The contraction process of living Vorticella sp. has been investigated by image processing using a high-speed video camera. In order to express the temporal change in the stalk length resulting from the contraction, a damped spring model and a nucleation and growth model are applied. A double exponential is deduced from a conventional damped spring model, while a stretched exponential is newly proposed from a nucleation and growth model. The stretched exponential function is more suitable for the curve fitting and suggests a more particular contraction mechanism in which the contraction of the stalk begins near the cell body and spreads downwards along the stalk. The index value of the stretched exponential is evaluated in the range from 1 to 2 in accordance with the model in which the contraction undergoes through nucleation and growth in a one-dimensional space.
The contraction process of living Vorticella sp. in polymer solutions with various viscosities has been investigated by image processing using a high-speed video camera. The viscosity of the external fluid ranges from 1 to 5 mPa·s for different polymer additives such as hydroxypropyl cellulose, polyethylene oxide, and Ficoll. The temporal change in the contraction length of Vorticella sp. in various macromolecular solutions is fitted well by a stretched exponential function based on the nucleation and growth model. The maximum speed of the contractile process monotonically decreases with an increase in the external viscosity, in accordance with power law behavior. The index values approximate to 0.5 and this suggests that the viscous energy dissipated by the contraction of Vorticella sp. is constant in a macromolecular environment.
We have investigated the flow birefringence property and assembly process of desmin, a muscle specific intermediate protein. Solution of non-polar desmin filaments showed birefringence when aligned in the sheared flow. The amount of birefringence of desmin filaments was considerably lower when compared with that of F-actin solution. Assembly of desmin from soluble state was followed by the birefringence measurements. At any desmin concentrations examined, the degree of flow birefringence increased rapidly just after the addition of the assembly buffer and reached a saturated level within 30 min. The time to reach half-maximal values of flow birefringence slightly but definitely depended on the initial soluble desmin concentrations. The plotting of the initial velocity of the assembly against the soluble desmin concentrations showed a slope of 1.4. This result suggested that the assembly process detected by flow birefringence measurements followed second-order kinetics, and the process corresponded to the second step of the three stage model for type III intermediate filament assembly proposed by Herrmann and his colleagues; the annealing of unit length filaments into filaments.
The power stroke model was criticized and a model was proposed for muscle contraction mechanism (Mitsui, 1999). The proposed model was further developed and calculations based on the model well reproduced major experimental data on the steady filament sliding (Mitsui and Ohshima, 2008) and on the transient phenomena (Mitsui, Takai and Ohshima, 2011). In this review more weight is put on explanation of the basic ideas of the model, especially logical necessity of the model, leaving mathematical details to the above-mentioned papers. A thermodynamic relationship that any models based upon the sliding filament theory should fulfill is derived. The model which fulfills the thermodynamic relationship is constructed on the assumption that a myosin head bound to an actin filament forms a complex with three actin molecules. In shortening muscles, the complex moves along the actin filament changing the partner actin molecules with steps of about 5.5 nm. This process is made possible through cooperative interaction among crossbridges. The ATP hydrolysis energy is liberated by fraction at each step through chemical reactions between myosin and actin molecules. The cooperativity among crossbridges disappears in length-clamped muscles, in agreement with experimental observations that the crossbridge produces force independently in the isometric tetanus state. The distance of the head movement per ATP hydrolysis cycle is expected to be about 5.5 nm or a few times of it under the condition of the in vitro single head experiments. Calculation results are surveyed illustrating that they are in good agreement with major experimental observations.
Upon DNA damage, the cell fate decision between survival and apoptosis is largely regulated by p53-related networks. Recent experiments found a series of discrete p53 pulses in individual cells, which led to the hypothesis that the cell fate decision upon DNA damage is controlled by counting the number of p53 pulses. Under this hypothesis, Sun et al. (2009) modeled the Bax activation switch in the apoptosis signal transduction pathway that can rigorously “count” the number of uniform p53 pulses. Based on experimental evidence, here we use variable p53 pulses with Sun et al.'s model to investigate how the variability in p53 pulses affects the rigor of the cell fate decision by the pulse number. Our calculations showed that the experimentally anticipated variability in the pulse sizes reduces the rigor of the cell fate decision. In addition, we tested the roles of the cooperativity in PUMA expression by p53, finding that lower cooperativity is plausible for more rigorous cell fate decision. This is because the variability in the p53 pulse height is more amplified in PUMA expressions with more cooperative cases.
Atomic force microscopy (AFM) enables the pick up of a single protein molecule to apply a mechanical force. This technique, called “force spectroscopy,” provides unique information about the intermediates and free energy landscape of the mechanical unfolding of proteins. In this review, we introduce the AFM-based single molecule force spectroscopy of proteins and describe recent studies that answer some fundamental questions such as “is the mechanical resistance of proteins isotropic?”, “what is the structure of the transition state in mechanical unfolding?”, and “is mechanical unfolding related to biological functions?”
Escherichia coli cells swim toward a favorable environment by chemotaxis. The chemotaxis system regulates the swimming behavior of the bacteria by controlling the rotational direction of their flagellar motors. Extracellular stimuli sensed by chemoreceptors are transduced to an intracellular signal molecule, phosphorylated CheY (CheY-P), that switches the rotational direction of the flagellar motors from counterclockwise (CCW) to clockwise (CW) or from CW to CCW. Many studies have focused on identifying the proteins involved in the chemotaxis system, and findings on the structures and intracellular localizations of these proteins have largely elucidated the molecular pathway. On the other hand, quantitative evaluations of the chemotaxis system, including the process of intracellular signaling by the propagation of CheY-P and the rotational switching of flagellar motor by binding of CheY-P molecules, are still uncertain. For instance, scientific consensus has held that the flagellar motors of an E. coli cell switch rotational direction asynchronously. However, recent work shows that the rotational switching of any two different motors on a single E. coli cell is highly coordinated; a sub-second switching delay between motors is clearly correlated with the relative distance of each motor from the chemoreceptor patch located at one pole of the cell. In this review of previous studies and our recent findings, we discuss the regulatory mechanism of the multiple flagellar motors on an individual E. coli cell and the intracellular signaling process that can be inferred from this coordinated switching.
The fluctuation theorem (FT), which is a recent achievement in non-equilibrium statistical mechanics, has been suggested to be useful for measuring the driving forces of motor proteins. As an example of this application, we performed single-molecule experiments on F1-ATPase, which is a rotary motor protein, in which we measured its rotary torque by taking advantage of FT. Because fluctuation is inherent nature in biological small systems and because FT is a non-destructive force measurement method using fluctuation, it will be applied to a wide range of biological small systems in future.
F1-ATPase (F1) is the smallest rotary motor protein that couples ATP hydrolysis/synthesis to rotary motion in a highly reversible manner. F1 is unique compared with other motor proteins because of its high efficiency and reversibility in converting chemical energy into mechanical work. To determine the energy conversion mechanism of F1-ATPase, we developed a novel single-molecule manipulation technique with magnetic tweezers and determined the timing of Pi release, which was the last unknown piece of the chemomechanical coupling scheme of F1. The established fundamental chemomechanical coupling scheme provides evidence to explain the high reversibility between catalysis and mechanical work.
Comparison and classification of protein structures are fundamental means to understand protein functions. Due to the computational difficulty and the ever-increasing amount of structural data, however, it is in general not feasible to perform exhaustive all-against-all structure comparisons necessary for comprehensive classifications. To efficiently handle such situations, we have previously proposed a method, now called GIRAF. We herein describe further improvements in the GIRAF protein structure search and alignment method. The GIRAF method achieves extremely efficient search of similar structures of ligand binding sites of proteins by exploiting database indexing of structural features of local coordinate frames. In addition, it produces refined atom-wise alignments by iterative applications of the Hungarian method to the bipartite graph defined for a pair of superimposed structures. By combining the refined alignments based on different local coordinate frames, it is made possible to align structures involving domain movements. We provide detailed accounts for the database design, the search and alignment algorithms as well as some benchmark results.
Formin homology proteins (formins) are actin nucleation factors which remain bound to the growing barbed end and processively elongate actin filament (F-actin). Recently, we have demonstrated that a mammalian formin mDia1 rotates along the long-pitch helix of F-actin during processive elongation (helical rotation) by single-molecule fluorescence polarization. We have also shown processive depolymerization of mDia1-bound F-actin during which helical rotation was visualized. In the cell where F-actins are highly cross-linked, formins should rotate during filament elongation. Therefore, when formins are tightly anchored to cellular structures, formins may not elongate F-actin. Adversely, helical rotation of formins might affect the twist of F-actin. Formins could thus control actin elongation and regulate stability of cellular actin filaments through helical rotation. On the other hand, ADP-actin elongation at the mDia1-bound barbed end turned out to become decelerated by profilin, in marked contrast to its remarkably positive effect on mDia1-mediated ATP-actin elongation. This deceleration is caused by enhancement of the off-rate of ADP-actin. While mDia1 and profilin enhance the ADP-actin off-rate, they do not apparently increase the ADP-actin on-rate at the barbed end. These results imply that G-actin-bound ATP and its hydrolysis may be part of the acceleration mechanism of formin-mediated actin elongation.
In response to an increased level of Zn2+, Synechococcus sp. PCC 7942 expresses SmtA, a metallothionein-like metal-chelating protein, while Synechocystis sp. PCC 6803 expresses ZiaA, a transporter of Zn2+. The gene expression of these proteins is regulated by repressor protein, SmtB and ZiaR, respectively. In spite of contributing to different response systems, both repressor proteins belong to the ArsR family and are highly homologous to each other. To understand the different systems responsible for dealing with excess Zn2+, we examined the cis-elements in the promoter regions of smtA and ziaA, as well as the binding affinities of recombinant SmtB and ZiaR proteins. The operator/promoter region of smtA included two palindromic sequences and that of ziaA included one. Electrophoretic mobility shift assay revealed that SmtB formed four different complexes with the operator/promoter region of smtA, whereas it formed only two different complexes with the corresponding region of ziaA. For ZiaR, the corresponding results were quite the same as those for SmtB. Furthermore, the complex formation between SmtB and operator/promoter regions is inhibited in the presence of Zn2+ at higher concentrations than 16 mM. On the other hand, the corresponding Zn2+ concentration is 128 mM. These results demonstrate that the degrees of protein-DNA complex formation between repressor proteins and the operator/promoter regions of regulated genes depend on the structures of the operator/promoter regions, and the effects of Zn2+ on the dissociation of these complexes are mainly associated with the structures of the repressors.
Bioluminescence has attracted considerable attention in the area of biophysics, primarily because the phenomenon can fundamentally be interpreted as the conversion of chemical to light energy. Although the molecular mechanisms underlying luminescence have been studied extensively in fireflies and bacteria, few studies have been undertaken in luminous fungi. This relative lack of information is likely due to the absence of a common and species-specific reaction-type in the luminous fungi examined to date. We recently succeeded in extracting, for the first time, a luminiferous substance from the fungus Mycena chlorophos. The substance was purified and characterized according to its chemical and optical properties. It is hoped that this information will facilitate the clarification of a novel molecular mechanism in fungal bioluminescence systems.
Herein, the absorption maximum of bacteriorhodopsin (bR) is calculated using our recently developed method in which the whole protein can be treated quantum mechanically at the level of INDO/S-CIS//ONIOM (B3LYP/6-31G(d,p): AMBER). The full quantum mechanical calculation is shown to reproduce the so-called opsin shift of bR with an error of less than 0.04 eV. We also apply the same calculation for 226 different bR mutants, each of which was constructed by replacing any one of the amino acid residues of the wild-type bR with Gly. This substitution makes it possible to elucidate the extent to which each amino acid contributes to the opsin shift and to estimate the inter-residue synergistic effect. It was found that one of the most important contributions to the opsin shift is the electron transfer from Tyr185 to the chromophore upon excitation. We also indicate that some aromatic (Trp86, Trp182) and polar (Ser141, Thr142) residues, located in the vicinity of the retinal polyene chain and the b-ionone ring, respectively, play an important role in compensating for the large blue-shift induced by both the counterion residues (Asp85, Asp212) and an internal water molecule (W402) located near the Schiff base linkage. In particular, the effect of Trp86 is comparable to that of Tyr185. In addition, Ser141 and Thr142 were found to contribute to an increase in the dipole moment of bR in the excited state. Finally, we provide a complete energy diagram for the opsin shift together with the contribution of the chromophore-protein steric interaction.
We predict protein structure using our recently developed free energy function for describing protein stability, which is focused on solvation thermodynamics. The function is combined with the current most reliable sampling methods, i.e., fragment assembly (FA) and comparative modeling (CM). The prediction is tested using 11 small proteins for which high-resolution crystal structures are available. For 8 of these proteins, sequence similarities are found in the database, and the prediction is performed with CM. Fairly accurate models with average Ca root mean square deviation (RMSD) ~ 2.0 Å are successfully obtained for all cases. For the rest of the target proteins, we perform the prediction following FA protocols. For 2 cases, we obtain predicted models with an RMSD ~ 3.0 Å as the best-scored structures. For the other case, the RMSD remains larger than 7 Å. For all the 11 target proteins, our scoring function identifies the experimentally determined native structure as the best structure. Starting from the predicted structure, replica exchange molecular dynamics is performed to further refine the structures. However, we are unable to improve its RMSD toward the experimental structure. The exhaustive sampling by coarse-grained normal mode analysis around the native structures reveals that our function has a linear correlation with RMSDs < 3.0 Å. These results suggest that the function is quite reliable for the protein structure prediction while the sampling method remains one of the major limiting factors in it. The aspects through which the methodology could further be improved are discussed.
Free-energy landscape is an important quantity to study large-scale motions of a biomolecular system because it maps possible pathways for the motions. When the landscape consists of thermodynamically stable states (low-energy basins), which are connected by narrow conformational pathways (i.e., bottlenecks), the narrowness slows the inter-basin round trips in conformational sampling. This results in inaccuracy of free energies for the basins. This difficulty is not cleared out even when an enhanced conformational sampling is fairly performed along a reaction coordinate. In this study, to enhance the inter-basin round trips we introduced a virtual state that covers the narrow pathways. The probability distribution function for the virtual state was controlled based on detailed balance condition for the inter-state transitions (transitions between the real-state basins and the virtual state). To mimic the free-energy landscape of a real biological system, we introduced a simple model where a wall separates two basins and a narrow hole is pierced in the wall to connect the basins. The sampling was done based on Monte Carlo (MC). We examined several hole-sizes and inter-state transition probabilities. For a small hole-size, a small inter-state transition probability produced a sampling efficiency 100 times higher than a conventional MC does. This result goes against ones intuition, because one considers generally that the sampling efficiency increases with increasing the transition probability. The present method is readily applicable to enhanced conformational sampling such as multicanonical or adaptive umbrella sampling, and extendable to molecular dynamics.
CRM1 (also known as exportin 1 or Xpo1) is the most versatile nuclear export receptor (exportin) that carries a broad range of proteins and ribonucleoproteins from the nucleus to the cytoplasm through the nuclear pore complex. The majority of the export substrates of CRM1 contain a short peptide sequence, so-called leucine-rich nuclear export signal (NES), which typically harbor four or five characteristically spaced hydrophobic residues. The transport directionality is determined by the small GTPase Ran and Ran-binding proteins that control the binding and dissociation of cargo. Here we review recent structural studies that advanced understanding of how NES is specifically recognized by CRM1 in the nucleus, and how NES is rapidly dissociated from CRM1 in the cytoplasm.
Most bacteria that swim are propelled by flagellar filaments, which are driven by a rotary motor powered by proton flux. The motor consists of the rotor and the stator. The stator consists of about 8 MotA-Mot B complex. There seems to be no definite information about the structure between the rotor and the stator, and it is examined whether the experimental data can be explained based upon the following assumptions. (a) There is viscoelastic medium between the rotor and the stator. (b) MotA-MotB complex has an electric dipole moment and produces shear stress in the electric field by a proton in the channel. Calculation results based upon these assumptions are in good agreement with the following experimental observations. (1) One revolution of the flagellar rotation consists of a constant number of steps. (2) The rotation velocity of the rotor is proportional to the transmembrane potential difference. (3) When the rotational velocity of a flagellum is changed by adjusting the viscosity of the outer fluid, the torque for the cell to rotate a flagellum is practically constant but sharply decreases when the rotational velocity increases over a critical value. (4) The rotation direction remains the same when the sign of the electrochemical potential gradient is reversed. (5) The cell produces constant torque to rotate the flagellum even when the cell is rotated by externally applied torque. (6) A simple switch mechanism is proposed for chemotaxis.
So far stimuli-responsive polymer gels and their application to smart materials have been widely studied. On the other hand, as a novel biomimetic gel, we developed gels with an autonomous self-oscillating function like a heart muscle, which was firstly reported in 1996. We designed the self-oscillating polymers and gels by utilizing the oscillating reaction, called the Belousov-Zhabotinsky (BZ) reaction. The self-oscillating polymer is composed of a poly(N-isopropylacrylamide) network in which the catalyst for the BZ reaction is covalently immobilized. In the presence of the reactants, the polymer gel undergoes spontaneous cyclic swelling-deswelling changes without any on-off switching of external stimuli. Potential applications of the self-oscillating polymers and gels include several kinds of functional material systems, such as biomimetic actuators and mass transport surface. In this review, recent progress on the polymer gels is introduced.
The rodent hippocampus possesses theta (θ) and beta (β) rhythms, which occur intermittently as bursts. Both rhythms are related to spatial memory processing in a novel environment. θ rhythm is related to spatial memory encoding process. β rhythm is related to the match/mismatch process. In the match/mismatch process, rodent hippocampus detects a representation matching sensory inputs of the current place among the retrieved internal representations of places. Long-term synaptic potentiation (LTP) is induced in both processes. The cholinergic agent carbachol induces intermittent θ and β oscillations in in vitro slices similar to in vivo bursts. LTP is facilitated during the generation of θ oscillation, suggesting that the facilitation of LTP is dependent upon the phases of intermittent burst (burst phases) of the oscillation. However, whether this is the case for β oscillation has not yet been studied. In the present study, LTP-inducing θ-burst stimulation was administered at the different burst phases of carbachol-induced β oscillations (CIBO), and the synaptic changes were measured at CA3-CA3 pyramidal cell synapses (CA3 synapse) and at CA3-CA1 pyramidal cell synapses (CA1 synapse). At the CA3 synapse, the largest magnitude of LTP was induced at the late burst phases of CIBO. At the CA1 synapse, LTP was induced only at the late burst phases. Modulation of LTP was suppressed when CIBO was blocked by the application of atropine at both synapses. The results suggest that the bursts of hippocampal β rhythm can determine the optimal temporal period for completing with the match/mismatch process.