Tomoki P. Terada

Observation of Kaonic Hydrogen K α X Rays

We have measured the shift and width of the kaonic hydrogen 1s state due to the {ovr K}N strong interaction. We have observed, for the first time, distinct K-series kaonic hydrogen x rays with good signal-to-noise ratio in the energy spectrum. The measured energy shift and width were determined to be {Delta}E(1s)=-323{plus_minus}63(stat){plus_minus}11(syst)eV (repulsive) and {Gamma}(1s)=407{plus_minus}208(stat){plus_minus}100(syst)eV,respectively. {copyright} {ital 1997} {ital The American Physical Society}

Nucleotide binding to the chaperonin GroEL: non-cooperative binding of ATP analogs and ADP, and cooperative effect of ATP

Chaperonin-assisted protein folding proceeds through cycles of ATP binding and hydrolysis by GroEL, which undergoes a large structural change by the ATP binding or hydrolysis. One of the main concerns of GroEL is the mechanism of the productive and cooperative structural change of GroEL induced by the nucleotide. We studied the cooperative nature of GroEL by nucleotide titration using isothermal titration calorimetry and fluorescence spectroscopy. Our results indicated that the binding of ADP and ATP analogs to a single ring mutant (SR1), as well as that to GroEL, was non-cooperative. Only ATP induces an apparently cooperative conformational change in both proteins. Furthermore, the fluorescence changes of pyrene-labeled GroEL indicated that GroEL has two kinds of nucleotide binding sites. The fluorescence titration result fits well with a model in which two kinds of binding sites are both non-cooperative and independent of each other. These results suggest that the binding and hydrolysis of ATP may be necessary for the cooperative transition of GroEL.

Monomer-Shuffling and Allosteric Transition in KaiC Circadian Oscillation

Circadian rhythms in living organisms have long been attributed solely to a transcription-translation loop comprising a negative or positive feedback. The rhythms in cyanobacteria are known to be modulated by kaiC, kaiA and kaiB genes. It was recently shown, however, that their product proteins KaiC, KaiA and KaiB are sufficient to reconstitute the circadian rhythm in the phosphorylation level of KaiC in vitro. It has since been unclear why such an oscillatory behavior can occur in the absence of the apparent transcription-translation feedback. In the meantime, it has been reported that the monomer exchange between KaiC hexamers occurs in a phosphorylation-dependent manner, which suggests that the monomer shuffling is also involved in the circadian rhythm (H. Kageyama et al., Mol. Cell, 23, 161 (2006)). To further clarify the role of the monomer shuffling, we have performed a computational modeling of interactions among Kai proteins assuming the allosteric transition of KaiC hexamer as well as the monomer shuffling. The results show that the existence of both monomer shuffling and allosteric transition can synchronize the phosphorylation level of the KaiC hexamers, and stabilizes its oscillation.

Unidirectional Brownian motion observed in an in silico single molecule experiment of an actomyosin motor

The actomyosin molecular motor, the motor composed of myosin II and actin filament, is responsible for muscle contraction, converting chemical energy into mechanical work. Although recent single molecule and structural studies have shed new light on the energy-converting mechanism, the physical basis of the molecular-level mechanism remains unclear because of the experimental limitations. To provide a clue to resolve the controversy between the lever-arm mechanism and the Brownian ratchet-like mechanism, we here report an in silico single molecule experiment of an actomyosin motor. When we placed myosin on an actin filament and allowed myosin to move along the filament, we found that myosin exhibits a unidirectional Brownian motion along the filament. This unidirectionality was found to arise from the combination of a nonequilibrium condition realized by coupling to the ATP hydrolysis and a ratchet-like energy landscape inherent in the actin-myosin interaction along the filament, indicating that a Brownian ratchet-like mechanism contributes substantially to the energy conversion of this molecular motor.

Time scales in epigenetic dynamics and phenotypic heterogeneity of embryonic stem cells.

A remarkable feature of the self-renewing population of embryonic stem cells (ESCs) is their phenotypic heterogeneity: Nanog and other marker proteins of ESCs show large cell-to-cell variation in their expression level, which should significantly influence the differentiation process of individual cells. The molecular mechanism and biological implication of this heterogeneity, however, still remain elusive. We address this problem by constructing a model of the core gene-network of mouse ESCs. The model takes account of processes of binding/unbinding of transcription factors, formation/dissolution of transcription apparatus, and modification of histone code at each locus of genes in the network. These processes are hierarchically interrelated to each other forming the dynamical feedback loops. By simulating stochastic dynamics of this model, we show that the phenotypic heterogeneity of ESCs can be explained when the chromatin at the Nanog locus undergoes the large scale reorganization in formation/dissolution of transcription apparatus, which should have the timescale similar to the cell cycle period. With this slow transcriptional switching of Nanog, the simulated ESCs fluctuate among multiple transient states, which can trigger the differentiation into the lineage-specific cell states. From the simulated transitions among cell states, the epigenetic landscape underlying transitions is calculated. The slow Nanog switching gives rise to the wide basin of ESC states in the landscape. The bimodal Nanog distribution arising from the kinetic flow running through this ESC basin prevents transdifferentiation and promotes the definite decision of the cell fate. These results show that the distribution of timescales of the regulatory processes is decisively important to characterize the fluctuation of cells and their differentiation process. The analyses through the epigenetic landscape and the kinetic flow on the landscape should provide a guideline to engineer cell differentiation.

Thermodynamics of nucleotide binding to the chaperonin GroEL studied by isothermal titration calorimetry: evidence for noncooperative nucleotide binding.

We characterized the thermodynamics of binding reactions of nucleotides ADP and ATPgammaS (a nonhydrolyzable analog of ATP) to GroEL in a temperature range of 5 degrees C to 35 degrees C by isothermal titration calorimetry. Analysis with a noncooperative binding model has shown that the bindings of nucleotides are driven enthalpically with binding constants of 7x103 M-1 and 4x104 M-1 for ADP and ATPgammaS, respectively, at 26 degrees C and that the heat capacity change DeltaCp is about 100 cal/mol.K for both the nucleotides. The stoichiometries of binding were about 8 and 9 molecules for ADP and ATPgammaS, respectively, per GroEL tetradecamer at 5 degrees C, and both increased with temperature to reach about 14 (ADP) and 12 (ATPgammaS) for both nucleotides at 35 degrees C. The absence of initial increase of binding heat as well as Hill coefficient less than 1.2, which were obtained from the fitting to the model curve by assuming positive cooperativity, showed that there was virtually no positive cooperativity in the nucleotide bindings. Incorporating a difference in affinity for the nucleotide (ADP and ATPgammaS) between the two rings of GroEL into the noncooperative binding model improved the goodness of fitting and the difference in the affinity increased with decreasing temperature.

Conformational change of the actomyosin complex drives the multiple stepping movement

Actin-myosin (actomyosin) generates mechanical force by consuming ATP molecules. We apply the energy landscape perspective to address a controversial issue as to whether the myosin head moves with multiple steps after a single ATP hydrolysis or only a single mechanical event of the lever-arm swinging follows a single ATP hydrolysis. Here we propose a theoretical model in which the refolding of the partially unfolded actomyosin complex and the movement of the myosin head along the actin filament are coupled. A single ATP hydrolysis is followed by the formation of a high free-energy partially unfolded actomyosin complex, which then gradually refolds with a concomitant multiple stepping movement on the way to the lowest free-energy rigor state. The model quantitatively explains the single-molecular observation of the multiple stepping movement and is consistent with structural observations of the disorder in the actomyosin-binding process. The model also explains the observed variety in dwell time before each step, which is not accounted for by previous models, such as the lever-arm or ratchet models.

Synchronization of Circadian Oscillation of Phosphorylation Level of KaiC In Vitro

In recent experimental reports, robust circadian oscillation of the phosphorylation level of KaiC has been reconstituted by incubating three cyanobacterial proteins, KaiA, KaiB, and KaiC, with ATP in vitro. This reconstitution indicates that protein-protein interactions and the associated ATP hydrolysis suffice to generate the oscillation, and suggests that the rhythm arising from this protein-based system is the circadian clock pacemaker in cyanobacteria. The mechanism of this reconstituted oscillation, however, remains elusive. In this study, we extend our previous model of oscillation by explicitly taking two phosphorylation sites of KaiC into account and we apply the extended model to the problem of synchrony of two oscillatory samples mixed at different phases. The agreement between the simulated and observed data suggests that the combined mechanism of the allosteric transition of KaiC hexamers and the monomer shuffling between them plays a key role in synchronization among KaiC hexamers and hence underlies the population-level oscillation of the ensemble of Kai proteins. The predicted synchronization patterns in mixtures of unequal amounts of two samples provide further opportunities to experimentally check the validity of the proposed mechanism. This mechanism of synchronization should be important in vivo for the persistent oscillation when Kai proteins are synthesized at random timing in cyanobacterial cells.

Folding pathway of a multidomain protein depends on its topology of domain connectivity

Significance Theoretical and experimental efforts during the last two decades have revealed many important features of protein folding. However, these efforts have largely focused on single-domain proteins, and additional methods and concepts have been awaited to understand the rich variety of multidomain proteins. To understand the relationship between the topology and folding pathways of multidomain proteins, we developed an Ising-like structure-based model. Our simulated results showed that a dominant pathway is selected in an example protein, dihydrofolate reductase (DHFR), which prevents the rapid decrease in conformation entropy during the course of folding. However, in its circular permutant, for which the topological complexity of wild-type DHFR is resolved, two pathways coexist, resulting in a complex folding behavior. How do the folding mechanisms of multidomain proteins depend on protein topology? We addressed this question by developing an Ising-like structure-based model and applying it for the analysis of free-energy landscapes and folding kinetics of an example protein, Escherichia coli dihydrofolate reductase (DHFR). DHFR has two domains, one comprising discontinuous N- and C-terminal parts and the other comprising a continuous middle part of the chain. The simulated folding pathway of DHFR is a sequential process during which the continuous domain folds first, followed by the discontinuous domain, thereby avoiding the rapid decrease in conformation entropy caused by the association of the N- and C-terminal parts during the early phase of folding. Our simulated results consistently explain the observed experimental data on folding kinetics and predict an off-pathway structural fluctuation at equilibrium. For a circular permutant for which the topological complexity of wild-type DHFR is resolved, the balance between energy and entropy is modulated, resulting in the coexistence of the two folding pathways. This coexistence of pathways should account for the experimentally observed complex folding behavior of the circular permutant.

Coupling of Lever Arm Swing and Biased Brownian Motion in Actomyosin

An important unresolved problem associated with actomyosin motors is the role of Brownian motion in the process of force generation. On the basis of structural observations of myosins and actins, the widely held lever-arm hypothesis has been proposed, in which proteins are assumed to show sequential structural changes among observed and hypothesized structures to exert mechanical force. An alternative hypothesis, the Brownian motion hypothesis, has been supported by single-molecule experiments and emphasizes more on the roles of fluctuating protein movement. In this study, we address the long-standing controversy between the lever-arm hypothesis and the Brownian motion hypothesis through in silico observations of an actomyosin system. We study a system composed of myosin II and actin filament by calculating free-energy landscapes of actin-myosin interactions using the molecular dynamics method and by simulating transitions among dynamically changing free-energy landscapes using the Monte Carlo method. The results obtained by this combined multi-scale calculation show that myosin with inorganic phosphate (Pi) and ADP weakly binds to actin and that after releasing Pi and ADP, myosin moves along the actin filament toward the strong-binding site by exhibiting the biased Brownian motion, a behavior consistent with the observed single-molecular behavior of myosin. Conformational flexibility of loops at the actin-interface of myosin and the N-terminus of actin subunit is necessary for the distinct bias in the Brownian motion. Both the 5.5–11 nm displacement due to the biased Brownian motion and the 3–5 nm displacement due to lever-arm swing contribute to the net displacement of myosin. The calculated results further suggest that the recovery stroke of the lever arm plays an important role in enhancing the displacement of myosin through multiple cycles of ATP hydrolysis, suggesting a unified movement mechanism for various members of the myosin family.