The splitting of water photoelectrochemically into hydrogen and oxygen represents a promising technology for converting solar energy to fuel. The main challenge is to ensure that photogenerated holes efficiently oxidize water, which generally requires modification of the photoanode with an oxygen evolution catalyst (OEC) to increase the photocurrent and reduce the onset potential. However, because excess OEC material can hinder light absorption and decrease photoanode performance, its deposition needs to be carefully controlled--yet it is unclear which semiconductor surface sites give optimal improvement if targeted for OEC deposition, and whether sites catalysing water oxidation also contribute to competing charge-carrier recombination with photogenerated electrons. Surface heterogeneity exacerbates these uncertainties, especially for nanostructured photoanodes benefiting from small charge-carrier transport distances. Here we use super-resolution imaging, operated in a charge-carrier-selective manner and with a spatiotemporal resolution of approximately 30 nanometres and 15 milliseconds, to map both the electron- and hole-driven photoelectrocatalytic activities on single titanium oxide nanorods. We then map, with sub-particle resolution (about 390 nanometres), the photocurrent associated with water oxidation, and find that the most active sites for water oxidation are also the most important sites for charge-carrier recombination. Site-selective deposition of an OEC, guided by the activity maps, improves the overall performance of a given nanorod--even though more improvement in photocurrent efficiency correlates with less reduction in onset potential (and vice versa) at the sub-particle level. Moreover, the optimal catalyst deposition sites for photocurrent enhancement are the lower-activity sites, and for onset potential reduction the optimal sites are the sites with more positive onset potential, contrary to what is obtainable under typical deposition conditions. These findings allow us to suggest an activity-based strategy for rationally engineering catalyst-improved photoelectrodes, which should be widely applicable because our measurements can be performed for many different semiconductor and catalyst materials.

Binding and unbinding of transcription regulators at operator sites constitute a primary mechanism for gene regulation. While many cellular factors are known to regulate their binding, little is known on how cells can modulate their unbinding for regulation. Using nanometer-precision single-molecule tracking, we study the unbinding kinetics from DNA of two metal-sensing transcription regulators in living Escherichia coli cells. We find that they show unusual concentration-dependent unbinding kinetics from chromosomal recognition sites in both their apo- and holo-forms. Unexpectedly, their unbinding kinetics further varies with the extent of chromosome condensation, and more surprisingly, varies in opposite ways for their apo-repressor vs. holo-activator forms. These findings suggest likely broadly relevant mechanisms for facile switching between transcription activation and deactivation in vivo and in coordinating transcription regulation of resistance genes with the cell cycle.

Metalloregulators regulate transcription in response to metal ions. Many studies have provided insights into how transcription is activated upon metal binding by MerR-family metalloregulators. In contrast, how transcription is turned off after activation is unclear. Turning off transcription promptly is important, however, as the cells would not want to continue expressing metal resistance genes and thus waste energy after metal stress is relieved. Using single-molecule FRET measurements we studied the dynamic interactions of the copper efflux regulator (CueR), a Cu þ -responsive MerR-family metalloregulator, with DNA. Besides quantifying its DNA binding and unbinding kinetics, we discovered that CueR spontaneously flips its binding orientation at the recognition site. CueR also has two different binding modes, corresponding to interactions with specific and nonspecific DNA sequences, which would facilitate recognition localization. Most strikingly, a CueR molecule coming from solution can directly substitute for a DNA-bound CueR or assist the dissociation of the incumbent CueR, both of which are unique examples for any DNA-binding protein. The kinetics of the direct protein substitution and assisted dissociation reactions indicate that these two unique processes can provide efficient pathways to replace a DNA-bound holo-CueR with apo-CueR, thus turning off transcription promptly and facilely.single-molecule imaging | protein-DNA interaction dynamics B acteria often dwell in environments with high concentrations of metals. Some of these metals are essential, but many are toxic. Even the essential metals, for example iron and copper, can become detrimental above a certain concentration inside cells. Many biological processes are thus present to regulate and maintain intracellular metal homeostasis (1-9). One of them is through metalloregulators, which respond to metal ions and regulate the transcription of genes that protect the bacteria from metal-induced stress (5-7, 10). The MerR-family metalloregulators respond to many metal ions with high selectivity and sensitivity, such as Hg 2þ and Cu 2þ (5,11,12).All MerR-family metalloregulators are homodimeric proteins. They regulate transcription via a DNA distortion mechanism (5, 13-16). They recognize specific dyad-symmetric DNA sequences within a promoter, and both their apo and holo forms bind DNA tightly. In the absence of metal, the metalloregulator bends the DNA; in this configuration RNA polymerase (RNAp) cannot interact with both −10 and −35 sequences properly and transcription is repressed. Upon binding metal, the metalloregulator changes its conformation and further unwinds the DNA slightly to allow proper RNAp interactions with the −10 and −35 sequences; transcription is then activated.Although the mechanisms of transcription activation by MerRfamily metalloregulators are well-studied (5, 13-16), little is yet known about how transcription activation is reversed. Turning off transcription promptly is important, however, as the cells would not want to conti...

The exciton-dopant energy transfer time in colloidal Mn-doped CdS/ZnS core/shell nanocrystals was investigated via pump-probe transient absorption measurements. The energy transfer time was obtained from the comparative analysis of the dynamics of exciton relaxation in undoped and doped nanocrystal samples with well-defined doping radius and concentration. For this purpose, the transient absorption was measured in the near-IR region probing primarily intraband transition of excitons without signal saturation even at high exciton densities, which is particularly useful for studying the energy transfer in a wide range of exciton densities. With an increasing Mn doping concentration, exciton-dopant energy transfer became faster, effectively competing with both radiative and nonradiative relaxation of exciton. In the spherical Mn-doped CdS/ZnS core/shell nanocrystals studied here, the time scale of energy transfer between a single pair of exciton and Mn 2+ ion was determined to be ∼60 ps.

Dynamic protein-DNA interactions constitute highly robust cellular machineries to fulfill cellular functions. A vast number of studies have focused on how DNA-binding proteins search for and interact with their target DNA segments and on what cellular cues can regulate protein binding, for which protein concentration is a most obvious one. In contrast, how protein unbinding could be regulated by protein concentration has evaded attention because protein unbinding from DNA is typically a unimolecular reaction and thus concentration independent. Recent single-molecule studies from multiple research groups have uncovered that protein concentration can facilitate the unbinding of DNA-bound proteins, revealing regulation of protein unbinding as another mechanistic paradigm for gene regulation. In this Account, we review these recent in vitro and in vivo single-molecule experiments that uncovered the concentration-facilitated protein unbinding by multiple types of DNA-binding proteins, including sequence-nonspecific DNA-binding proteins (e.g., nucleoid-associated proteins, NAP), sequence-specific DNA-binding proteins (e.g., metal-responsive transcription regulators CueR and ZntR), sequence-neutral single-stranded DNA-binding proteins (e.g., Replication protein A, RPA), and DNA polymerases. For the in vitro experiments, Marko's group investigated the exchange of GFP-tagged DNA-bound NAPs with nontagged NAPs in solution of increasing concentration using single-molecule magnetic-tweezers fluorescence microscopy. The faster fluorescence intensity decrease with higher nontagged NAP concentrations suggests that DNA-bound NAPs undergo faster exchange with higher free NAP concentrations. Chen's group used single-molecule fluorescence resonance energy transfer measurements to study the unbinding of CueR from its cognate oligomeric DNA. The average microscopic dwell times of DNA-bound states become shorter with increasing CueR concentrations in the surroundings, demonstrating that free CueR proteins can facilitate the unbinding of the incumbent one on DNA through either assisted dissociation or direct substitution. Greene's group studied the unbinding of RPAs from single-stranded DNA using total internal reflection fluorescence microscopy and DNA curtain techniques. The fluorescence intensity versus time traces show faster decay with higher wild-type RPA concentrations, indicating that DNA-bound RPAs can undergo a concentration-facilitated exchange when encountering excess free RPA. van Oijen's group investigated the leading/lagging-strand polymerase exchange events in the bacteriophage T7 and E. coli replication systems using a combination of single-molecule fluorescence microscopy and DNA-flow-stretching assay. The processivity was observed to have larger decrease when the concentration of the Y526F polymerase mutant increases, indicating that the unbinding of the polymerase is also concentration-dependent. Using stroboscopic imaging and single-molecule tracking, Chen's group further advanced their study into living bacterial ...

INTRODUCTIONGeneration of hot electrons in metallic or semiconducting materials and hot electron-induced chemistry on their surfaces have been actively investigated for decades. For instance, optically excited hot electrons with excess energy above the Fermi level of metals demonstrated its capability to transfer part of the electron energy to the adsorbate molecules and excite their internal modes initiating various surface chemistries. 1À6 In semiconductors, hot electrons excited above the conduction band edge have been shown to be beneficial in catalysis and photovoltaic applications via fast interfacial charge transfer that can effectively compete with intraband relaxation. 7À9 Recently, a number of groups reported efficient interfacial charge transfer of optically excited hot electrons in semiconductor nanocrystals, 10À12 demonstrating the potential of harvesting hot electrons in nanocrystals that are widely employed in catalysis and photovoltaic applications. Hot electrons with larger excess energy will generally experience a lower energy barrier for interfacial charge transfer, and their wave functions can reach further away from the nanocrystals, making them potentially useful for photocatalytic and photovoltaic applications.Hot electrons in semiconductor nanocrystals can be excited via optical excitation in a number of different ways. The simplest way is the above-band-gap excitation with sufficient excess energy that can excite the electrons high in the conduction band. Hot electrons with higher excess energy generally require excitation with the higher-energy photons in the UV region, which is increasingly less convenient to work with. The lowerenergy photons can also excite hot electrons with large excess energy via multiphoton excitation. 13À15 However, multiphoton excitation is usually much less efficient than single photon excitation and requires high excitation intensity.

Optically induced ultrafast demagnetization and its recovery in superparamagnetic colloidal iron oxide (Fe3O4) nanocrystals have been investigated via time-resolved Faraday rotation measurements. Optical excitation with near-infrared laser pulse resulted in ultrafast demagnetization in approximately 100 fs via the destruction of ferrimagnetic ordering. The degree of demagnetization increased with the excitation density, and the complete demagnetization reached at approximately 10% excitation density. The magnetization recovered on two time scales, several picoseconds and hundreds of picoseconds, which can be associated with the initial reestablishment of the ferrimagnetic ordering and the electronic relaxation back to the ground state, respectively. The amplitude of the slower recovery component increased with the size of the nanocrystals, suggesting the size-dependent ferrimagnetic ordering throughout the volume of the nanocrystal.

Metalloregulators respond to metal ions to regulate transcription of metal homeostasis genes. MerR-family metalloregulators act on σ 70 -dependent suboptimal promoters and operate via a unique DNA distortion mechanism in which both the apo and holo forms of the regulators bind tightly to their operator sequence, distorting DNA structure and leading to transcription repression or activation, respectively. It remains unclear how these metalloregulator−DNA interactions are coupled dynamically to RNA polymerase (RNAP) interactions with DNA for transcription regulation. Using single-molecule FRET, we study how the copper efflux regulator (CueR)-a Cu + -responsive MerR-family metalloregulator-modulates RNAP interactions with CueR's cognate suboptimal promoter PcopA, and how RNAP affects CueR−PcopA interactions. We find that RNAP can form two noninterconverting complexes at PcopA in the absence of nucleotides: a dead-end complex and an open complex, constituting a branched interaction pathway that is distinct from the linear pathway prevalent for transcription initiation at optimal promoters. Capitalizing on this branched pathway, CueR operates via a "biased sampling" instead of "dynamic equilibrium shifting" mechanism in regulating transcription initiation; it modulates RNAP's binding-unbinding kinetics, without allowing interconversions between the deadend and open complexes. Instead, the apo-repressor form reinforces the dominance of the dead-end complex to repress transcription, and the holo-activator form shifts the interactions toward the open complex to activate transcription. RNAP, in turn, locks CueR binding at PcopA into its specific binding mode, likely helping amplify the differences between apo-and holo-CueR in imposing DNA structural changes. Therefore, RNAP and CueR work synergistically in regulating transcription.single-molecule FRET | protein-DNA interaction dynamics | MerR-family regulators | metal-responsive transcription regulation M aintaining cellular metal homeostasis is essential for bacteria, which often dwell in environments with high concentrations of metals. Some of these metals are purely toxic to bacteria, such as Cd and Hg. Many others are required for cellular function, such as Zn and Cu, but can be toxic in excess. Cells have thus developed many ways to regulate intracellular metal concentrations (1-9). Metal-responsive transcriptional regulation is one of them, where metalloregulators respond to intracellular metal ions and regulate transcription of metal efflux, uptake, or other metal homeostasis genes (4-9).In Gram-negative bacteria, MerR-family metalloregulators act on σ 70 -dependent suboptimal promoters to repress or activate transcription of metal resistance genes (7,8). These suboptimal promoters have elongated spacing, 19-20 bp (Fig. 1), compared with the optimal 17 ± 1 bp, between the −35 and −10 elements. This elongated spacing causes a misalignment of these two recognition elements, impairing proper interactions with the RNA polymerase (RNAP) and leading to a weak basal leve...