Correction: Electrochemically shape-controlled synthesis of great stellated dodecahedral Au nanocrystals with high-index facets for nitrogen reduction to ammonia.

Correction for 'Electrochemically shape-controlled synthesis of great stellated dodecahedral Au nanocrystals with high-index facets for nitrogen reduction to ammonia' by Yu-Chen Jiang et al., Chem. Commun., 2020, DOI: 10.1039/d0cc04326e.

Affinity of Skp to OmpC revealed by single-molecule detection.

Outer membrane proteins (OMPs) are essential to gram-negative bacteria, and molecular chaperones prevent the OMPs from aggregation in the periplasm during the OMPs biogenesis. Skp is one of the molecular chaperones for this purpose. Here, we combined single-molecule fluorescence resonance energy transfer and fluorescence correlation spectroscopy to study the affinity and stoichiometric ratio of Skp in its binding with OmpC at the single-molecule level. The half concentration of the Skp self-trimerization (C1/2) was measured to be (2.5 ± 0.7) × 102 nM. Under an Skp concentration far below the C1/2, OmpC could recruit Skp monomers to form OmpC·Skp3. The affinity to form the OmpC·Skp3 complex was determined to be (5.5 ± 0.4) × 102 pM with a Hill coefficient of 1.6 ± 0.2. Under the micromolar concentrations of Skp, the formation of OmpC·(Skp3)2 was confirmed, and the dissociation constant of OmpC·(Skp3)2 was determined to be 1.2 ± 0.4 µM. The precise information will help us to quantitatively depict the role of Skp in the biogenesis of OMPs.

Electrochemically shape-controlled synthesis of great stellated dodecahedral Au nanocrystals with high-index facets for nitrogen reduction to ammonia.

Au great stellated dodecahedra (GSD), one of the Kepler-Poinsot solids, are synthesized by an electrochemical double-step potential method in a choline chloride-urea based deep eutectic solvent. The as-synthesized Au GSD are bound by high-index {331} facets and exhibit excellent electrocatalytic performance for the nitrogen reduction reaction with a high NH3 yield rate (49.96 µg h-1 cm-2) and faradaic efficiency (28.59%) under ambient conditions.

Target search and recognition mechanisms of glycosylase AlkD revealed by scanning FRET-FCS and Markov state models.

DNA glycosylase is responsible for repairing DNA damage to maintain the genome stability and integrity. However, how glycosylase can efficiently and accurately recognize DNA lesions across the enormous DNA genome remains elusive. It has been hypothesized that glycosylase translocates along the DNA by alternating between a fast but low-accuracy diffusion mode and a slow but high-accuracy mode when searching for DNA lesions. However, the slow mode has not been successfully characterized due to the limitation in the spatial and temporal resolutions of current experimental techniques. Using a newly developed scanning fluorescence resonance energy transfer (FRET)-fluorescence correlation spectroscopy (FCS) platform, we were able to observe both slow and fast modes of glycosylase AlkD translocating on double-stranded DNA (dsDNA), reaching the temporal resolution of microsecond and spatial resolution of subnanometer. The underlying molecular mechanism of the slow mode was further elucidated by Markov state model built from extensive all-atom molecular dynamics simulations. We found that in the slow mode, AlkD follows an asymmetric diffusion pathway, i.e., rotation followed by translation. Furthermore, the essential role of Y27 in AlkD diffusion dynamics was identified both experimentally and computationally. Our results provided mechanistic insights on how conformational dynamics of AlkD-dsDNA complex coordinate different diffusion modes to accomplish the search for DNA lesions with high efficiency and accuracy. We anticipate that the mechanism adopted by AlkD to search for DNA lesions could be a general one utilized by other glycosylases and DNA binding proteins.

Molecular mechanism of networking among DegP, Skp and SurA in periplasm for biogenesis of outer membrane proteins.

The biogenesis of outer membrane proteins (OMPs) is an extremely challenging process. In the periplasm of Escherichia coli, a group of quality control factors work together to exercise the safe-guard and quality control of OMPs. DegP, Skp and SurA are the three most prominent ones. Although extensive investigations have been carried out, the molecular mechanism regarding the networking among these proteins remains mostly mysterious. Our group has previously studied the molecular interactions of OMPs with SurA and Skp, using single-molecule detection (SMD). In this work, again using SMD, we studied how OmpC, a representative of OMPs, interacts with DegP, Skp and SurA collectively. Several important discoveries were made. The self-oligomerization of DegP to form hexamer occurs over hundred micromolars. When OmpC is in a monomer state at a low concentration, the OmpC·DegP6 and OmpC·DegP24 complexes form when the DegP concentration is around sub-micromolars and a hundred micromolars, respectively. High OmpC concentration promotes the binding affinity of DegP to OmpC by ∼100 folds. Skp and SurA behave differently when they interact synergistically with DegP in the presence of substrate. DegP can degrade SurA-protected OmpC, but Skp-protected OmpC forms the ternary complex OmpC·(Skp3)n·DegP6 (n = 1,2) to resist the DegP-mediated degradation. Combined with previous results, we were able to depict a comprehensive picture regarding the molecular mechanism of the networking among DegP, Skp and SurA in the periplasm for the OMPs biogenesis under physiological and stressed conditions.

Excavated cubic platinum-iridium alloy nanocrystals with high-index facets as highly efficient electrocatalysts in N2 fixation to NH3.

Excavated cubic Pt93Ir7 alloy nanocrystals enclosed by high-index {710} facets exhibit excellent electrocatalytic properties for the nitrogen reduction reaction (NRR) with a high faradaic efficiency (40.8%) and NH3 production rate (28 µg h-1 cm-2). The presence of Ir on the Pt stepped surface suppresses the hydrogen evolution reaction (HER) and accelerates the NRR.

Single-molecule study on conformational dynamics of M.HhaI.

We found that apo DNA methyltransferase M.HhaI under the physiological salt concentration does not possess the structure characterized by X-ray crystallography; instead, it interchanges between prefolded and unfolded states. Only after binding to the substrate, it transforms into a crystal-structure-like state. Flipping rates of its catalytic loop were directly measured.

Single-Molecule Detection Reveals Different Roles of Skp and SurA as Chaperones.

Skp and SurA are both periplasmic chaperones involved in the biogenesis of Escherichia coli ß-barrel outer membrane proteins (OMPs). It is commonly assumed that SurA plays a major role whereas Skp is a minor factor. However, there is no molecular evidence for whether their roles are redundant. Here, by using different dilution methods, we obtained monodisperse and aggregated forms of OmpC and studied their interactions with Skp and SurA by single-molecule fluorescence resonance energy transfer and fluorescence correlation spectroscopy. We found that Skp can dissolve aggregated OmpC while SurA cannot convert aggregated OmpC into the monodisperse form and the conformations of OmpC recognized by the two chaperones as well as their stoichiometries of binding are different. Our study demonstrates the functional distinctions between Skp and SurA. In particular, the role of Skp is not redundant and is probably more significant under stress conditions.

Urea hydrogen bond donor-mediated synthesis of high-index faceted platinum concave nanocubes grown on multi-walled carbon nanotubes and their enhanced electrocatalytic activity.

We have reported, for the first time, in situ growth of high-index {hk0} faceted concave Pt nanocubes on multi-walled carbon nanotubes (CNTs) via an electrochemical method in choline chloride-urea (ChCl-U) based deep eutectic solvents (DESs). Mechanistic studies indicate that a urea hydrogen bond donor (HBD) plays a key role in the formation of concave Pt nanocubes, in which the urea HBD preferentially adsorbs onto the {100} faces and blocks the growth of nanocrystals along the 〈100〉 axis. The as-prepared concave Pt nanocubes are characterized to be enclosed mainly with high-index {710}, {610} and {510} facets. It has been determined that the concave cubic Pt/CNT exhibits higher catalytic activity and stability than the flower-like Pt/CNT and commercial Pt/C catalysts, and this is ascribed to its high density of surface atomic steps and the synergistic effect between the CNT and Pt nanocubes.

Determination of Equilibrium Constant and Relative Brightness in FRET-FCS by Including the Third-Order Correlations.

Fluorescence correlation spectroscopy (FCS) encodes the information on the equilibrium constant (K), the relative fluorescence brightness of fluorophore (Q), and the forward and backward reaction rate constants (k+ and k-) on a physical or chemical relaxation. However, it has been a long-standing problem to completely resolve the FCS data to get the thermodynamic and kinetic information. Recently, we have solved the problem for fluorescence autocorrelation spectroscopy (FACS). Here, we extend the method to fluorescence cross-correlation spectroscopy (FCCS), which appears when FCS is coupled with fluorescence resonance energy transfer (FRET). Among 12 total second-order and third-order pre-exponential factors in a relaxation process probed by the FRET-FCS technique, 3 are independent. We presented and discussed 3 sets of explicit solutions to use these pre-exponential factors to calculate K and Q. Together with the relaxation time, the acquired K will allow people to obtain k+ and k-, so that the goal of deciphering the FRET-FCS data will be fully reached. The theory is verified by extensive computer simulations and tested experimentally on a system of oligonucleotide hybridization.

Seeds and Potentials Mediated Synthesis of High-Index Faceted Gold Nanocrystals with Enhanced Electrocatalytic Activities.

Because high-index facets (HIFs) possess high surface energy, the metal nanoparticles enclosed with HIFs are eliminated during their growth in a conventional shape-controlled synthesis due to the thermodynamics that drives the particles minimizing their total surface energy. This study develops a double-step potential method to prepare an unprecedentedly stellated Au nanocrystals (NCs) bounded by high-index {711} and {331} facets in deep eutectic solvent (DES) medium. The formation of Au NCs bounded by HIFs was systematically studied. It has demonstrated that the shapes of Au NCs are strongly dependent on the size of seeds and the growth potentials as well as the urea adsorbates in the DES. By adjusting the size of seeds and the growth potentials, the stellated Au NCs can be transformed into concave hexoctahedra (HOH) with high-index {421} facets and concave trisoctahedra (TOH) with high-index {991} facets. The electrocatalytic activities of the as-prepared Au NCs are evaluated by glucose oxidation. Thanks to HIFs having high density of atomic steps and kinks, the stellated, TOH, and HOH Au NCs exhibit higher electrocatalytic activity than that of the polycrystalline Au electrode, demonstrating that the steps and kinks serve as the active sites and play an important role in glucose electro-oxidation.

Determination of Equilibrium Constant and Relative Brightness in Fluorescence Correlation Spectroscopy by Considering Third-Order Correlations.

Fluorescence correlation spectroscopy (FCS) is a powerful tool to investigate molecular diffusion and relaxations, which may be utilized to study many problems such as molecular size and aggregation, chemical reaction, molecular transportation and motion, and various kinds of physical and chemical relaxations. This article focuses on a problem related to using the relaxation term to study a reaction. If two species with different fluorescence photon emission efficiencies are connected by a reaction, the kinetic and equilibrium properties will be manifested in the relaxation term of the FCS curve. However, the conventional FCS alone cannot simultaneously determine the equilibrium constant (K) and the relative fluorescence brightness (Q), both of which are indispensable in the extraction of thermodynamic and kinetic information from the experimental data. To circumvent the problem, an assumption of Q = 0 is often made for the weak fluorescent species, which may lead to numerous errors when the actual situation is not the case. We propose to combine the third-order FCS with the conventional second-order FCS to determine K and Q without invoking other resources. The strategy and formalism are verified by computer simulations and demonstrated in a classical example of the hairpin DNA-folding process.

Scanning Single-Molecule Fluorescence Correlation Spectroscopy Enables Kinetics Study of DNA Hairpin Folding with a Time Window from Microseconds to Seconds.

Single-molecule fluorescence measurements have been widely used to explore kinetics and dynamics of biological systems. Among them, single-molecule imaging (SMI) is good at tracking processes slower than tens of milliseconds, whereas fluorescence correlation spectroscopy (FCS) is good at probing processes faster than submilliseconds. However, there is still shortage of simple yet effective single-molecule fluorescence method to cover the time-scale between submilliseconds and tens of milliseconds. To effectively bridge this millisecond gap, we developed a single-molecule fluorescence correlation spectroscopy (smFCS) method that works on surface-immobilized single molecules through surface scanning. We validated it by monitoring the classical DNA hairpin folding process. With a wide time window from microseconds to seconds, the experimental data are well fitted to the two-state folding model. All relevant molecular parameters, including the relative fluorescence brightness, equilibrium constant, and reaction rate constants, were uniquely determined.

Accurate placement of substrate RNA by Gar1 in H/ACA RNA-guided pseudouridylation.

H/ACA RNA-guided ribonucleoprotein particle (RNP), the most complicated RNA pseudouridylase so far known, uses H/ACA guide RNA for substrate capture and four proteins (Cbf5, Nop10, L7Ae and Gar1) for pseudouridylation. Although it was shown that Gar1 not only facilitates the product release, but also enhances the catalytic activity, the chemical role that Gar1 plays in this complicated machinery is largely unknown. Kinetics measurement on Pyrococcus furiosus RNPs at different temperatures making use of fluorescence anisotropy showed that Gar1 reduces the catalytic barrier through affecting the activation entropy instead of enthalpy. Site-directed mutagenesis combined with molecular dynamics simulations demonstrated that V149 in the thumb loop of Cbf5 is critical in placing the target uridine to the right position toward catalytic D85 of Cbf5. The enzyme elegantly aligns the position of uridine in the catalytic site with the help of Gar1. In addition, conversion of uridine to pseudouridine results in a rigid syn configuration of the target nucleotide in the active site and causes Gar1 to pull out the thumb. Both factors guarantee the efficient release of the product.

Periplasmic quality control in biogenesis of outer membrane proteins.

The ß-barrel outer membrane proteins (OMPs) are integral membrane proteins that reside in the outer membrane of Gram-negative bacteria and perform a diverse range of biological functions. Synthesized in the cytoplasm, OMPs must be transported across the inner membrane and through the periplasmic space before they are assembled in the outer membrane. In Escherichia coli, Skp, SurA and DegP are the most prominent factors identified to guide OMPs across the periplasm and to play the role of quality control. Although extensive genetic and biochemical analyses have revealed many basic functions of these periplasmic proteins, the mechanism of their collaboration in assisting the folding and insertion of OMPs is much less understood. Recently, biophysical approaches have shed light on the identification of the intricate network. In the present review, we summarize recent advances in the characterization of these key factors, with a special emphasis on the multifunctional protein DegP. In addition, we present our proposed model on the periplasmic quality control in biogenesis of OMPs.

EF-G catalyzes tRNA translocation by disrupting interactions between decoding center and codon-anticodon duplex.

During translation, elongation factor G (EF-G) catalyzes the translocation of tRNA2-mRNA inside the ribosome. Translocation is coupled to a cycle of conformational rearrangements of the ribosomal machinery, and how EF-G initiates translocation remains unresolved. Here we performed systematic mutagenesis of Escherichia coli EF-G and analyzed inhibitory single-site mutants of EF-G that preserved pretranslocation (Pre)-state ribosomes with tRNAs in A/P and P/E sites (Pre-EF-G). Our results suggest that the interactions between the decoding center and the codon-anticodon duplex constitute the barrier for translocation. Catalysis of translocation by EF-G involves the factor's highly conserved loops I and II at the tip of domain IV, which disrupt the hydrogen bonds between the decoding center and the duplex to release the latter, hence inducing subsequent translocation events, namely 30S head swiveling and tRNA2-mRNA movement on the 30S subunit.

Dynamics of spontaneous flipping of a mismatched base in DNA duplex.

DNA base flipping is a fundamental theme in DNA biophysics. The dynamics for a B-DNA base to spontaneously flip out of the double helix has significant implications in various DNA-protein interactions but are still poorly understood. The spontaneous base-flipping rate obtained previously via the imino proton exchange assay is most likely the rate of base wobbling instead of flipping. Using the diffusion-decelerated fluorescence correlation spectroscopy together with molecular dynamics simulations, we show that a base of a single mismatched base pair (T-G, T-T, or T-C) in a double-stranded DNA can spontaneously flip out of the DNA duplex. The extrahelical lifetimes are on the order of 10 ms, whereas the intrahelical lifetimes range from 0.3 to 20 s depending on the stability of the base pairs. These findings provide detailed understanding on the dynamics of DNA base flipping and lay down foundation to fully understand how exactly the repair proteins search and locate the target mismatched base among a vast excess of matched DNA bases.

Identification of FkpA as a key quality control factor for the biogenesis of outer membrane proteins under heat shock conditions.

The outer membrane proteins (OMPs) of Gram-negative bacterial cells, as well as the mitochondrion and chloroplast organelles, possess unique and highly stable ß-barrel structures. Biogenesis of OMPs in Escherichia coli involves such periplasmic chaperones as SurA and Skp. In this study, we found that the ΔsurA Δskp double-deletion strain of E. coli, although lethal and defective in the biogenesis of OMPs at the normal growth temperature, is viable and effective at the heat shock temperature. We identified FkpA as the multicopy suppressor for the lethal phenotype of the ΔsurA Δskp strain. We also demonstrated that the deletion of fkpA from the ΔsurA cells resulted in only a mild decrease in the levels of folded OMPs at the normal temperature but a severe decrease as well as lethality at the heat shock temperature, whereas the deletion of fkpA from the Δskp cells had no detectable effect on OMP biogenesis at either temperature. These results strongly suggest a functional redundancy between FkpA and SurA for OMP biogenesis under heat shock stress conditions. Mechanistically, we found that FkpA becomes a more efficient chaperone for OMPs under the heat shock condition, with increases in both binding rate and affinity. In light of these observations and earlier reports, we propose a temperature-responsive OMP biogenesis mechanism in which the degrees of functional importance of the three chaperones are such that SurA > Skp > FkpA at the normal temperature but FkpA ≥ SurA > Skp at the heat shock temperature.

Single-molecule detection reveals knot sliding in TrmD denaturation.

An increasing number of proteins are found to contain a knot in their polypeptide chain. Although some studies have looked into the folding mechanism of knotted proteins, why and how these complex topologies form are still far from being fully answered. Moreover, no experimental information about how the knot moves during the protein-folding process is available. Herein, by combining single-molecule fluorescence resonance energy transfer (smFRET) experiments with molecular dynamics (MD) simulations, we performed a detailed study to characterize the knot in the denatured state of TrmD, a knotted tRNA (guanosine-1) methyltransferase from Escherichia coli, as a model system. We found that the knot still existed in the unfolded state of TrmD, consistent with the results for two other knotted proteins, YibK and YbeA. More interestingly, both smFRET experiments and MD simulations revealed that the knot slid towards the C-terminal during the unfolding process, which could be explained by the relatively strong interactions between the ß-sheet core at the N terminal of the native knot region. The size of the knot in the unfolded state is not larger than that in the native state. In addition, the knot slid in a "downhill" mode with simultaneous chain collapse in the denatured state.