Byung-Kuk Yoo

Confinement induces actin flow in a meiotic cytoplasm

In vivo, F-actin flows are observed at different cell life stages and participate in various developmental processes during asymmetric divisions in vertebrate oocytes, cell migration, or wound healing. Here, we show that confinement has a dramatic effect on F-actin spatiotemporal organization. We reconstitute in vitro the spontaneous generation of F-actin flow using Xenopus meiotic extracts artificially confined within a geometry mimicking the cell boundary. Perturbations of actin polymerization kinetics or F-actin nucleation sites strongly modify the network flow dynamics. A combination of quantitative image analysis and biochemical perturbations shows that both spatial localization of F-actin nucleators and actin turnover play a decisive role in generating flow. Interestingly, our in vitro assay recapitulates several symmetry-breaking processes observed in oocytes and early embryonic cells.

Picosecond primary structural transition of the heme is retarded after nitric oxide binding to heme proteins

We investigated the ultrafast structural transitions of the heme induced by nitric oxide (NO) binding for several heme proteins by subpicosecond time-resolved resonance Raman and femtosecond transient absorption spectroscopy. We probed the heme iron motion by the evolution of the iron-histidine Raman band intensity after NO photolysis. Unexpectedly, we found that the heme response and iron motion do not follow the kinetics of NO rebinding. Whereas NO dissociation induces quasi-instantaneous iron motion and heme doming (< 0.6 ps), the reverse process results in a much slower picosecond movement of the iron toward the planar heme configuration after NO binding. The time constant for this primary domed-to-planar heme transition varies among proteins (∼30 ps for myoglobin and its H64V mutant, ∼15 ps for hemoglobin, ∼7 ps for dehaloperoxidase, and ∼6 ps for cytochrome c) and depends upon constraints exerted by the protein structure on the heme cofactor. This observed phenomenon constitutes the primary structural transition in heme proteins induced by NO binding.

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.

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

Significance The nature of the active site in (photo) catalysis is fundamental to our understanding of the processes involved, and to their control. Four-dimensional ultrafast electron microscopy (UEM) provides a dynamic probe for catalytic active site in photocatalytic materials thanks to its unprecedented resolution both in time (femtosecond) and space (angstrom). In this contribution, we visualize the femtosecond atomic movement at the titanium active center in a single-site photocatalyst. UEM allows us to investigate the structural dynamics of the radiation sensitive specimen by measuring time-resolved diffraction intensities from different lattice planes. These findings contribute fundamental insights for developing advanced photocatalysts and suggest broad ranges of applications. 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 Ti4+=O2− double bond transformation to a Ti3+−O1− 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 Ti3+−O1− 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.

Quaternary structure controls ligand dynamics in soluble guanylate cyclase

Background: NO and CO dynamics are compared in soluble guanylate cyclase and isolated heme domain. Results: CO geminately rebinds to the isolated heme domain β1(190), contrary to entire sGC. Photo-oxidation of β1(190) heme may occur. Conclusion: The isolated heme domain β1(190) has a different reactivity than full-length sGC. Significance: The structural strains between domains in the full-length protein are crucial for its functioning. Soluble guanylate cyclase (sGC) is the mammalian endogenous nitric oxide (NO) receptor. The mechanisms of activation and deactivation of this heterodimeric enzyme are unknown. For deciphering them, functional domains can be overexpressed. We have probed the dynamics of the diatomic ligands NO and CO within the isolated heme domain β1(190) of human sGC by piconanosecond absorption spectroscopy. After photo-excitation of nitrosylated sGC, only NO geminate rebinding occurs in 7.5 ps. In β1(190), both photo-dissociation of 5c-NO and photo-oxidation occur, contrary to sGC, followed by NO rebinding (7 ps) and back-reduction (230 ps and 2 ns). In full-length sGC, CO geminate rebinding to the heme does not occur. In contrast, CO geminately rebinds to β1(190) with fast multiphasic process (35, 171, and 18 ns). We measured the bimolecular association rates kon = 0.075 ± 0.01 × 106 m−1·s−1 for sGC and 0.83 ± 0.1 × 106 m−1·s−1 for β1(190). These different dynamics reflect conformational changes and less proximal constraints in the isolated heme domain with respect to the dimeric native sGC. We concluded that the α-subunit and the β1(191–619) domain exert structural strains on the heme domain. These strains are likely involved in the transmission of the energy and relaxation toward the activated state after Fe2+-His bond breaking. This also reveals the heme domain plasticity modulated by the associated domains and subunit.

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 β(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 β(1)(200) sGC heme domain and is the same in sGC and in the NO-sensor from Clostridium botulinum.

Absorption Band III Kinetics Probe the Picosecond Heme Iron Motion Triggered by Nitric Oxide Binding to Hemoglobin and Myoglobin

To study the ultrafast movement of the heme iron induced by nitric oxide (NO) binding to hemoglobin (Hb) and myoglobin (Mb), we probed the picosecond spectral evolution of absorption band III (∼760 nm) and vibrational modes (iron-histidine stretching, ν(4) and ν(7) in-plane modes) in time-resolved resonance Raman spectra. The time constants of band III intensity kinetics induced by NO rebinding (25 ps for hemoglobin and 40 ps for myoglobin) are larger than in Soret bands and Q-bands. Band III intensity kinetics is retarded with respect to NO rebinding to Hb and to Mb. Similarly, the ν((Fe-His)) stretching intensity kinetics are retarded with respect to the ν(4) and ν(7) heme modes and to Soret absorption. In contrast, band III spectral shift kinetics do not coincide with band III intensity kinetics but follows Soret kinetics. We concluded that, namely, the band III intensity depends on the heme iron out-of-plane position, as theoretically predicted ( Stavrov , S. S. Biopolymers 2004 , 74 , 37 - 40 ).

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

Significance Soluble guanylate cyclase is the mammalian endogenous nitric oxide (NO) receptor that controls numerous signaling physiological processes. Time-resolved spectroscopy allowed us to probe the dynamics of the heme coordination after NO release and binding. After photodissociation of NO, all heme transitions are identified within the time range of 1 ps to 0.2 s, notably the bond breaking and reformation between the heme iron and proximal His, which are major events for the activation/deactivation processes. It is thus possible to demonstrate that the structural allosteric transition occurs in the time range 1–50 μs, which remarkably matches the time range observed for hemoglobin, the prototypic protein for allostery. These findings relate not only to NO signaling but also to general allostery in heme proteins. 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 × 108 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.

Rebinding of Proximal Histidine in the Cytochrome c' from Alcaligenes xylosoxidans Acts as a Molecular Trap for Nitric Oxide

Transient absorption spectra on cytochrome c’ and their kinetics were recorded to identify the formation of 5-coordinate (5c)-NO and 5c-His hemes from 4c-heme (99% and 1% amplitudes; 7-ps and 100-ps time constants, respectively). We demonstrate that proximal histidine precludes NO rebinding at the proximal site.

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.