The H-NOX protein structure adapts to different mechanisms in sensors interacting with nitric oxide.

Some classes of bacteria within phyla possess protein sensors identified as homologous to the heme domain of soluble guanylate cyclase, the mammalian NO-receptor. Named H-NOX domain (Heme-Nitric Oxide or OXygen-binding), their heme binds nitric oxide (NO) and O2 for some of them. The signaling pathways where these proteins act as NO or O2 sensors appear various and are fully established for only some species. Here, we investigated the reactivity of H-NOX from bacterial species toward NO with a mechanistic point of view using time-resolved spectroscopy. The present data show that H-NOXs modulate the dynamics of NO as a function of temperature, but in different ranges, changing its affinity by changing the probability of NO rebinding after dissociation in the picosecond time scale. This fundamental mechanism provides a means to adapt the heme structural response to the environment. In one particular H-NOX sensor the heme distortion induced by NO binding is relaxed in an ultrafast manner (∼15 ps) after NO dissociation, contrarily to other H-NOX proteins, providing another sensing mechanism through the H-NOX domain. Overall, our study links molecular dynamics with functional mechanism and adaptation.

Computational identification of a systemic antibiotic for gram-negative bacteria.

Discovery of antibiotics acting against Gram-negative species is uniquely challenging due to their restrictive penetration barrier. BamA, which inserts proteins into the outer membrane, is an attractive target due to its surface location. Darobactins produced by Photorhabdus, a nematode gut microbiome symbiont, target BamA. We reasoned that a computational search for genes only distantly related to the darobactin operon may lead to novel compounds. Following this clue, we identified dynobactin A, a novel peptide antibiotic from Photorhabdus australis containing two unlinked rings. Dynobactin is structurally unrelated to darobactins, but also targets BamA. Based on a BamA-dynobactin co-crystal structure and a BAM-complex-dynobactin cryo-EM structure, we show that dynobactin binds to the BamA lateral gate, uniquely protruding into its ß-barrel lumen. Dynobactin showed efficacy in a mouse systemic Escherichia coli infection. This study demonstrates the utility of computational approaches to antibiotic discovery and suggests that dynobactin is a promising lead for drug development.

Nanoscale-Femtosecond Imaging of Evanescent Surface Plasmons on Silver Film by Photon-Induced Near-Field Electron Microscopy.

Surface plasmons on silver nanostructures have a broad range of tunable resonance properties in visible and near-infrared regimes, which possess wide applications in nanophotonics and optoelectronics. Here we use a femtosecond laser to excite surface plasmons on a silver film and trace the subsequent transient dynamics via photon-induced near-field electron microscopy (PINEM). A polarization experiment of PINEM demonstrates a conspicuous polarization dependence of the transient surface plasmon field on the silver film; however, unlike silver nanowires and nanorods, there is no polarization dependence for the PINEM intensity. This compelling finding suggests a thin film platform can be more easily used to identify the temporal and spatial overlaps between the pump laser and probe electron pulses in 4D ultrafast electron microscopy (UEM). Our work illustrates the femtosecond excitation and transient behavior of the surface plasmons on silver film and paves a universal, simple way for identifying the time zero in 4D UEM.

CaMn3IV O4 Cubane Models of the Oxygen-Evolving Complex: Spin Ground States S<9/2 and the Effect of Oxo Protonation.

We report the single crystal XRD and MicroED structure, magnetic susceptibility, and EPR data of a series of CaMn3IV O4 and YMn3IV O4 complexes as structural and spectroscopic models of the cuboidal subunit of the oxygen-evolving complex (OEC). The effect of changes in heterometal identity, cluster geometry, and bridging oxo protonation on the spin-state structure was investigated. In contrast to previous computational models, we show that the spin ground state of CaMn3IV O4 complexes and variants with protonated oxo moieties need not be S=9/2. Desymmetrization of the pseudo-C3 -symmetric Ca(Y)Mn3IV O4 core leads to a lower S=5/2 spin ground state. The magnitude of the magnetic exchange coupling is attenuated upon oxo protonation, and an S=3/2 spin ground state is observed in CaMn3IV O3 (OH). Our studies complement the observation that the interconversion between the low-spin and high-spin forms of the S2 state is pH-dependent, suggesting that the (de)protonation of bridging or terminal oxygen atoms in the OEC may be connected to spin-state changes.

Microcrystal Electron Diffraction Elucidates Water-Specific Polymorphism-Induced Emission Enhancement of Bis-arylacylhydrazone.

Aggregation-induced emission (AIE) phenomena have gained intense interest over the last decades because of its importance in solid-state emission. However, the elucidation of a working mechanism is difficult owing to the limited characterization methods on solid-state molecules, further complicated if dynamic structural changes occur. Here, a series of bis-arylacylhydrazones (BAHs) were synthesized, for which their AIE properties are only turned on by the reversible adsorption of water molecules. We used microcrystal electron diffraction (MicroED) to determine the molecular structures of two BAHs directly from bulk powders (without attempting to grow crystals) prepared in the absence or presence of water adsorption. This study reveals the unambiguous characterization of the dependence of crystal packing on the specific cocrystallization with hydrates. The structural analysis demonstrates that water molecules form strong hydrogen bonds with three neighboring BAH-1, resulting in the almost complete planarization and restriction of the intramolecular rotation of the molecule. MicroED plays an important role in providing a decisive clue for the reversible polymorphism changes induced by the adsorption of water molecules, regulating emissive properties.

Ultrafast dynamics of heme distortion in the O2-sensor of a thermophilic anaerobe bacterium.

Heme-Nitric oxide and Oxygen binding protein domains (H-NOX) are found in signaling pathways of both prokaryotes and eukaryotes and share sequence homology with soluble guanylate cyclase, the mammalian NO receptor. In bacteria, H-NOX is associated with kinase or methyl accepting chemotaxis domains. In the O2-sensor of the strict anaerobe Caldanaerobacter tengcongensis (Ct H-NOX) the heme appears highly distorted after O2 binding, but the role of heme distortion in allosteric transitions was not yet evidenced. Here, we measure the dynamics of the heme distortion triggered by the dissociation of diatomics from Ct H-NOX using transient electronic absorption spectroscopy in the picosecond to millisecond time range. We obtained a spectroscopic signature of the heme flattening upon O2 dissociation. The heme distortion is immediately (<1 ps) released after O2 dissociation to produce a relaxed state. This heme conformational change occurs with different proportions depending on diatomics as follows: CO < NO < O2. Our time-resolved data demonstrate that the primary structural event of allostery is the heme distortion in the Ct H-NOX sensor, contrastingly with hemoglobin and the human NO receptor, in which the primary structural events are respectively the motion of the proximal histidine and the rupture of the iron-histidine bond.

4D electron microscopy of T cell activation.

T cells can be controllably stimulated through antigen-specific or nonspecific protocols. Accompanying functional hallmarks of T cell activation can include cytoskeletal reorganization, cell size increase, and cytokine secretion. Photon-induced near-field electron microscopy (PINEM) is used to image and quantify evanescent electric fields at the surface of T cells as a function of various stimulation conditions. While PINEM signal strength scales with multiple of the biophysical changes associated with T cell functional activation, it mostly strongly correlates with antigen-engagement of the T cell receptors, even under conditions that do not lead to functional T cell activation. PINEM image analysis suggests that a stimulation-induced reorganization of T cell surface structure, especially over length scales of a few hundred nanometers, is the dominant contributor to these PINEM signal changes. These experiments reveal that PINEM can provide a sensitive label-free probe of nanoscale cellular surface structures.

Optical manipulation of magnetic vortices visualized in situ by Lorentz electron microscopy.

Understanding the fundamental dynamics of topological vortex and antivortex naturally formed in microscale/nanoscale ferromagnetic building blocks under external perturbations is crucial to magnetic vortex-based information processing and spintronic devices. All previous studies have focused on magnetic vortex-core switching via external magnetic fields, spin-polarized currents, or spin waves, which have largely prohibited the investigation of novel spin configurations that could emerge from the ground states in ferromagnetic disks and their underlying dynamics. We report in situ visualization of femtosecond laser quenching-induced magnetic vortex changes in various symmetric ferromagnetic Permalloy disks by using Lorentz phase imaging of four-dimensional electron microscopy that enables in situ laser excitation. Besides the switching of magnetic vortex chirality and polarity, we observed with distinct occurrence frequencies a plenitude of complex magnetic structures that have never been observed by magnetic field- or current-assisted switching. These complex magnetic structures consist of a number of newly created topological magnetic defects (vortex and antivortex) strictly conserving the topological winding number, demonstrating the direct impact of topological invariants on magnetization dynamics in ferromagnetic disks. Their spin configurations show mirror or rotation symmetry due to the geometrical confinement of the disks. Combined micromagnetic simulations with the experimental observations reveal the underlying magnetization dynamics and formation mechanism of the optical quenching-induced complex magnetic structures. Their distinct occurrence rates are pertinent to their formation-growth energetics and pinning effects at the disk edge. On the basis of these findings, we propose a paradigm of optical quenching-assisted fast switching of vortex cores for the control of magnetic vortex-based information recording and spintronic devices.

Time-resolved spectroscopy of the ensembled photoluminescence of nitrogen- and boron/nitrogen-doped carbon dots.

Carbon dots (CDs) have potential applications in various fields such as energy, catalysis, and bioimaging due to their strong and tuneable photoluminescence (PL), low toxicity, and robust chemical inertness. Although several PL mechanisms have been proposed, the origin of PL in CDs is still in debate because of the ensembled nature of the heterogeneous luminophores present in the CDs. To unravel the origin of PL in CDs, we performed time-resolved spectroscopy on two types of CDs: nitrogen-doped (N-CD) and boron-nitrogen co-doped (BN-CD). The PL decays were fitted by stretched exponential functions to estimate the distribution of the decay kinetics in the CDs, which have different PL lifetime distributions. Both CDs displayed main, blue emission decaying in 15 ns, which originates from the dominant molecular state. The analysis of the non-exponential PL decay using stretched exponential fits revealed that the functional surface luminophores are of less variety but of more environmental heterogeneity and have much lower populations in BN-CD than in N-CD.

Structural changes and picosecond to second dynamics of cytochrome c in interaction with nitric oxide in ferrous and ferric redox states.

Apart from its role in electron transfer, mitochondrial cytochrome c also plays a role in apoptosis and is subject to nitrosylation. The cleavage of the Fe-Met80 bond plays a role in several processes including the release of Cyt c from mitochondria or increase of its peroxidase activity. Nitrosylation of Cyt c precludes the reformation of the disrupted Fe-Met80 bond and was shown to occur during apoptosis. These physiological properties are associated with a conformational change of the heme center of Cyt c. Here, we demonstrate that NO binding induces pronounced heme conformational changes in the six-coordinate Cyt c-NO complex. Equilibrium and time-resolved Raman data reveal that the heme structural conformation depends both on the nature of the distal iron ligand (NO or Met80) and on the Fe2+ or Fe3+ heme redox state. Upon nitrosylation, the heme ruffling distortion is greatly enhanced for ferrous Cyt c. Contrastingly, the initial strong heme distortion in native ferric Cyt c almost disappears after NO binding. We measured the heme coordination dynamics in the picosecond to second time range and identified Met80 and NO rebinding phases using time-resolved Raman and absorption spectroscopies. Dissociation of NO instantly produces 5-coordinate heme with a domed structure which continues to rearrange within 15 ps, while the initial ruffling distortion disappears. The rates of Cyt c-NO complex formation measured by transient absorption are kon = 1.81 × 106 M-1 s-1 for ferric Cyt c and 83 M-1 s-1 for ferrous Cyt c. After NO dissociation and exit from the heme pocket, the rebinding of Met80 to the heme iron takes place 6 orders of magnitude more slowly (3-5 µs) than Met80 rebinding in the absence of NO (5 ps). Altogether, these data reveal the structural and dynamic properties of Cyt c in interaction with nitric oxide relevant for the molecular mechanism of apoptosis.

Photon-Induced Near-Field Electron Microscopy of Eukaryotic Cells.

Photon-induced near-field electron microscopy (PINEM) is a technique to produce and then image evanescent electromagnetic fields on the surfaces of nanostructures. Most previous applications of PINEM have imaged surface plasmon-polariton waves on conducting nanomaterials. Here, the application of PINEM on whole human cancer cells and membrane vesicles isolated from them is reported. We show that photons induce time-, orientation-, and polarization-dependent evanescent fields on the surfaces of A431 cancer cells and isolated membrane vesicles. Furthermore, the addition of a ligand to the major surface receptor on these cells and vesicles (epidermal growth factor receptor, EGFR) reduces the intensity of these fields in both preparations. We propose that in the absence of plasmon waves in biological samples, these evanescent fields reflect the changes in EGFR kinase domain polarization upon ligand binding.

On the dynamical nature of the active center in a single-site photocatalyst visualized by 4D ultrafast electron microscopy.

Understanding the dynamical nature of the catalytic active site embedded in complex systems at the atomic level is critical to developing efficient photocatalytic materials. Here, we report, using 4D ultrafast electron microscopy, the spatiotemporal behaviors of titanium and oxygen in a titanosilicate catalytic material. The observed changes in Bragg diffraction intensity with time at the specific lattice planes, and with a tilted geometry, provide the relaxation pathway: the Ti(4+)=O(2-) double bond transformation to a Ti(3+)-O(1-) single bond via the individual atomic displacements of the titanium and the apical oxygen. The dilation of the double bond is up to 0.8 Å and occurs on the femtosecond time scale. These findings suggest the direct catalytic involvement of the Ti(3+)-O(1-) local structure, the significance of nonthermal processes at the reactive site, and the efficient photo-induced electron transfer that plays a pivotal role in many photocatalytic reactions.

Observing in space and time the ephemeral nucleation of liquid-to-crystal phase transitions.

The phase transition of crystalline ordering is a general phenomenon, but its evolution in space and time requires microscopic probes for visualization. Here we report direct imaging of the transformation of amorphous titanium dioxide nanofilm, from the liquid state, passing through the nucleation step and finally to the ordered crystal phase. Single-pulse transient diffraction profiles at different times provide the structural transformation and the specific degree of crystallinity (η) in the evolution process. It is found that the temporal behaviour of η exhibits unique 'two-step' dynamics, with a robust 'plateau' that extends over a microsecond; the rate constants vary by two orders of magnitude. Such behaviour reflects the presence of intermediate structure(s) that are the precursor of the ordered crystal state. Theoretically, we extend the well-known Johnson-Mehl-Avrami-Kolmogorov equation, which describes the isothermal process with a stretched-exponential function, but here over the range of times covering the melt-to-crystal transformation.

Biochemical perturbations of the mitotic spindle in Xenopus extracts using a diffusion-based microfluidic assay.

A microfluidic device is a powerful tool to manipulate in a controlled manner at spatiotemporal scales for biological systems. Here, we describe a simple diffusion-based assay to generate and measure the effect of biochemical perturbations within the cytoplasm of cell-free extracts from Xenopus eggs. Our approach comprises a microliter reservoir and a model cytoplasm that are separated by a synthetic membrane containing sub-micrometric pores through which small molecules and recombinant proteins can diffuse. We have used this system to examine the perturbation of elements of the mitotic spindle, which is a microtubule-based bipolar structure involved in the segregation of the replicated genome to daughter cells during cell division. First, we used the small molecule inhibitor monastrol to target kinesin-5, a molecular motor that maintains the microtubule spindle bipolarity. Next, we explored the dynamics of the mitotic spindle by monitoring the exchange between unpolymerized and polymerized tubulin within microtubule fibers. These results show that a simple diffusion-based system can generate biochemical perturbations directly within a cell-free cytoplasm based on Xenopus egg extracts at the time scale of minutes. Our assay is therefore suitable for monitoring the dynamics of supramolecular assemblies within cell-free extracts in response to perturbations. This strategy opens up broad perspectives including phenotype screening or mechanistic studies of biological assembly processes and could be applied to other cell-free extracts such as those derived from mammalian or bacterial cells.

Motion of proximal histidine and structural allosteric transition in soluble guanylate cyclase.

We investigated the changes of heme coordination in purified soluble guanylate cyclase (sGC) by time-resolved spectroscopy in a time range encompassing 11 orders of magnitude (from 1 ps to 0.2 s). After dissociation, NO either recombines geminately to the 4-coordinate (4c) heme (τG1 = 7.5 ps; 97 ± 1% of the population) or exits the heme pocket (3 ± 1%). The proximal His rebinds to the 4c heme with a 70-ps time constant. Then, NO is distributed in two approximately equal populations (1.5%). One geminately rebinds to the 5c heme (τG2 = 6.5 ns), whereas the other diffuses out to the solution, from where it rebinds bimolecularly (τ = 50 µs with [NO] = 200 µM) forming a 6c heme with a diffusion-limited rate constant of 2 × 10(8) M(-1)⋅s(-1). In both cases, the rebinding of NO induces the cleavage of the Fe-His bond that can be observed as an individual reaction step. Saliently, the time constant of bond cleavage differs depending on whether NO binds geminately or from solution (τ5C1 = 0.66 µs and τ5C2 = 10 ms, respectively). Because the same event occurs with rates separated by four orders of magnitude, this measurement implies that sGC is in different structural states in both cases, having different strain exerted on the Fe-His bond. We show here that this structural allosteric transition takes place in the range 1-50 µs. In this context, the detection of NO binding to the proximal side of sGC heme is discussed.

Reactivity and dynamics of H2S, NO, and O2 interacting with hemoglobins from Lucina pectinata.

Hemoglobin HbI from the clam Lucina pectinata is involved in H2S transport, whereas homologous heme protein HbII/III is involved in O2 metabolism. Despite similar tertiary structures, HbI and HbII/III exhibit very different reactivity toward heme ligands H2S, O2, and NO. To investigate this reactivity at the heme level, we measured the dynamics of ligand interaction by time-resolved absorption spectroscopy in the picosecond to nanosecond time range. We demonstrated that H2S can be photodissociated from both ferric and ferrous HbI. H2S geminately rebinds to ferric and ferrous out-of-plane iron with time constants (τgem) of 12 and 165 ps, respectively, with very different proportions of photodissociated H2S exiting the protein (24% in ferric and 80% in ferrous HbI). The Gln(E7)His mutation considerably changes H2S dynamics in ferric HbI, indicating the role of Gln(E7) in controling H2S reactivity. In ferric HbI, the rate of diffusion of H2S from the solvent into the heme pocket (kentry) is 0.30 µM(-1) s(-1). For the HbII/III-O2 complex, we observed mainly a six-coordinate vibrationally excited heme-O2 complex with O2 still bound to the iron. This explains the low yield of O2 photodissociation and low koff from HbII/III, compared with those of HbI and Mb. Both isoforms behave very differently with regard to NO and O2 dynamics. Whereas the amplitude of geminate rebinding of O2 to HbI (38.5%) is similar to that of myoglobin (34.5%) in spite of different distal heme sites, it appears to be much larger for HbII/III (77%). The distal Tyr(B10) side chain present in HbII/III increases the energy barrier for ligand escape and participates in the stabilization of bound O2 and NO.

Picosecond binding of the His ligand to four-coordinate heme in cytochrome c': a one-way gate for releasing proximal NO.

We provide a direct demonstration of a "kinetic trap" mechanism in the proximal 5-coordinate heme-nitrosyl complex (5c-NO) of cytochrome c' from Alcaligenes xylosoxidans (AXCP) in which picosecond rebinding of the endogenous His ligand following heme-NO dissociation acts as a one-way gate for the release of proximal NO into solution. This demonstration is based upon picosecond transient absorption changes following NO photodissociation of the proximal 5c-NO AXCP complex. We have determined the absolute transient absorption spectrum of 4-coordinate ferrous heme to which NO rebinds with a time constant τ(NO) = 7 ps (k(NO) = 1.4 × 10(11) s(-1)) and shown that rebinding of the proximal histidine to the 4-coordinate heme takes place with a time constant τ(His) = 100 ± 10 ps (k(His) = 10(10) s(-1)) after the release of NO from the proximal heme pocket. This rapid His reattachment acts as a one-way gate for releasing proximal NO by precluding direct proximal NO rebinding once it has left the proximal heme pocket and requiring NO rebinding from solution to proceed via the distal heme face.

Association of the Neuronal Cell Adhesion Molecule (NrCAM) Gene Variants with Personality Traits and Addictive Symptoms in Methamphetamine Use Disorder.

OBJECTIVE: 1) To investigate the relationship between NrCAM polymorphisms and methamphetamine abuse in an ethnically homogenous Korean population. 2) To further support our findings by investigating the association among NrCAM gene variants, certain personality traits, and addictive symptoms of methamphetamine abusers. METHODS: Thirty-seven male methamphetamine abusers (age=43.3±7.8) and30 non-users (16 men, 14 women; age=59.8±10.4) were recruited. Ten single nucleotide polymorphisms (SNPs) in the NrCAM gene were assayed to compare genotype distributions between the 2 groups. Personality characteristics were measured using the Temperament and Character Inventory (TCI) and the NEO Personality Inventory, Revised (NEO PI-R). Addictive symptoms were assessed using the Diagnostic Interview for Genetic Studies (DIGS) and reviews of the subject's medical records. RESULTS: Among the 10 SNPs in the NrCAM gene, the frequency of the TA genotype at rs1990162 was significantly lower in methamphetamine abusers compared to non-users (p=0.042). In the 3 NrCAM gene SNPs (rs381318, rs2072546, and rs6954366), the distribution of genotypes and alleles were significantly associated with some traits in the TCI and NEO PI-R. Genotypes and alleles at 5 gene SNPs (rs2142325, rs381318, rs1269621, rs1269634, and rs1990162) were associated with certain addictive symptom dimensions in the patients. CONCLUSION: These findings support the idea that NrCAM is associated with genetic susceptibility of methamphetamine abuse and is also associated with certain personality characteristics that may increase disturbed addictive behavior.

Direct observation of conformation-dependent pathways in the excited-state proton transfer of 7-hydroxyquinoline in bulk alcohols.

The excited-state proton transfer (ESPT) of 7-hydroxyquinoline (7HQ) in bulk alcoholic solvents has been explored with variation of protic hydrogen atoms as well as alcohols. By measuring time-resolved kinetic profiles at two different excitation wavelengths, we have observed conformation-specific pathways in the ESPT of 7HQ. There are two possible rotamers of 7HQ according to the configuration of its hydroxyl group, which are trans and cis. On one hand, trans-7HQ cannot undergo proton transfer within its excited-state lifetime because the internal rotation of the hydroxyl group to form cis-7HQ hardly occurs. On the other hand, some cis-7HQ molecules exist as cyclic complexes of 7HQ·(alcohol)(2) at the moment of excitation, and the cyclic complexes can undergo ESPT rapidly via tunneling. However, the other cis-7HQ molecules should undergo solvent reorganization to form cyclic 7HQ·(alcohol)(2) complexes prior to intrinsic ESPT, and the solvent reorganization becomes the rate-determining step. In contrast to proton transfer, where intrinsic ESPT and solvent reorganization have been observed separately in time-resolved kinetic profiles, intrinsic deuteron transfer is too slow to be distinguished kinetically from solvent reorganization.

Picosecond to second dynamics reveals a structural transition in Clostridium botulinum NO-sensor triggered by the activator BAY-41-2272.

Soluble guanylate cyclase (sGC) is the mammalian endogenous nitric oxide (NO) receptor that synthesizes cGMP upon NO activation. In synergy with the artificial allosteric effector BAY 41-2272 (a lead compound for drug design in cardiovascular treatment), sGC can also be activated by carbon monoxide (CO), but the structural basis for this synergistic effect are unknown. We recorded in the unusually broad time range from 1 ps to 1 s the dynamics of the interaction of CO binding to full length sGC, to the isolated sGC heme domain ß(1)(200) and to the homologous bacterial NO-sensor from Clostridium botulinum. By identifying all phases of CO binding in this full time range and characterizing how these phases are modified by BAY 41-2272, we show that this activator induces the same structural changes in both proteins. This result demonstrates that the BAY 41-2272 binding site resides in the ß(1)(200) sGC heme domain and is the same in sGC and in the NO-sensor from Clostridium botulinum.