOF surfaces would allow for catalysis involving large substrates. In both cases, the catalytic efficiency and selectivity depend not only on enzyme integrity/concentration but also orientation. However, it has been a challenge to determine the orientation of enzymes that are supported on solid matrices, which is even more challenging for enzymes immobilized/trapped in MOFs due to the interferences of the MOF background signals. To address such challenge, we demonstrate in this work the utilization of site-directed spin labeling in combination with Electron Paramagnetic Resonance spectroscopy, which allows for the first time the characterization of the orientation of enzymes trapped on MOF surfaces. The obtained insights are fundamentally important for MOF-based enzyme immobilization design and understanding enzyme orientation once trapped in solid matrices or even cellular confinement conditions.

Insights on the Structure, Molecular Weight and Activity of an Antibacterial Protein-Polymer Hybrid.

Protein-polymer conjugates are attractive biomaterials which combine the functions of both proteins and polymers. The bioactivity of these hybrid materials, however, is often reduced upon conjugation. It is important to determine and monitor the protein structure and active site availability in order to optimize the polymer composition, attachment point, and abundance. The challenges in probing these insights are the large size and high complexity in the conjugates. Herein, we overcome the challenges by combining electron paramagnetic resonance (EPR) spectroscopy and atomic force microscopy (AFM) and characterize the structure of antibacterial hybrids formed by polyethylene glycol (PEG) and an antibacterial protein. We discovered that the primary reasons for activity loss were PEG blocking the substrate access pathway and/or altering protein surface charges. Our data indicated that the polymers tended to stay away from the protein surface and form a coiled conformation. The structural insights are meaningful for and applicable to the rational design of future hybrids.

Characterization and mechanism of phosphoinositide 3-kinases (PI3Ks) members in insulin-induced changes of protein metabolism in yellow catfish Pelteobagrus fulvidraco.

In the present study, seven phosphoinositide 3-kinase (PI3K) members (PI3KCa, PI3KCb, PI3KCd, PI3KCg, PI3KC2a, PI3KC2b and PI3KC3, respectively) were isolated and characterized from yellow catfish Pelteobagrus fulvidraco, and their roles in insulin-induced changes of protein metabolism were determined. These seven PI3Ks can be divided into three classes, class I (including PI3KCa, PI3KCb, PI3KCd and PI3KCg), class II (including PI3KC2a and PI3KC2b) and class III (only including PI3KC3). Compared with mammals, all of these members share similar domain structure. Their mRNAs were widely expressed across ten tested tissues (liver, white muscle, spleen, brain, gill, mesenteric fat, intestine, heart, kidney and ovary), but at variable levels. In the in vivo study, insulin treatment significantly increased hepatic protein content at 3h, accompanied with reduced plasma total amino acid contents and liver ALT activity, and with increased total RNA content and the mRNA levels of PI3KCb, PI3KC2a, AKT2, mTORC1 and S6K1 in liver. At 6h and 12h, insulin injection showed no significant effect on liver protein content and plasma total amino acid, but reduced liver ALT activity and increased liver total RNA and the mRNA levels of AKT2, mTORC1 and S6K1 in liver at 6h. In the in vitro study, insulin incubation also tended to increase protein content of hepatocytes, accompanied with reduced cell medium total amino acid contents and hepatocytes ALT activity, and increased total RNA content and the mRNA levels of PI3KCb, PI3KC2a, AKT2, mTORC1 and S6K1 in hepatocytes. However, insulin treatment showed no significant effect on GDH activity and mRNA expression of PI3KCa, PI3KCd, PI3KCg, PI3KC2b, PI3KC3 and eEF2 both in the in vivo and in vitro studies. Effects of insulin on the mRNA levels of eIF-4E and 4E-BP1 were different between the in vivo and in vitro studies, and also time-dependent. Compared to single insulin group, insulin+wortmannin group increased ALT activity at 6h but reduced T-RNA content at 6 and 12h. AKT2 and S6K1 mRNA levels at 6 and 12h, mRNA levels of mTORC1, 4E-BP1 and eEF2 at 3 and 6h, and EIF-4E mRNA levels at 3 and 12h, PI3KCb and PI3KC2a mRNA levels were significantly lower in insulin+wortmannin group than those in single insulin group. Thus, our study demonstrated that among seven PI3K members, PI3KCb and PI3KC2a were more sensitive to the insulin signaling pathway, and insulin stimulated hepatic protein synthesis in yellow catfish through PI3K signaling pathway.

Lysozyme-phenolics bioconjugates with antioxidant and antibacterial bifunctionalities: Structural basis underlying the dual-function.

This work aims at adopting an Electron Paramagnetic Resonance (EPR) spectroscopic technique to help understanding protein-phenolic conjugation and final functionalities relationship as well as the underlying structural basis of antioxidant and antibacterial dual functionalities. Specifically, lysozyme (Lys) was conjugated with two natural phenolic acids, i.e. rosmarinic acid (RA) and gentisic acid (GA, our previous work) with obviously different molecular features. Lys-RA displayed 8.6- and 4.0-times enhanced antioxidant stoichiometry compared to the native Lys and ones with GA, respectively, due to the stronger antioxidant activity of RA. However, RA conjugation mitigated both enzymatic and antibacterial activities of Lys-RA conjugates. Such inhibition effect is attributed to the greater structural and surface property changes of Lys upon conjugating with RA. Furthermore, the polyphenol conjugation related structural basis of disturbance, reactivity and selectivity were explored via site-directed spin labeling (SDSL)-EPR. A dynamic picture of reactivity and selectivity of phenolics conjugation on Lys was proposed.

Molecular interaction mechanism and structure-activity relationships of protein-polyphenol complexes revealed by side-directed spin labeling-electron paramagnetic resonance (SDSL-EPR) spectroscopy.

Deciphering interactions between bioactive protein and polyphenols are critical for designing and controlling functional protein-polyphenol complexes. Herein, using the site-directed spin labeled T4 lysozyme (T4L) and rosmarinic acid (RA) as a model system, we combined electron paramagnetic resonance spectra to investigate molecular interaction mechanism of the protein-polyphenol complexes in structural or conformational details. Experimental results show that molecular interactions between T4L and RA are a process from order to disorder. TEM images display that the complexes finally assemble into quasi-spherical colloidal particles. When T4L/RA ratio is 1:1, the complexes exhibit the optimized enzymatic and antioxidant dual-functionalities due to the synergetic effect and protection mechanism. However, with excess addition of RA, the enzymatic and antioxidant activities of the complexes started to attenuate because the catalytic active site and bioactive hydroxyl groups were buried. The revealed high-resolution interaction process could help better understand the corresponding alterations between structure and functionalities.

Metal-Organic Materials (MOMs) Enhance Proteolytic Selectivity, Efficiency, and Reusability of Trypsin: A Time-Resolved Study on Proteolysis.

Proteases are involved in essential biological functions in nature and have become drug targets recently. In spite of the promising progress, two challenges, (i) the intrinsic instability and (ii) the difficulty in monitoring the catalytic process in real time, still hinder the further understanding and engineering of protease functionalities. These challenges are caused by the lack of proper materials/approaches to stabilize proteases and monitor proteolytic products (truncated polypeptides) in real time in a highly heterogeneous reaction mixture. This work combines metal-organic materials (MOMs), site-directed spin labeling-electron paramagnetic resonance (SDSL-EPR) spectroscopy, and mass spectrometry (MS) to overcome both barriers. A model protease, trypsin, which cleaves the peptide bonds at lysine or arginine residues, was immobilized on a Ca-MOM via aqueous-phase, one-pot cocrystallization, which allows for trypsin protection and ease of separation from its proteolytic products. Time-resolved EPR and MS were employed to monitor the populations, rotational motion, and sequences of the cleaved peptide truncations of a model protein substrate as the reaction proceeded. Our data suggest a significant (at least 5-10 times) enhancement in the catalytic efficiency (kcat/km) of trypsin@Ca-MOM and excellent reusability as compared to free trypsin in solution. Surprisingly, entrapping trypsin in Ca-MOMs results in cleavage site/region selectivity against the protein substrate, as compared to the near nonselective cleavage of all lysine and arginine residues of the substrate in solution. Remarkably, immobilizing trypsin allows for the separation and, thus, MS study on the sequences of truncated peptides in real time, leading to a time-resolved "movie" of trypsin proteolysis. This work demonstrates the use of MOMs and cocrystallization to enhance the selectivity, catalytic efficiency, and stability of trypsin, suggesting the possibility of tuning the catalytic performance of a general protease using MOMs.

Storage Stability of 6FDA-DMB Polyamic Acid Solution Detected by Gel Permeation Chromatography Coupled with Multiple Detectors.

Polyamic acid (PAA) is the precursor of polyimide (PI), and its solution's properties have a direct influence on the final performances of PI resins, films, or fibers. The viscosity loss of a PAA solution over time is notorious. A stability evaluation and revelation of the degradation mechanism of PAA in a solution based on variations of molecular parameters other than viscosity with storage time is necessary. In this study, a PAA solution was prepared through the polycondensation of 4,4'-(hexafluoroisopropene) diphthalic anhydride (6FDA) and 4,4'-diamino-2,2'-dimethylbiphenyl (DMB) in DMAc. The stability of a PAA solution stored at different temperatures (-18, -12, 4, and 25 °C) and different concentrations (12 wt% and 0.15 wt%) was systematically investigated by measuring the molecular parameters, including Mw, Mn, Mw/Mn, Rg, and [η], using gel permeation chromatography coupled with multiple detectors (GPC-RI-MALLS-VIS) in a mobile phase 0.02 M LiBr/0.20 M HAc/DMF. The stability of PAA in a concentrated solution decreased, as shown by the reduction ratio of Mw from 0%, 7.2%, and 34.7% to 83.8% and that of Mn from 0%, 4.7%, and 30.0% to 82.4% with an increase of temperature from -18, -12, and 4 to 25 °C, respectively, after storage for 139 days. The hydrolysis of PAA in a concentrated solution was accelerated at high temperatures. Notably, at 25 °C, the diluted solution was much less stable than the concentrated one and exhibited an almost linear degradation rate within 10 h. The Mw and Mn decreased rapidly by 52.8% and 48.7%, respectively, within 10 h. Such faster degradation was caused by a greater water ratio and less entanglement of chains in the diluted solution. The degradation of (6FDA-DMB) PAA in this study did not follow the chain length equilibration mechanism reported in literature, given that both Mw and Mn declined simultaneously during storage.

Unveiling the orientation and dynamics of enzymes in unstructured artificial compartments of metal-organic frameworks (MOFs).

Confining enzymes in well-shaped MOF compartments is a promising approach to mimic the cellular environment of enzymes and determine enzyme structure-function relationship therein. Under the cellular crowding, however, enzymes can also be confined in unstructured spaces that are close to the shapes/outlines of the enzyme. Therefore, for a better understanding of enzymes in their physiological environments, it is necessary to study enzymes in these unstructured spaces. However, practically it is challenging to create compartments that are close to the outline of an enzyme and probe enzyme structural information therein. Here, for proof-of-principle, we confined a model enzyme, lysozyme, in the crystal defects of a MOF via co-crystallization, where lysozyme served as the nuclei for MOF crystal scaffolds to grow on so that unstructured spaces close to the outline of lysozyme are created, and determined enzyme relative orientation and dynamics. This effort is important for understanding enzymes in near-native environments and guiding the rational design of biocatalysts that mimic how nature confines enzymes.

Design, synthesis and characterization of lysozyme-gentisic acid dual-functional conjugates with antibacterial/antioxidant activities.

Both microbiological and chemical food spoilages remain to be the major challenges in the food industry's efforts to combat food waste and loss because of the lack of high efficacy food preservatives. In this study, dual-functional conjugates that simultaneously suppress both lipid oxidation and microorganism growth are fabricated by covalently conjugating natural antioxidant gentisic acid (GA) on native antibacterial lysozyme (Lys). The mixing ratio of Lys and GA determines the particle size, morphology, antioxidant activity, and antimicrobial performance of the ensuing conjugates. With more of GA being grafted, a drastic decrease in the net surface charge with the concomitant occurrence of aggregations are observed in the conjugates. The maximum antioxidant activity and antibacterial performance of the conjugates is achieved when Lys:GA molar ratio is 1:112. The findings could guide the rational design of future functional food ingredients that combine multiple natural bioactive compounds to effectively intervene food waste and loss.

Expanding the "Library" of Metal-Organic Frameworks for Enzyme Biomineralization.

Metal-organic frameworks (MOFs) are advanced platforms for enzyme immobilization. Enzymes can be entrapped via either diffusion (into pre-formed MOFs) or co-crystallization. Enzyme co-crystallization with specific metals/ligands in the aqueous phase, also known as biomineralization, minimizes the enzyme loss compared to organic phase co-crystallization, removes the size limitation on enzymes and substrates, and can potentially broaden the application of enzyme@MOF composites. However, not all enzymes are stable/functional in the presence of excess metal ions and/or ligands currently available for co-crystallization. Furthermore, most current biomineralization-based MOFs have limited (acid) pH stability, making it necessary to explore other metal-ligand combinations that can also immobilize enzymes. Here, we report our discovery on the combination of five metal ions and two ligands that can form biocomposites with two model enzymes differing in size and hydrophobicity in the aqueous phase under ambient conditions. Surprisingly, most of the formed composites are single- or multiphase crystals, even though the reaction phase is aqueous, with the rest as amorphous powders. All 20 enzyme@MOF composites showed good to excellent reusability and were stable under weakly acidic pH values. The stability under weakly basic conditions depended upon the selection of enzyme and metal-ligand combinations, yet for both enzymes, 3-4 MOFs offered decent stability under basic conditions. This work initiates the expansion of the current "library" of metal-ligand selection for encapsulating/biomineralizing large enzymes/enzyme clusters, leading to customized encapsulation of enzymes according to enzyme stability, functionality, and optimal pH.

Protocol for resolving enzyme orientation and dynamics in advanced porous materials via SDSL-EPR.

Enzyme encapsulation in metal-organic frameworks (MOFs)/covalent-organic frameworks (COFs) provides advancement in biocatalysis, yet the structural basis underlying the catalytic performance is challenging to probe. Here, we present an effective protocol to determine the orientation and dynamics of enzymes in MOFs/COFs using site-directed spin labeling and electron paramagnetic resonance spectroscopy. The protocol is demonstrated using lysozyme and can be generalized to other enzymes. For complete information on the generation and use of this protocol, please refer to Pan et al. (2021a).

Cascade/Parallel Biocatalysis via Multi-enzyme Encapsulation on Metal-Organic Materials for Rapid and Sustainable Biomass Degradation.

Multiple-enzyme cooperation simultaneously is an effective approach to biomass conversion and biodegradation. The challenge, however, lies in the interference of the involved enzymes with each other, especially when a protease is needed, and thus, the difficulty in reusing the enzymes; while extracting/synthesizing new enzymes costs energy and negative impact on the environment. Here, we present a unique approach to immobilize multiple enzymes, including a protease, on a metal-organic material (MOM) via co-precipitation in order to enhance the reusability and sustainability. We prove our strategy on the degradation of starch-containing polysaccharides (require two enzymes to degrade) and food proteins (require a protease to digest) before the quantification of total dietary fiber. As compared to the widely adopted "official" method, which requires the sequential addition of three enzymes under different conditions (pH/temperature), the three enzymes can be simultaneously immobilized on the surface of our MOM crystals to allow for contact with the large substrates (starch), while MOMs offer sufficient protection to the enzymes so that the reusability and long-term storage are improved. Furthermore, the same biodegradation can be carried out without adjusting the reaction condition, further reducing the reaction time. Remarkably, the simultaneous presence of all enzymes enhances the reaction efficiency by a factor of ∼3 as compared to the official method. To our best knowledge, this is the first experimental demonstration of using aqueous-phase co-precipitation to immobilize multiple enzymes for large-substrate biocatalysis. The significantly enhanced efficiency can potentially impact the food industry by reducing the labor requirement and enhancing enzyme cost efficiency, leading to reduced food cost. The reduced energy cost of extracting enzymes and adjusting reaction conditions minimize the negative impact on the environment. The strategy to prevent protease damage in a multi-enzyme system can be adapted to other biocatalytic reactions involving proteases.

Protein Detection using Quadratic Fit Analysis Near Dirac Point of Graphene Field Effect Biosensors.

Although graphene-based biosensors provid extreme sensitivity for the detection of atoms, gases, and biomolecules, the specificity of graphene biosensors to the target molecules requires surface decoration of graphene with bifunctional linkers such pyrene derivatives. Here, we demonstrate that the pyrene functionalization influences graphene's electrical properties by yielding partial formation of bilayer graphene which was confirmed by Raman 2D spectrum. Based on this observation, we introduce quadratic fit analysis of the nonlinear electrical behavior of pyrene-functionalized graphene near the Dirac point. Compared to the conventional linear fit analysis of the transconductance at a distance from the Dirac point, the quadratic fit analysis of the nonlinear transconductance near the Dirac point increased the overall protein detection sensitivity by a factor of 5. Furthermore, we show that both pyrene linkers and gating voltage near the Dirac point play critical roles in sensitive and reliable detection of proteins' biological activities with the graphene biosensors.

Size-Tunable Metal-Organic Framework-Coated Magnetic Nanoparticles for Enzyme Encapsulation and Large-Substrate Biocatalysis.

Immobilizing enzymes on nanoparticles (NPs) enhances the cost-efficiency of biocatalysis; however, when the substrates are large, it becomes difficult to separate the enzyme@NP from the products while avoiding leaching or damage of enzymes in the reaction medium. Metal-organic framework (MOF)-coated magnetic NPs (MNPs) offer efficient magnetic separation and enhanced enzyme protection; however, the involved enzymes/substrates have to be smaller than the MOF apertures. A potential solution to these challenges is coprecipitating metal/ligand with enzymes on the MNP surface, which can partially bury (protect) the enzyme below the composite surface while exposing the rest of the enzyme to the reaction medium for catalysis of larger substrates. Here, to prove this concept, we show that using Ca2+ and terephthalic acid (BDC), large-substrate enzymes can be encapsulated in CaBDC-MOF layers coated on MNPs via an enzyme-friendly, aqueous-phase one-pot synthesis. Interestingly, we found that using MNPs as the nuclei of crystallization, the composite size can be tuned so that nanoscale composites were formed under low Ca2+/BDC concentrations, while microscale composites were formed under high Ca2+/BDC concentrations. While the microscale composites showed significantly enhanced reusability against a non-structured large substrate, the nanoscale composites displayed enhanced catalytic efficiency against a rigid, crystalline-like large substrate, starch, likely due to the improved diffusivity of the nanoscale composites. To our best knowledge, this is the first report on aqueous-phase one-pot synthesis of size-tunable enzyme@MOF/MNP composites for large-substrate biocatalysis. Our platform can be applied to immobilize other large-substrate enzymes with enhanced reusability and tunable sizes.

Enzyme Immobilization on Graphite Oxide (GO) Surface via One-Pot Synthesis of GO/Metal-Organic Framework Composites for Large-Substrate Biocatalysis.

Although enzyme immobilization has improved many areas, biocatalysis involving large-size substrates is still challenging for immobilization platform design because of the protein damage under the often "harsh" reaction conditions required for these reactions. Our recent efforts indicate the potential of using Metal-Organic Frameworks (MOFs) to partially confine enzymes on the surface of MOF-based composites while offering sufficient substrate contact. Still, improvements are required to expand the feasible pH range and the efficiency of contacting substrates. In this contribution, we discovered that Zeolitic Imidazolate Framework (ZIF) and a new calcium-carboxylate based MOF (CaBDC) can both be coprecipitated with a model large-substrate enzyme, lysozyme (lys), to anchor the enzyme on the surface of graphite oxide (GO). We observed lys activity against its native substrate, bacterial cell walls, indicating lys was confined on composite surface. Remarkably, lys@GO/CaBDC displayed a stronger catalytic efficiency at pH 6.2 as compared to pH 7.4, indicating CaBDC is a good candidate for biocatalysis under acidic conditions as compared to ZIFs which disassemble under pH < 7. Furthermore, to understand the regions of lys being exposed to the reaction medium, we carried out a site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy study. Our data showed a preferential orientation of lys in GO/ZIF composite, whereas a random orientation in GO/CaBDC. This is the first report on immobilizing solution-state large-substrate enzymes on GO surface using two different MOFs via one-pot synthesis. These platforms can be generalized to other large-substrate enzymes to carry out catalysis under the optimal buffer/pH conditions. The orientation of enzyme at the molecular level on composite surfaces is critical for guiding the rational design of new composites.

Spatial Distribution and Solvent Polarity-Triggered Release of a Polypeptide Incorporated into Invertible Micellar Assemblies.

Extracting, stabilizing, or delivering biomacromolecules such as proteins and peptides in organic phases have potential applications in biocatalysis, protein extraction, and food antioxidation. However, most current delivery/stabilization platforms face various limitations such as protein/peptide molecular size, platform stability/reusability, and/or potential damage to the cargos. A potential solution to these problems is micellar self-assemblies from amphiphilic invertible polymers, which have recently been demonstrated to be powerful as molecular hosts to deliver both small molecular drugs and functional polypeptides in the aqueous phase. To better understand the function of biomacromolecules and predict the usefulness of the formed invertible micellar assemblies (IMAs) as biomacromolecular hosts in organic phases, it is critical to characterize the spatial distribution, structure, and dynamics of biomacromolecules in the IMA including those upon release. However, the background signals of the IMAs limit the application of most peptide characterization approaches. In this work, we overcome the technical barriers by using site-directed spin labeling electron paramagnetic resonance to probe the spatial arrangement and release of a model, the hemagglutinin (HA) peptide, in the IMAs formed from two different amphiphilic invertible polymers. By site-specifically probing three residues along the peptide chain, for the first time, we depict the possible spatial distribution of HA within the IMAs. By triggering the disassembly of the IMAs with a thermodynamically good solvent (in this study, acetone), we detailed the stability of IMAs in toluene and the peptide release conditions once the polarity of the medium changes. Our findings are important for the application of peptides/proteins at the polar-nonpolar interface or using this interface to extract or deliver biomacromolecules. Our work also demonstrates the power of SDSL-EPR on probing peptide or micelle dynamics, which can be generalized to understand proteins or other biomacromolecules in micellar polymer assemblies in varied applications.

A Mixed-Metal Porphyrinic Framework Promoting Gas-Phase CO2 Photoreduction without Organic Sacrificial Agents.

A photoactive porphyrinic metal-organic framework (MOF) has been prepared by exchanging Ti into a Zr-based MOF precursor. The resultant mixed-metal Ti/Zr porphyrinic MOF demonstrates much-improved efficiency for gas-phase CO2 photoreduction into CH4 and CO under visible-light irradiation using water vapor compared to the parent Zr-MOF. Insightful studies have been conducted to probe the photocatalysis processes. This work provides the first example of gas-phase CO2 photoreduction into methane without organic sacrificial agents on a MOF platform, thereby paving an avenue for developing MOF-based photocatalysts for application in CO2 photoreduction and other types of photoreactions.