Characterization of human XPD helicase activity with single-molecule magnetic tweezers.

XPD helicase is a DNA-unwinding enzyme involved in DNA repair. As part of TFIIH, XPD opens a repair bubble in DNA for access by proteins in the nucleotide excision repair pathway. XPD uses the energy from ATP hydrolysis to translocate in the 5' to 3' direction on one strand of duplex DNA, displacing the opposite strand in the process. We used magnetic tweezers assays to measure the double-stranded DNA unwinding and single-stranded DNA translocation activities of human XPD in isolation. In our experimental setup, hXPD exhibited low unwinding processivity of ∼14 bp and slow unwinding rate of ∼0.3 bp/s. Measurements of the ssDNA translocation activity demonstrated that hXPD translocated on ssDNA at a similar rate as unwinding, revealing that slow rate was an intrinsic property of the hXPD translocation. Individual unwinding and translocation events were composed of pauses and runs with a distribution of lengths and rates. Analysis of these events unveiled similar mean run lengths and rates for unwinding and translocation, indicating that the unwinding behavior was a direct reflection of the translocation activity. The analysis also revealed that hXPD spent similar time stalling and unwinding/translocating. The detailed basal activity of hXPD reported here provides a baseline for future studies on how hXPD activity is regulated by other TFIIH components.

Microstructure optimization strategy of ZnIn2S4/rGO composites toward enhanced and tunable electromagnetic wave absorption properties.

Although microstructure optimization is an effective strategy to improve and regulate electromagnetic wave (EMW) absorption properties, preparing microwave absorbents with enhanced EMW absorbing performance and tuned absorption band by a simple method remains challenging. Herein, ZnIn2S4/reduced graphene oxide (rGO) composites with flower-like and cloud-like morphologies were fabricated by a convenient hydrothermal method. The ZnIn2S4/rGO composites with different morphologies realize efficient EMW absorption and tunable absorption bands, covering a wide frequency range. The flower-like structure has an optimal reflection loss (RL) of up to -49.2 dB with a maximum effective absorption bandwidth (EAB) of 5.7 GHz, and its main absorption peaks are concentrated in the C and Ku bands. The minimal RL of the cloud-like structure can reach -36.3 dB, and the absorption peak shifts to the junction of X and Ku bands. The distinguished EMW absorption capacity originates from the uniquely optimized microstructure, complementary effect of ZnIn2S4 and rGO in dielectric constant, and synergy of various loss mechanisms, such as interfacial polarization, dipole polarization, conductive loss, and multiple reflections. This study proposes a guide for the structural optimization of an ideal EMW absorber to achieve efficient and tunable EMW absorption performance.

One-Pot synthesis of flavones catalyzed by an Au-mediated covalent organic framework.

Covalent organic frameworks (COFs) are excellent candidates for rationally designed metal-coordinated catalysts due to their porous structures and adjustable organic building blocks. In this work, a two-dimensional (2D) COF with novel fxt topology was synthesized. The newly devised COF had been fully characterized by a range of spectroscopic and microscopic techniques. The COF was further metallized by the gold species to form a heterogeneous catalyst that enabled the one-pot synthesis of flavone and its derivatives. The Au@COF catalyst showed high catalytic activity and good recyclability. This work demonstrates the great potential of metallized COFs with unique well-defined pores in organic catalysis.

Synthesis of a Novel Spherical-Shell-Structure Polymerized Ionic Liquid Microsphere PILM/Au/Al(OH)3 Catalyst for Benzyl Alcohol Oxidation.

In order to selectively oxidize benzyl alcohol, a novel noble metal catalyst based on polymer ionic liquids with a core-shell structure was created. First, polymer ionic liquid microspheres (PILMs) were prepared by free radical polymerization. Second, the in situ adsorption of Au nanoparticles on the surface of PILMs was accomplished, thanks to the strong electrostatic interaction between N atoms and metal ions on the diazole ring of PILMs. Additionally, the introduction of Al(OH)3 prevented the aggregation of Au nanoparticles and promoted the catalytic reaction. Finally, the PILM/Au/Al(OH)3 catalyst with a core-shell structure was formed. The effectiveness of the PILM/Au/Al(OH)3 catalyst was assessed by varying the catalyst's type, quantity, amount of Au, amount of H2O2, temperature, and reaction time. After five cycles of experiments, the catalyst was effective and reusable. In addition, the potential catalytic mechanism of the catalyst in the oxidation of benzyl alcohol was proposed.

Characterization of human XPD helicase activity with Single Molecule Magnetic Tweezers.

XPD helicase is a DNA unwinding enzyme involved in multiple cellular processes. As part of TFIIH, XPD opens a repair bubble in DNA for access by proteins in the nucleotide excision repair pathway. XPD uses the energy from ATP hydrolysis to translocate in the 5-prime to 3-prime direction on one strand of duplex DNA, displacing the opposite strand in the process. We used magnetic tweezers assays to measure the double-stranded DNA (dsDNA) unwinding and single-stranded DNA (ssDNA) translocation activities of human XPD by itself. In our experimental setup, hXPD exhibits low unwinding processivity of ~14 bp and slow overall unwinding rate of ~0.3 bp/s. Individual unwinding and translocation events were composed of fast and slow runs and pauses. Analysis of these events gave similar mean run sizes and rates for unwinding and translocation, suggesting that unwinding is a reflection of translocation. The analysis also revealed that hXPD spent similar time stalling and unwinding. hXPD translocated on ssDNA at a similar overall rate as that of unwinding, pointing to an active helicase. However, we observed modest effects of DNA sequence on stalling and unwinding initiation position. Considering the slow unwinding rate, high probability of base pair separation at the ssDNA/dsDNA fork, and the observed DNA sequence dependences, we propose that hXPD is most likely a partially active helicase. Our results provide detailed information on the basal activity of hXPD which enhances our mechanistic understanding of hXPD activity.

Fe-based MOFs@Pd@COFs with spatial confinement effect and electron transfer synergy of highly dispersed Pd nanoparticles for Suzuki-Miyaura coupling reaction.

Controlling the spatial confinement effect and highly dispersed Pd nanoparticles (NPs) can help to improve applicability in catalysis, energy conversion, and separation. However, the nonspatial confinement effect, agglomeration of Pd NPs of catalyst and harsh reaction conditions have become the urgent problems to be solved in Suzuki-Miyaura cross-coupling reaction. Herein, we report the first application of a new MOFs@COFs by using core with metal organic frameworks (MOFs) NH2-MIL-101(Fe) and shell with covalent organic frameworks (COFs) for loading Pd NPs. The quickly formation of a transition state, the highly dispersed Pd NPs and the advancedly spatial confinement effect were achieved by coupling Fe base synergistic active components, electron-oriented anchoring with controlling pore scale, respectively. Most notably, as a proof-of-concept application, the high catalytic activity of NH2-MIL-101(Fe)@Pd@COFs(3 + 3) in catalysis is elucidated for Suzuki-Miyaura coupling reaction by the broad scope of the reactants and the preeminent yields of the products, together with excellent stability and recoverability. With this strategy, the mechanism of Suzuki-Miyaura coupling reaction was verified by examining the catalytic activity. We hope that our approach can further facilitate the study of the design and use of functional MOFs@Pd@COFs materials.

Comparison of Single and Multiple Turnovers of SecYEG in Escherichia coli.

Precursor proteins are translocated across the cytoplasmic membrane in Escherichia coli by the general secretory, or Sec, pathway. The main components of the pathway are the integral membrane heterotrimeric SecYEG complex and the peripheral membrane ATPase, SecA. In this study, we have applied an in vitro assay using inverted cytoplasmic membrane vesicles to investigate the complex cycle that leads to translocation. We compared the apparent rate constants for nine precursors under two experimental conditions, single turnover and multiple turnovers. For each precursor, the rate constant for a single turnover was higher than for multiple turnovers, indicating that a different step limits the rate under the two conditions. We conclude that the rate-limiting step for a single turnover is an early step in the initial phase of transit through the channel, whereas the rate of multiple turnovers is limited by the resetting of the translocon. The presence of the chaperone SecB during multiple turnovers increased the maximal amplitude translocated for the three precursor species tested, pGBP, pPhoA, and proOmpA, and also increased the apparent rate constants for both pGBP and pPhoA. The rate constant for proOmpA was decreased by the presence of SecB.IMPORTANCE Vastly different experimental techniques and conditions have been used to study export in E. coli We demonstrated that altering experimental conditions can change the step that is observed during study. Investigators should consider specific experimental conditions when comparing data from different laboratories, as well as when comparing data from different experiments within a laboratory. We have shown that each precursor species has inherent properties that determine the translocation rate; thus generalizations from studies of a single species must be made with caution. A summary of advantages and disadvantages in use of nine precursors is presented.

Protein Translocation Activity in Surface-Supported Lipid Bilayers.

Surface-supported lipid bilayers are used widely throughout the nanoscience community as cellular membrane mimics. For example, they are frequently employed in single-molecule atomic force microscopy (AFM) studies to shed light on membrane protein conformational dynamics and folding. However, in AFM as well as in other surface-sensing techniques, the close proximity of the supporting surface raises questions about preservation of the biochemical activity. Employing the model translocase from the general secretory (Sec) system of Escherichia coli, here we quantify the activity via two biochemical assays in surface-supported bilayers. The first assesses ATP hydrolysis and the second assesses polypeptide translocation across the membrane via protection from added protease. Hydrolysis assays revealed distinct levels of activation ranging from medium (translocase-activated) to high (translocation-associated) that were similar to traditional solution experiments and further identified an adenosine triphosphatase population exhibiting characteristics of conformational hysteresis. Translocation assays revealed turn over numbers that were comparable to solution but with a 10-fold reduction in apparent rate constant. Despite differences in kinetics, the chemomechanical coupling (ATP hydrolyzed per residue translocated) only varied twofold on glass compared to solution. The activity changed with the topographic complexity of the underlying surface. Rough glass coverslips were favored over atomically flat mica, likely due to differences in frictional coupling between the translocating polypeptide and surface. Neutron reflectometry and AFM corroborated the biochemical measurements and provided structural characterization of the submembrane space and upper surface of the bilayer. Overall, the translocation activity was maintained for the surface-adsorbed Sec system, albeit with a slower rate-limiting step. More generally, polypeptide translocation activity measurements yield valuable quantitative metrics to assess the local environment about surface-supported lipid bilayers.

Direct visualization of the E. coli Sec translocase engaging precursor proteins in lipid bilayers.

Escherichia coli exports proteins via a translocase comprising SecA and the translocon, SecYEG. Structural changes of active translocases underlie general secretory system function, yet directly visualizing dynamics has been challenging. We imaged active translocases in lipid bilayers as a function of precursor protein species, nucleotide species, and stage of translocation using atomic force microscopy (AFM). Starting from nearly identical initial states, SecA more readily dissociated from SecYEG when engaged with the precursor of outer membrane protein A as compared to the precursor of galactose-binding protein. For the SecA that remained bound to the translocon, the quaternary structure varied with nucleotide, populating SecA2 primarily with adenosine diphosphate (ADP) and adenosine triphosphate, and the SecA monomer with the transition state analog ADP-AlF3. Conformations of translocases exhibited precursor-dependent differences on the AFM imaging time scale. The data, acquired under near-native conditions, suggest that the translocation process varies with precursor species.

[HDMF]Cl-based DES as highly efficient extractants and catalysts for oxidative desulfurization of model oil.

N,N-Dimethylformamide hydrochloric acid/XMCl n ([HDMF]Cl/XMCl n , M = Zn or Fe, n = 2 or 3) was synthesized by stirring the mixture of [HDMF]Cl and metal chloride. [HDMF]Cl-based DES was characterized by FT-IR spectroscopy, ESI-MS and 1H-NMR spectroscopy. The oxidative desulfurization activity was investigated using [HDMF]Cl/0.2FeCl3 and [HDMF]Cl/ZnCl2 as the extractant and catalyst, and hydrogen peroxide (H2O2) as the oxidant. The desulfurization rate can reach up to 98.08% and 99.2% for DBT using [HDMF]Cl/0.2FeCl3 and [HDMF]Cl/ZnCl2, respectively. After recycling for 7 times, the removal rate of DBT still can reach more than 97%.

Single-molecule observation of nucleotide induced conformational changes in basal SecA-ATP hydrolysis.

SecA is the critical adenosine triphosphatase that drives preprotein transport through the translocon, SecYEG, in Escherichia coli. This process is thought to be regulated by conformational changes of specific domains of SecA, but real-time, real-space measurement of these changes is lacking. We use single-molecule atomic force microscopy (AFM) to visualize nucleotide-dependent conformations and conformational dynamics of SecA. Distinct topographical populations were observed in the presence of specific nucleotides. AFM investigations during basal adenosine triphosphate (ATP) hydrolysis revealed rapid, reversible transitions between a compact and an extended state at the ~100-ms time scale. A SecA mutant lacking the precursor-binding domain (PBD) aided interpretation. Further, the biochemical activity of SecA prepared for AFM was confirmed by tracking inorganic phosphate release. We conclude that ATP-driven dynamics are largely due to PBD motion but that other segments of SecA contribute to this motion during the transition state of the ATP hydrolysis cycle.

Stoichiometry of SecYEG in the active translocase of Escherichia coli varies with precursor species.

We have established a reconstitution system for the translocon SecYEG in proteoliposomes in which 55% of the accessible translocons are active. This level corresponds to the fraction of translocons that are active in vitro when assessed in their native environment of cytoplasmic membrane vesicles. Assays using these robust reconstituted proteoliposomes and cytoplasmic membrane vesicles have revealed that the number of SecYEG units involved in an active translocase depends on the precursor undergoing transfer. The active translocase for the precursor of periplasmic galactose-binding protein contains twice the number of heterotrimeric units of SecYEG as does that for the precursor of outer membrane protein A.

Dynamic structure of the translocon SecYEG in membrane: direct single molecule observations.

Purified SecYEG was reconstituted into liposomes and studied in near-native conditions using atomic force microscopy. These SecYEG proteoliposomes were active in translocation assays. Changes in the structure of SecYEG as a function of time were directly visualized. The dynamics observed were significant in magnitude (∼1-10 Å) and were attributed to the two large loops of SecY linking transmembrane helices 6-7 and 8-9. In addition, we identified a distribution between monomers and dimers of SecYEG as well as a smaller population of higher order oligomers. This work provides a new vista of the flexible and dynamic structure of SecYEG, an intricate and vital membrane protein.

Maximal efficiency of coupling between ATP hydrolysis and translocation of polypeptides mediated by SecB requires two protomers of SecA.

SecA is the ATPase that provides energy for translocation of precursor polypeptides through the SecYEG translocon in Escherichia coli during protein export. We showed previously that when SecA receives the precursor from SecB, the ternary complex is fully active only when two protomers of SecA are bound. Here we used variants of SecA and of SecB that populate complexes containing two protomers of SecA to different degrees to examine both the hydrolysis of ATP and the translocation of polypeptides. We conclude that the low activity of the complexes with only one protomer is the result of a low efficiency of coupling between ATP hydrolysis and translocation.

Sites of interaction of a precursor polypeptide on the export chaperone SecB mapped by site-directed spin labeling.

Export of protein into the periplasm of Escherichia coli via the general secretory system requires that the transported polypeptides be devoid of stably folded tertiary structure. Capture of the precursor polypeptides before they fold is achieved by the promiscuous binding to the chaperone SecB. SecB delivers its ligand to export sites through its specific binding to SecA, a peripheral component of the membrane translocon. At the translocon the ligand is passed from SecB to SecA and subsequently through the SecYEG channel. We have previously used site-directed spin labeling and electron paramagnetic resonance spectroscopy to establish a docking model between SecB and SecA. Here we report use of the same strategy to map the pathway of a physiologic ligand, the unfolded form of precursor galactose-binding protein, on SecB. Our set of SecB variants each containing a single cysteine, which was used in the previous study, has been expanded to 48 residues, which cover 49% of the surface of SecB. The residues on SecB involved in contacts were identified as those that, upon addition of the unfolded polypeptide ligand, showed changes in spectral line shape consistent with restricted motion of the nitroxide. We conclude that the bound precursor makes contact with a large portion of the surface of the small chaperone. The sites on SecB that interact with the ligand are compared with the previously identified sites that interact with SecA and a model for transfer of the ligand is discussed.

Mapping of the docking of SecA onto the chaperone SecB by site-directed spin labeling: insight into the mechanism of ligand transfer during protein export.

Export of protein into the periplasm of Escherichia coli via the general secretory system is achieved by action of a ternary complex comprising the polypeptide ligand, the chaperone SecB and SecA, a peripheral component of the membrane translocon, which is itself an ATPase. The unfolded ligand is captured initially by SecB and must be transferred to SecA and subsequently through the membrane translocon into the periplasm. We have taken the first steps in the elucidation of the mechanism of this dynamic transfer by determining the interface of interaction between SecB and SecA. Site-directed spin labeling and electron paramagnetic resonance spectroscopy were combined to identify which of the residues on SecB showed changes in spectral line shape upon addition of SecA. In all, 43% of the surface of SecB was covered by the 41 positions examined. A model of docking between SecB and SecA is proposed based on the pattern of amino acid residues on SecB shown to make contacts when in complex with SecA. This model in combination with previously published biochemical data provides insight into the transfer of the unfolded polypeptide from the chaperone SecB to SecA.

Asymmetric binding between SecA and SecB two symmetric proteins: implications for function in export.

SecB, a small tetrameric chaperone in Escherichia coli, facilitates export of precursor polypeptides from the cytoplasm to the periplasmic space. During this process, SecB displays two modes of binding. As a chaperone, it binds promiscuously to precursors to maintain them in a non-native conformation. SecB also demonstrates specific recognition of, and binding to, SecA. SecB with the precursor tightly bound enters an export-active complex with SecA and must pass the ligand to SecA at the translocon in the membrane. Here we use variants of SecA and SecB to further probe these interactions. We show that, unexpectedly, the binding between the two symmetric molecules is asymmetric and that the C-terminal alpha-helices of SecB bind in the interfacial region of the SecA dimer. We suggest that disruption of this interface by SecB facilitates conformational changes of SecA that are crucial to the transfer of the precursor from SecB to SecA.

Optimization of micellar liquid chromatographic separation of polycyclic aromatic hydrocarbons with the addition of second organic additive.

The micellar liquid chromatographic (MLC) separations of polycyclic aromatic hydrocarbons (PAHs) were optimized for three micellar systems, cetyltrimethylammonium chloride (CTAC), dodecyltrimethylammonium chloride (DTAC), and sodium dodecylsulfate (SDS), with 1-pentanol as the only organic additive. A difference in the separation was observed between CTAC and SDS/DTAC. Under each optimized separation conditions, CTAC-modified mobile phase provides the least desirable separation, which is attributed to its longer carbon tail (C16 vs. C12). In addition to 1-pentanol, the main organic additive, a second organic additive (3% 1-propanol) in the micelle-modified mobile phase was found to enhance the resolution of PAH chromatographic peaks. However, the extent of the enhancement varies for the different micellar systems, with the greatest resolution improvement seen for CTAC, and little effect for shorter-tail SDS and DTAC. This study shows the potential use of second organic additive (1-propanol), to the main nonpolar additive (1-pentanol), in facilitating the MLC separation of larger nonpolar compounds.

High-performance liquid chromatographic separation of polycyclic aromatic hydrocarbons using pyridinium chloride as a selective fluorescence quencher to aid detection.

The first use of pyridinium chloride (PC), as a selective fluorescence quenching agent of alternant polycyclic aromatic hydrocarbons (PAHs), under HPLC separation conditions is reported. PC was found to be superior to nitromethane, the only reported PAH selective quencher used in HPLC. The mobile phase addition of 0.03 M PC greatly simplifies the observed fluorescence-detected chromatograms for complex PAH mixtures, facilitating PAH identification. Stern-Volmer quenching constants (K(sv)) for PAHs were calculated from the chromatograms obtained under isocratic and gradient conditions and found to be similar. The K(sv) values were shown to be useful in establishing peak purity.