A Mechanistic Study of Trichoderma reesei Cel7B Catalyzed Glycosidic Bond Cleavage

An ONIOM study is performed to illustrate the mechanism of Trichoderma reesei Cel7B catalyzed <i>p</i>-nitrophenyl lactoside hydrolysis. In both the glycosylation and deglycosylation steps, the reaction proceeds in a concerted way, meaning the nucleophilic attack and the glycosidic bond cleavage occur simultaneously. The glycosylation step is rate limiting with a barrier of 18.9 kcal/mol, comparable to the experimental value derived from the <i>k</i>_cat measured in this work. The function of four residues R108, Y146, Y170, and D172, which form a hydrogen-bond network involving the substrate, is studied by conservative mutations. The mutants, including R108K, Y146F, Y170F, and D172N, decrease the enzyme activity by about 150–8000-fold. Molecular dynamics simulations show that the mutations disrupt the hydrogen-bond network, cause the substrate to deviate from active binding and hinder either the proton transfer from E201 to O₄(+1) or the nucleophilic attack from E196 to C₁(−1)

Protein Apparent Dielectric Constant and Its Temperature Dependence from Remote Chemical Shift Effects

A NMR protocol is introduced that permits accurate measurement of minute, remote chemical shift perturbations (CSPs), caused by a mutation-induced change in the electric field. Using protein GB3 as a model system, ¹H^N CSPs in K19A and K19E mutants can be fitted to small changes in the electric field at distal sites in the protein using the Buckingham equation, yielding an apparent dielectric constant ε_a of 8.6 ± 0.8 at 298 K. These CSPs, and their derived ε_a value, scale strongly with temperature. For example, CSPs at 313 K are about ∼30% smaller than those at 278 K, corresponding to an effective ε_a value of about 7.3 at 278 K and 10.5 at 313 K. Molecular dynamics simulations in explicit solvent indicate that solvent water makes a significant contribution to ε_a

Water-Soluble Conjugated Polymer as a Fluorescent Probe for Monitoring Adenosine Triphosphate Level Fluctuation in Cell Membranes during Cell Apoptosis and in Vivo

Adenosine triphosphate (ATP) is used as the energy source in cells and plays crucial roles in various cellular events. The cellular membrane is the protective barrier for the cytoplasm of living cells and involved in many essential biological processes. Many fluorescent probes for ATP have been successfully developed, but few of these probes were appropriate for visualizing ATP level fluctuation in cell membranes during the apoptotic cell death process. Herein, we report the synthesis of a new water-soluble cationic polythiophene derivative that can be utilized as a fluorescent sensor for detecting ATP in cell membranes. Poly­((3-((4-methylthiophen-3-yl)­oxy)­propyl)­triphenylphosphonium chloride) (PMTPP) exhibits high sensitivity and good selectivity to ATP, and the detection limit is 27 nM. The polymer shows low toxicity to live cells and excellent photostability in cell membranes. PMTPP was practically utilized for real-time monitoring of ATP levels in the cell membrane through fluorescence microscopy. We have demonstrated that the ATP levels in cell membranes increased during the apoptotic cell death process. The probe was also capable of imaging ATP levels in living mice

Water-Soluble Conjugated Polymer as a Fluorescent Probe for Monitoring Adenosine Triphosphate Level Fluctuation in Cell Membranes during Cell Apoptosis and in Vivo

Adenosine triphosphate (ATP) is used as the energy source in cells and plays crucial roles in various cellular events. The cellular membrane is the protective barrier for the cytoplasm of living cells and involved in many essential biological processes. Many fluorescent probes for ATP have been successfully developed, but few of these probes were appropriate for visualizing ATP level fluctuation in cell membranes during the apoptotic cell death process. Herein, we report the synthesis of a new water-soluble cationic polythiophene derivative that can be utilized as a fluorescent sensor for detecting ATP in cell membranes. Poly­((3-((4-methylthiophen-3-yl)­oxy)­propyl)­triphenylphosphonium chloride) (PMTPP) exhibits high sensitivity and good selectivity to ATP, and the detection limit is 27 nM. The polymer shows low toxicity to live cells and excellent photostability in cell membranes. PMTPP was practically utilized for real-time monitoring of ATP levels in the cell membrane through fluorescence microscopy. We have demonstrated that the ATP levels in cell membranes increased during the apoptotic cell death process. The probe was also capable of imaging ATP levels in living mice

Palladacycle from Cyclometalation of the Unsubstituted Cyclopentadienyl Ring in Ferrocene: Synthesis, Characterization, Theoretical Studies, and Application to Suzuki–Miyaura Reaction

The ferrocenylimines of general formula [(η⁵-C₅H₅)­Fe­(η⁵-C₅H₄)-CH₂NCH-C­(R)CH-C₆H₅] with R = H (<b>2a</b>) and CH₃ (<b>2b</b>) were conveniently prepared from ferrocenylmethylamine. Reaction of <b>2a</b>,<b>b</b> with lithium tetrachloropalladate in methanol in the presence of anhydrous sodium acetate resulted in the formation of the di-μ-chloro-bridged heteroannular cyclopalladated complexes <b>3a</b>,<b>b</b> via the unsubstituted ferrocenyl C–H bond activation of the related ligands. Treatment of <b>3a</b>,<b>b</b> with triphenylphosphine gave Pd­{[(η⁵-C₅H₄)­Fe­(η⁵-C₅H₄)­CH₂NCH-CHCH-C₆H₅]}­ClPPh₃ (<b>4a</b>) and Pd­{[(η⁵-C₅H₄)­Fe­(η⁵-C₅H₄)-CH₂NCH-C­(CH₃)CH-C₆H₅]}­ClPPh₃ (<b>4b</b>), respectively. The crystal structures of <b>4a</b>,<b>b</b> confirmed the formation of a carbon–palladium bond by using a carbon atom in the unsubstituted cyclopentadienyl ring. Additionally, theoretical studies using density functional theory calculations were carried out in order to account for the regioselectivity of cyclometalation. As for the catalysts, using 0.1% of palladacycles <b>4a</b>,<b>b</b> in the presence of K₃PO₄·7H₂O as base exhibited excellent yields in the Suzuki–Miyaura coupling reaction of aryl bromides with phenylboronic acid

Palladacycle from Cyclometalation of the Unsubstituted Cyclopentadienyl Ring in Ferrocene: Synthesis, Characterization, Theoretical Studies, and Application to Suzuki–Miyaura Reaction

The ferrocenylimines of general formula [(η⁵-C₅H₅)­Fe­(η⁵-C₅H₄)-CH₂NCH-C­(R)CH-C₆H₅] with R = H (<b>2a</b>) and CH₃ (<b>2b</b>) were conveniently prepared from ferrocenylmethylamine. Reaction of <b>2a</b>,<b>b</b> with lithium tetrachloropalladate in methanol in the presence of anhydrous sodium acetate resulted in the formation of the di-μ-chloro-bridged heteroannular cyclopalladated complexes <b>3a</b>,<b>b</b> via the unsubstituted ferrocenyl C–H bond activation of the related ligands. Treatment of <b>3a</b>,<b>b</b> with triphenylphosphine gave Pd­{[(η⁵-C₅H₄)­Fe­(η⁵-C₅H₄)­CH₂NCH-CHCH-C₆H₅]}­ClPPh₃ (<b>4a</b>) and Pd­{[(η⁵-C₅H₄)­Fe­(η⁵-C₅H₄)-CH₂NCH-C­(CH₃)CH-C₆H₅]}­ClPPh₃ (<b>4b</b>), respectively. The crystal structures of <b>4a</b>,<b>b</b> confirmed the formation of a carbon–palladium bond by using a carbon atom in the unsubstituted cyclopentadienyl ring. Additionally, theoretical studies using density functional theory calculations were carried out in order to account for the regioselectivity of cyclometalation. As for the catalysts, using 0.1% of palladacycles <b>4a</b>,<b>b</b> in the presence of K₃PO₄·7H₂O as base exhibited excellent yields in the Suzuki–Miyaura coupling reaction of aryl bromides with phenylboronic acid

Characterization of the Dielectric Constant in the <i>Trichoderma reesei</i> Cel7B Active Site

An attempt is made to evaluate the dielectric constant of the <i>Trichoderma reesei</i> Cel7B active site. Through kinetic measurements, the p<i>K</i>_a value of the catalytic acid E201 is determined. Mutations (away from E201) with net charge changes are introduced to perturb the E201 p<i>K</i>_a. It is shown that the mutation with a +1 charge change (including G225R, G230R, and A335R) decreases the p<i>K</i>_a of E201, whereas the mutation with a −1 charge change (including Q149E, A222D, G225D, and G230D) increases the p<i>K</i>_a. This effect is consistent with the electrostatic interaction between the changed charge and the E201 side chain. The fitting of the experimental data yields an apparent dielectric constant of 25–80. Molecular dynamics simulations with explicit water molecules indicate that the high solvent accessibility of the active site contributes largely to the high dielectric constant. ONIOM calculations show that high dielectric constant benefits the catalysis through decreasing the energy of the transition state relative to that of the enzyme substrate complex

Herbal Medicine-Inspired Carbon Quantum Dots with Antibiosis and Hemostasis Effects for Promoting Wound Healing

Bleeding and bacterial infections are crucial factors affecting wound healing. The usage of herbal medicine-derived materials holds great potential for promoting wound healing. However, the uncertain intrinsic effective ingredients and unclear mechanism of action remain great concerns. Herein, inspired by the herbal medicine Ligusticum wallichii, we reported the synthesis of tetramethylpyrazine-derived carbon quantum dots (TMP-CQDs) for promoting wound healing. Of note, the use of TMP as the precursor instead of L. wallichii ensured the repeatability and homogeneity of the obtained products. Furthermore, TMP-CQDs exhibited high antibacterial activity. Mechanically, TMP-CQDs inhibited the DNA repair, biosynthesis, and quorum sensing of the bacteria and induced intracellular reactive oxygen species (ROS). Moreover, TMP-CQDs could accelerate blood coagulation through activating factor VIII and promoting platelet aggregation. Effective wound healing was achieved by using TMP-CQDs in the Staphylococcus aureus-infected mouse skin wound model. This study sheds light on the development of herbal medicine-inspired materials as effective therapeutic drugs