The cysteine S-H stretching mode reports on long-range conformational changes in pyruvate oxidase from E. coli.

The pyruvate oxidases from Escherichia coli (EcPOX) and Lactobacillus plantarum (LpPOX) are both thiamin-dependent flavoenzymes. Their sequence and structure are closely related, and they catalyse similar reactions-but they differ in their activity pattern: LpPOX is always highly active, EcPOX only when activated by lipids or limited proteolysis, both involving the protein's C-terminal 23 residues (the 'α-peptide'). Here, we relate the redox-induced infrared (IR) difference spectrum of EcPOX to its unusual activation mechanism. The IR difference spectrum of EcPOX is marked by contributions from the protein backbone, reflecting major conformational changes. A rare sulfhydryl (-SH) difference signal indicates changes in the vicinity of cysteines. We could pin the Cys-SH difference signal to Cys88 and Cys494, both being remote from the moving α-peptide and the redox-active flavin cofactor. Yet, when the α-peptide is proteolytically removed, the Cys-SH difference signal disappears, together with several difference signals in the amide range. The remaining IR signature of the permanently activated EcPOXΔ23 is strikingly similar to the simpler signature of LpPOX. The loss of the α-peptide 'transforms' the catalytically complex EcPOX into the catalytically 'simpler' LpPOX.

Versatile Vibrational Energy Sensors for Proteins.

Vibrational energy transfer (VET) is emerging as key mechanism for protein functions, possibly playing an important role for energy dissipation, allosteric regulation, and enzyme catalysis. A deep understanding of VET is required to elucidate its role in such processes. Ultrafast VIS-pump/IR-probe spectroscopy can detect pathways of VET in proteins. However, the requirement of having a VET donor and a VET sensor installed simultaneously limits the possible target proteins and sites; to increase their number we compare six IR labels regarding their utility as VET sensors. We compare these labels in terms of their FTIR, and VET signature in VET donor-sensor dipeptides in different solvents. Furthermore, we incorporated four of these labels in PDZ3 to assess their capabilities in more complex systems. Our results show that different IR labels can be used interchangeably, allowing for free choice of the right label depending on the system under investigation and the methods available.

Through bonds or contacts? Mapping protein vibrational energy transfer using non-canonical amino acids.

Vibrational energy transfer (VET) is essential for protein function. It is responsible for efficient energy dissipation in reaction sites, and has been linked to pathways of allosteric communication. While it is understood that VET occurs via backbone as well as via non-covalent contacts, little is known about the competition of these two transport channels, which determines the VET pathways. To tackle this problem, we equipped the ß-hairpin fold of a tryptophan zipper with pairs of non-canonical amino acids, one serving as a VET injector and one as a VET sensor in a femtosecond pump probe experiment. Accompanying extensive non-equilibrium molecular dynamics simulations combined with a master equation analysis unravel the VET pathways. Our joint experimental/computational endeavor reveals the efficiency of backbone vs. contact transport, showing that even if cutting short backbone stretches of only 3 to 4 amino acids in a protein, hydrogen bonds are the dominant VET pathway.

Proton-Coupled Electron Transport in Two Distinct CYBASC Paralogs of Arabidopsis thaliana: A Comparative Characterization of Highly Conserved Tyrosine and Lysine Residues.

CYBASC proteins are ascorbate (AscH-) reducible, diheme b-containing integral membrane cytochrome b561 proteins (cytb561), which are proposed to be involved in AscH- recycling and facilitation of iron absorption. Two distinct CYBASC paralogs from the plant Arabidopsis thaliana, Atcytb561-A (A-paralog) and Atcytb561-B (B-paralog), have been found to differ in their visible-spectral characteristics and their interaction with AscH- and ferric iron chelates. A previously determined crystal structure of the B-paralog provides the first insights into the structural organization of a CYBASC member and implies hydrogen bonding between the substrate AscH- and the conserved lysine residues at positions 77 (B-K77) and 81 (B-K81). The function of the highly conserved tyrosine at position 70 (B-Y70) is not obvious in the crystal structure, but its localization indicates the possible involvement in proton-coupled electron transfer. Here we show that B-Y70 plays a major role in the modulation of the oxidation-reduction midpoint potential of the high-potential heme, EM(bH), as well as in AscH- oxidation. Our results support the involvement of the functionally conserved B-K77 in the stabilization of the dianion Asc2-. These findings are supported by the crystal structure of the B-paralog, but a comparative biochemical and biophysical characterization of the A- and B-paralogs implied distinct and more complex functions of the corresponding residues A-Y69 and A-K76 in the A-paralog. Our results emphasize the need for a high-resolution crystal structure of the A-paralog to illuminate the differences in functional organization between the two paralogs.

Site-Resolved Observation of Vibrational Energy Transfer Using a Genetically Encoded Ultrafast Heater.

Allosteric information transfer in proteins has been linked to distinct vibrational energy transfer (VET) pathways in a number of theoretical studies. Experimental evidence for such pathways, however, is sparse because site-selective injection of vibrational energy into a protein, that is, localized heating, is required for their investigation. Here, we solved this problem by the site-specific incorporation of the non-canonical amino acid ß-(1-azulenyl)-l-alanine (AzAla) through genetic code expansion. As an exception to Kasha's rule, AzAla undergoes ultrafast internal conversion and heating after S1 excitation while upon S2 excitation, it serves as a fluorescent label. We equipped PDZ3, a protein interaction domain of postsynaptic density protein 95, with this ultrafast heater at two distinct positions. We indeed observed VET from the incorporation sites in the protein to a bound peptide ligand on the picosecond timescale by ultrafast IR spectroscopy. This approach based on genetically encoded AzAla paves the way for detailed studies of VET and its role in a wide range of proteins.

Delicate Structural Control of Si-SiOx-C Composite via High-Speed Spray Pyrolysis for Li-Ion Battery Anodes.

Despite the high theoretical capacity, silicon (Si) anodes in lithium-ion batteries have difficulty in meeting the commercial standards in various aspects. In particular, the huge volume change of Si makes it very challenging to simultaneously achieve high initial Coulombic efficiency (ICE) and long-term cycle life. Herein, we report spray pyrolysis to prepare Si-SiOx composite using an aqueous precursor solution containing Si nanoparticles, citric acid, and sodium hydroxide (NaOH). In the precursor solution, Si nanoparticles are etched by NaOH with the production of [SiO4]4-. During the dynamic course of spray pyrolysis, [SiO4]4- transforms to SiOx matrix and citric acid decomposes to carbon surface layer with the assistance of NaOH that serves as a decomposition catalyst. As a result, a Si-SiOx composite, in which Si nanodomains are homogeneously embedded in the SiOx matrix with carbon surface layer, is generated by a one-pot process with a residence time of only 3.5 s in a flow reactor. The optimal composite structure in terms of Si domain size and Si-to-O ratio exhibited excellent electrochemical performance, such as reversible capacity of 1561.9 mAh g-1 at 0.06C rate and ICE of 80.2% and 87.9% capacity retention after 100 cycles at 1C rate.

UV-VIS absorption spectroscopy: Lambert-Beer reloaded.

UV-VIS absorption spectroscopy is used in almost every spectroscopy laboratory for routine analysis or research. All spectroscopists rely on the Lambert-Beer Law but many of them are less aware of its limitations. This tutorial discusses typical problems in routine spectroscopy that come along with technical limitations or careless selection of experimental parameters. Simple rules are provided to avoid these problems.

Controlled Prelithiation of Silicon Monoxide for High Performance Lithium-Ion Rechargeable Full Cells.

Despite the recent considerable progress, the reversibility and cycle life of silicon anodes in lithium-ion batteries are yet to be improved further to meet the commercial standards. The current major industry, instead, adopts silicon monoxide (SiOx, x ≈ 1), as this phase can accommodate the volume change of embedded Si nanodomains via the silicon oxide matrix. However, the poor Coulombic efficiencies (CEs) in the early period of cycling limit the content of SiOx, usually below 10 wt % in a composite electrode with graphite. Here, we introduce a scalable but delicate prelithiation scheme based on electrical shorting with lithium metal foil. The accurate shorting time and voltage monitoring allow a fine-tuning on the degree of prelithiation without lithium plating, to a level that the CEs in the first three cycles reach 94.9%, 95.7%, and 97.2%. The excellent reversibility enables robust full-cell operations in pairing with an emerging nickel-rich layered cathode, Li[Ni0.8Co0.15Al0.05]O2, even at a commercial level of initial areal capacity of 2.4 mAh cm(-2), leading to a full cell energy density 1.5-times as high as that of graphite-LiCoO2 counterpart in terms of the active material weight.

Dynamic Cross-Linking of Polymeric Binders Based on Host-Guest Interactions for Silicon Anodes in Lithium Ion Batteries.

We report supramolecular cross-linking of polymer binders via dynamic host-guest interactions between hyperbranched ß-cyclodextrin polymer and a dendritic gallic acid cross-linker incorporating six adamantane units for high-capacity silicon anodes. Calorimetric analysis in the solution phase indicates that the given host-guest complexation is a highly spontaneous and enthalpically driven process. These findings are further verified by carrying out gelation experiments in both aqueous and organic media. The dynamic cross-linking process enables intimate silicon-binder interaction, structural stability of electrode film, and controlled electrode-electrolyte interface, yielding enhanced cycling performance. Control experiments using both α, γ-CDp with different cavity sizes and a guest molecule incorporating a single adamantane unit verified that the enhanced cycle life originates from the host-guest interaction between ß-cyclodextrin and adamantane. The impact of the dynamic cross-linking is maximized at an optimal stoichiometry between the two components. Importantly, the present investigation proves that the molecular-level tuning of the host-guest interactions can be translated directly to the cycling performance of silicon anodes.

Limitations and high pressure behavior of MOF-5 for CO2 capture.

Porous network structures (e.g. metal-organic frameworks, MOFs) show considerable potential in dethroning monoethanol amine (MEA) from being the dominant scrubber for CO2 at the fossil-fuel-burning power generators. In contrast to their promise, structural stability and high-pressure behavior of MOFs are not well documented. We herein report moisture stability, mechanical properties and high-pressure compression on a model MOF structure, MOF-5. Our results show that MOF-5 can endure all tested pressures (0-225 bar) without losing its structural integrity, however, its moist air stability points at a 3.5 hour safety window (at 21.6 °C and 49% humidity) for an efficient CO2 capture. Isosteric heats of CO2 adsorption at high pressures show moderate interaction energy between CO2 molecules and the MOF-5 sorbent, which combined with the large sorption ability of MOF-5 in the studied pressure-temperature ranges show the viability of this sorbent for CO2 capturing purposes. The combination of the physicochemical methods we used suggests a generalized analytical standard for measuring viability in CO2 capture operations.

Fluorescence photoactivation by ligand exchange around the boron center of a BODIPY chromophore.

Chelation of the boron center of the borondipyrromethene (BODIPY) platform by a catecholate ligand results in effective fluorescence suppression. Electron transfer from the chelating unit to the adjacent chromophore upon excitation is responsible for fluorescence quenching. Under the influence of a photoacid generator, the catecholate chelator can be exchanged with a pair of methoxide ligands. This photoinduced transformation prevents electron transfer and efficiently activates the fluorescence of the BODIPY chromophore.

Superresolution imaging with switchable fluorophores based on oxazine auxochromes.

The spatial resolution of fluorescence microscopes is limited by diffraction to about half of the light wavelength, hampering the observation of many important intracellular processes. Recent emerging techniques have overcome that diffraction barrier using the temporal discrimination of close objects that are otherwise unresolved or blurred within the spatial resolution of the microscope. The key of these techniques is to switch the signal of fluorescence markers on and off exploiting their distinct molecular states, and detect and localize these markers at the single-molecule level. This underlying principle highlights the critical role of the photophysical properties of the probes, and the importance of finding adequate switching mechanisms. Here, we present strategies to achieve fluorescence modulation based on novel molecular assemblies containing a [1,3]oxazine as the two states, building block responsible for the transformation. Two different triggering events, based on the photochromic and halochromic properties of the oxazine, induce a large absorption and emission bathochromic shift of a pendant fluorophore, as the ultimate fluorescence switching event. The implementation of these approaches to achieve spatial resolution beyond the diffraction limit is also discussed.

Photoinduced fluorescence activation and nitric oxide release with biocompatible polymer nanoparticles.

A viable strategy to encapsulate a fluorophore/photochrome dyad and a nitric oxide photodonor within supramolecular assemblies of a cyclodextrin-based polymer in water was developed. The two photoresponsive guests do not interact with each other within their supramolecular container and can be operated in parallel under optical control. Specifically, the dyad permits the reversible switching of fluorescence on a microsecond timescale for hundreds of cycles, and the photodonor enables the irreversible release of nitric oxide. Furthermore, these supramolecular assemblies cross the membrane of human melanoma cancer cells and transport their cargo in the cytosol. The fluorescence of one component allows the visualization of the labeled cells, and its switchable character could, in principle, be used to acquire super-resolution images, while the release of nitric oxide from the other induces significant cell mortality. Thus, our design logic for the construction of biocompatible nanoparticles with dual functionality might evolve into the realization of valuable photoresponsive probes for imaging and therapeutic applications.

Fluorescence photoactivation by intermolecular proton transfer.

We designed a strategy to activate fluorescence under the influence of optical stimulations based on the intermolecular transfer of protons. Specifically, the illumination of a 2-nitrobenzyl derivative at an activating wavelength is accompanied by the release of hydrogen bromide. In turn, the photogenerated acid encourages the opening of an oxazine ring embedded within a halochromic compound. This structural transformation extends the conjugation of an adjacent coumarin fluorophore and enables its absorption at an appropriate excitation wavelength. Indeed, this bimolecular system offers the opportunity to activate fluorescence in liquid solutions, within rigid matrixes and inside micellar assemblies, relying on the interplay of activating and exciting beams. Furthermore, this strategy permits the permanent imprinting of fluorescent patterns on polymer films, the monitoring of proton diffusion within such materials in real time on a millisecond time scale, and the acquisition of images with spatial resolution at the nanometer level. Thus, our operating principles for fluorescence activation can eventually lead to the development of valuable photoswitchable probes for imaging applications and versatile mechanisms for the investigation of proton transport.

Insights into the isomerization of photochromic oxazines from the excitation dynamics of BODIPY-oxazine dyads.

We synthesized five BODIPY-oxazine dyads in one to four synthetic steps from known precursors. They differ in the nature of the unsaturated spacer linking the oxazine photochrome to either the conjugated framework or the boron center of the BODIPY fluorophore. Despite the π-character of the linkers, the two functional components are electronically isolated in the ground state and the BODIPY fluorophore maintains its absorption and, with one exception, emission properties unaltered. Instead, the photochemical response of the photochromic component is completely suppressed within all dyads. Rather than the expected opening of the oxazine ring, the laser excitation of these molecular assemblies results in the effective population of the BODIPY triplet in four of the five dyads. Control experiments with appropriate model compounds indicate that the local excitation of the oxazine component results first in intersystem crossing and then energy transfer to the BODIPY component. In fact, the transfer of energy from the triplet state of the former to the triplet state of the latter competes successfully with the opening of the oxazine ring and prevents the isomerization of the photochromic component. These observations demonstrate, for the very first time, that the photoinduced opening of these photochromic oxazines occurs along the potential energy surface of their triplet state. Such valuable mechanistic insights into their excitation dynamics can guide the design of novel members of this family of photochromic compounds with improved photochemical properties.

Fast fluorescence switching within hydrophilic supramolecular assemblies.

We designed a supramolecular strategy to modulate fluorescence in water under optical control. It is based on the entrapment of fluorophore-photochrome dyads within the hydrophobic interior of an amphiphilic polymer. The polymeric envelope around the dyads protects them from the aqueous environment, while imposing hydrophilic character on the overall supramolecular construct. In the resulting assemblies, the photochromic component can be operated reversibly on a microsecond timescale under the influence of ultraviolet stimulations. In turn, the reversible transformations control the emission intensity of the adjacent fluorophore. As a result, the fluorescence of such nanostructured constructs can be photomodulated for hundreds of cycles in water with microsecond switching speeds. Thus, our protocol for fast fluorescence switching in aqueous solutions can eventually lead to the realization of functional probes for the investigation of biological samples.

Fast and stable photochromic oxazines for fluorescence switching.

The stringent limitations imposed by diffraction on the spatial resolution of fluorescence microscopes demand the identification of viable strategies to switch fluorescence under optical control. In this context, the photoinduced and reversible transformations of photochromic compounds are particularly valuable. In fact, these molecules can be engineered to regulate the emission intensities of complementary fluorophores in response to optical stimulations. On the basis of this general design logic, we assembled a functional molecular construct consisting of a borondipyrromethene fluorophore and a nitrospiropyran photochrome and demonstrated that the emission of the former can be modulated with the interconversion of the latter. This fluorophore-photochrome dyad, however, has a slow switching speed and poor fatigue resistance. To improve both parameters, we developed a new family of photochromic switches based on the photoinduced opening and thermal closing of an oxazine ring. These compounds switch back and forth between ring-closed and -open isomers on nanosecond-microsecond timescales and tolerate thousands of switching cycles with no sign of degradation. In addition, the attachment of appropriate chromophoric fragments to their switchable oxazine ring can be exploited to either deactivate or activate fluorescence reversibly in response to illumination with a pair of exciting beams. Specifically, we assembled three dyads, each based on either a borondipyrromethene or a coumarin fluorophore and an oxazine photochrome, and modulated their fluorescence in a few microseconds with outstanding fatigue resistance. The unique photochemical and photophysical properties of our fluorophore-photochrome dyads can facilitate the development of switchable fluorophores for superresolution imaging and, ultimately, provide valuable molecular probes for the visualization of biological samples on the nanometer level.

Supramolecular strategies to construct biocompatible and photoswitchable fluorescent assemblies.

We designed and synthesized an amphiphilic copolymer with pendant hydrophobic decyl and hydrophilic poly(ethylene glycol) chains along a common poly(methacrylate) backbone. This macromolecular construct captures hydrophobic boron dipyrromethene fluorophores and hydrophobic spiropyran photochromes and transfers mixtures of both components in aqueous environments. Within the resulting hydrophilic supramolecular assemblies, the spiropyran components retain their photochemical properties and switch reversibly to the corresponding merocyanine isomers upon ultraviolet illumination. Their photoinduced transformations activate intermolecular electron and energy transfer pathways, which culminate in the quenching of the boron dipyrromethene fluorescence. As a result, the emission intensity of these supramolecular constructs can be modulated in aqueous environments under optical control. Furthermore, the macromolecular envelope around the fluorescent and photochromic components can cross the membrane of Chinese hamster ovarian cells and transport its cargo unaffected into the cytosol. Indeed, the fluorescence of these supramolecular constructs can be modulated also intracellularly by operating the photochromic component with optical inputs. In addition, cytotoxicity tests demonstrate that these supramolecular assemblies and the illumination conditions required for their operation have essentially no influence on cell viability. Thus, supramolecular events can be invoked to construct fluorescent and photoswitchable systems from separate components, while imposing aqueous solubility and biocompatibility on the resulting assemblies. In principle, this simple protocol can evolve into a general strategy to deliver and operate intracellularly functional molecular components under optical control.

Photoswitchable fluorescent dyads incorporating BODIPY and [1,3]oxazine components.

We designed and synthesized three compounds incorporating a BODIPY fluorophore and an oxazine photochrome within the same molecular skeleton and differing in the nature of the linker bridging the two functional components. The [1,3]oxazine ring of the photochrome opens in less than 6 ns upon laser excitation in two of the three fluorophore-photochrome dyads. This process generates a 3H-indolium cation with a quantum yield of 0.02-0.05. The photogenerated isomer has a lifetime of 1-3 µs and reverts to the original species with first-order kinetics. Both photochromic systems tolerate hundreds of switching cycles with no sign of degradation. The visible excitation of the dyads is accompanied by the characteristic fluorescence of the BODIPY component. However, the cationic fragment of their photogenerated isomers can accept an electron or energy from the excited fluorophore. As a result, the photoinduced transformation of the photochromic component within each dyad results in the effective quenching of the BODIPY emission. Indeed, the fluorescence of these photoswitchable compounds can be modulated on a microsecond time scale with excellent fatigue resistance under optical control. Thus, our operating principles and choice of functional components can ultimately lead to the development of valuable photoswitchable fluorescent probes for the super-resolution imaging of biological samples.

Fluorescence patterning in films of a photoswitchable BODIPY-spiropyran dyad.

A BODIPY-spiropyran dyad was embedded within poly(methyl methacrylate) films spin-coated on glass slides. Visible illumination of the resulting materials excites selectively the BODIPY fragment, which then deactivates radiatively by emitting light in the form of fluorescence. Ultraviolet irradiation promotes the isomerization of the spiropyran component to the corresponding merocyanine. This photoinduced transformation activates electron and energy transfer pathways from the fluorescent to the photochromic fragment. Consistently, the BODIPY fluorescence is effectively suppressed within the photogenerated isomer. As a result, ultraviolet illumination with a laser, producing a doughnut-shaped spot on the sample, confines the fluorescent species within the doughnut hole. This behavior is an essential requisite for the implementation of super-resolution imaging schemes based on fluorescence photodeactivation. Thus, the operating principles governing the photochemical and photophysical response of this molecular switch can ultimately lead to the development of innovative probes for fluorescence nanoscopy.