Adenosine triphosphate (ATP) is a versatile molecule used mainly for energy and a phosphate source. The hydrolysis of γ phosphate initiates the reactions and these reactions almost always start when ATP binds to protein. Therefore, there should be a mechanism to prevent spontaneous hydrolysis reaction and a mechanism to lead ATP to a pure energy source or to a phosphate source. To address these questions, we extensively analyzed the effect of protein to ATP conformation based on the sampling of the ATP solution conformations obtained from molecular dynamics simulation and the sampling of ATP structures bound to protein found in a protein structure database. The comparison revealed mainly the following three points; 1) The ribose ring in ATP molecule, which puckers in many ways in solution, tends to assume either C2′ exo or C2′ endo when it binds to protein. 2) The adenine ring in ATP molecule, which takes open-book motion with the two ring structures, has two distinct structures when ATP binds to protein. 3) The glycosyl-bond and the bond between phosphate and the ribose have unique torsion angles, when ATP binds to protein. The combination of torsion angles found in protein-bound forms is under-represented in ATP molecule in water. These findings suggest that ATP-binding protein exerts forces on ATP molecule to assume a conformation that is rarely found in solution, and that this conformation change should be a trigger for the reactions on ATP molecule.
The nucleotide turnover rates of bipolar myosin thick filament along which actin filament slides were measured by the displacement of prebound fluorescent ATP analog 2′(3′)-O-[N-[2-[(Cy3)]amindo]ethyl] carbamoyl]-adenosine 5′ triphosphate (Cy3-EDA-ATP) upon flash photolysis of caged ATP. The fluorescence of the thick filament where actin filament slides decayed with two exponential processes. The slower rate constant was the same as that without actin filament. Along bipolar myosin thick filament, actin filaments slide at a fast speed towards the central bare zone (forward), but more slowly away from the bare zone (backward). The displacement rate constant of fluorescent ATP from the myosin filament where actin filament moved forward was 5.0 s-1, whereas the rate constant where the actin filament slid backward was 1.7 s-1. These findings suggest that the slow ADP release rate is responsible for the slow backward sliding movement.
Flagellar motors embedded in bacterial membranes are molecular machines powered by specific ion flows. Each motor is composed of a stator and a rotor and the interactions of those components are believed to generate the torque. Na+ influx through the PomA/PomB stator complex of Vibrio alginolyticus is coupled to torque generation and is speculated to trigger structural changes in the cytoplasmic domain of PomA that interacts with a rotor protein in the C-ring, FliG, to drive the rotation. In this study, we tried to overproduce the cytoplasmic loop of PomA (PomA-Loop), but it was insoluble. Thus, we made a fusion protein with a small soluble tag (GB1) which allowed us to express and characterize the recombinant protein. The structure of the PomA-Loop seems to be very elongated or has a loose tertiary structure. When the PomA-Loop protein was produced in E. coli, a slight dominant effect was observed on motility. We conclude that the cytoplasmic loop alone retains a certain function.
Nucleotidyl-transfer reaction catalyzed by DNA polymerase is a fundamental enzymatic reaction for DNA synthesis. Until now, a number of structural and kinetic studies on DNA polymerases have proposed a two-metal-ion mechanism of the nucleotidyl-transfer reaction. However, the actual reaction process has never been visualized. Recently, we have followed the nucleotidyl-transfer reaction process by human DNA polymerase η using time-resolved protein crystallography. In sequence, two Mg2+ ions bind to the active site, the nucleophile 3′-OH is deprotonated, the deoxyribose at the primer end converts from C2′-endo to C3′-endo, and the nucleophile and the α-phosphate of the substrate dATP approach each other to form the new bond. In this process, we observed transient elements, which are a water molecule to deprotonate the 3′-OH and an additional Mg2+ ion to stabilize the intermediate state. Particularly, the third Mg2+ ion observed in this study may be a general feature of the two-metal-ion mechanism.
The influences of structural alterations of sphingomyelin (SM) on its interactions with cholesterol (chol) and on ordered phase formation were examined by density measurements and surface pressure vs. molecular area isotherm measurements. In addition, we quantitatively characterized the ordered phase formed in each SM and chol binary mixture on the basis of the molecular compressional modulus of SM (C-1mol). Density measurements demonstrated that the ordered phase formation in threo-SM (tSM)/chol and dihydrosphingomyelin (DHSM)/chol binary bilayers shows similar chol concentration-dependency to that of natural erythro-SM (eSM)/chol bilayers; the ordered phase formation was completed in the presence of 25 mol% chol. In contrast, SM bearing a triple bond in the place of a double bond (tripleSM) required a greater concentration of chol to completely transform the bilayer into the ordered phase (at 40 mol% chol). Surface pressure vs. molecular area isotherms showed that the DHSM molecule (C-1mol=290 mN/m) is more rigid than eSM (C-1mol=240 mN/m) above 30 mol% chol (in the ordered phase), although these values are similar (140-150 mN/m) in the absence of chol (liquid condensed phase). Most likely, the DHSM/chol mixture forms a more ordered membrane than the eSM/chol mixture does. Moreover, in the absence of chol, the rigidity of the tripleSM molecule (C-1mol=250 mN/m) is significantly higher as compared with that of the eSM molecule (C-1mol=150 mN/m), which is probably due to the presence of a triple bond.
The synthesis of ATP, the key reaction of biological energy metabolism, is accomplished by the rotary motor protein; FoF1-ATP synthase (FoF1). In vivo, FoF1, located on the cell membrane, carries out ATP synthesis by using the proton motive force. This heterologous energy conversion is supposed to be mediated by the mechanical rotation of FoF1; however, it still remained unclear. Recently, we developed the novel experimental setup to reproduce the proton motive force in vitro and succeeded in directly observing the proton-driven rotation of FoF1. In this review, we describe the interesting working principles determined so far for FoF1 and then introduce results from our recent study.
Channelrhodopsin (ChR) is a light-gated cation channel derived from green algae. Since the inward flow of cations triggers the neuron firing, neurons expressing ChRs can be optically controlled even within freely moving mammals. Although ChR has been broadly applied to neuroscience research, little is known about its molecular mechanisms. We determined the crystal structure of chimeric ChR at 2.3 Å resolution and revealed its molecular architecture. The integration of structural, electrophysiological, and computational analyses provided insight into the molecular basis for the channel function of ChR, and paved the way for the principled design of ChR variants with novel properties.
The bacterial flagellar hook acts as a universal joint to smoothly transmit torque produced by the motor to the filament. The hook protein FlgE assembles into a 55 nm tubular structure with the help of the hook cap (FlgD). FlgE consists of four domains, D0, Dc, D1 and D2, arranged from the inner to the outer part of the tubular structure of the hook. The Dc domain contributes to the structural stability of the hook, but it is unclear how this Dc domain is responsible for the universal joint mechanism. Here, we carried out a deletion analysis of the FlgE Dc domain. FlgEΔ4/5 with deletion of residues 30 to 49 was not secreted into the culture media. FlgEΔ5 and FlgEΔ6 with deletions of residues 40 to 49 and 50 to 59, respectively, still formed hooks, allowing the export apparatus to export the hook-filament junction proteins FlgK and FlgL and flagellin FliC. However, these deletions inhibited the replacement of the FlgD hook cap by FlgK at the hook tip, thereby abolishing filament formation. Deletion of residues 50 to 59 significantly affected hook morphology. These results suggest that the Dc domain is responsible not only for hook assembly but also for FlgE export, the interaction with FlgK, and the polymorphic supercoiling mechanism of the hook.
For genome stability, the proper segregation of chromosomes is required. The exquisite process of chromosome segregation has charmed a lot of cell- and molecular biologists into watching what happens inside a mitotic cell and how each molecule contributes to this process for the accomplishment of accurate cell division1. The process to partition the duplicated genome to the daughter cells in each cell division is mediated by a self-organized structure called the mitotic spindle. It is well known that the mitotic spindle is a multi-component macromolecular machine composed of microtubules, molecular motors (kinesins, cytoplasmic dynein), and other regulatory molecules (microtubule-associated proteins, kinases, etc.). In recent years, most of the protein components of the mitotic spindle have been identified and the functions of these proteins have been characterized using molecular perturbations2,3. Thus, the mechanisms for spindle assembly and chromosome segregation are being revealed rapidly. However, the chromosome segregation machinery is poorly understood from the mechanical point of view, such as how the mitotic spindle within a cell responds to a variety of mechanical forces, originating from cell-cell interactions or environmental fluctuations. Recent advances in the controlled mechanical perturbation have indicated that the mitotic spindle possesses a structural pliability, size adaptability to the applied external forces, and a strong self-organizing ability. Mechanical perturbations revealed also the mechanochemical regulation of chromosome segregation machinery, which responds to the applied forces. Here, we discuss the current progress in the biophysical research on the architectural and functional dynamics of the mitotic spindle.
Western blotting is a widely used technique for the detection and quantification of proteins and peptides. However, it is challenging to detect small peptides efficiently by the conventional Western blotting method with shaking, in part because the peptides readily detach from the blotted membrane. Although some modified Western blotting protocols have been developed to overcome this problem, it remains difficult to prevent peptide detachment from the membrane. In this study, we show that the previously developed vacuum-assisted detection method greatly improves the detection of small peptides without additional protocol modification. The vacuum-assisted method was developed to shorten the time required for all immunodetection steps, and all the Western blotting solutions penetrated the membrane quickly and efficiently by this method. By using this vacuum method, we succeeded in detecting small peptides that were completely undetectable by the conventional Western blotting method. We also confirmed that peptide detachment was induced even by gentle shaking in the case of the conventional method, and the detachment was accelerated when detergent was present in the buffer. Unlike in the conventional method, there is no need to shake the membrane in solution in the vacuum method. Therefore, it is thought that the small peptides could be detected sensitively only by the vacuum method.
Absorption spectra of visual pigments are adaptively tuned to optimize informational capacity in most visual systems. Our recent investigation of the eyes of the jumping spider reveals an apparent exception: the absorption characteristics of a visual pigment cause defocusing of the image, reducing visual acuity generally in a part of the retina. However, the amount of defocus can theoretically provide a quantitative indication of the distance of an object. Therefore, we proposed a novel mechanism for depth perception in jumping spiders based on image defocus. Behavioral experiments revealed that the depth perception of the spider depended on the wavelength of the ambient light, which affects the amount of defocus because of chromatic aberration of the lens. This wavelength effect on depth perception was in close agreement with theoretical predictions based on our hypothesis. These data strongly support the hypothesis that the depth perception mechanism of jumping spiders is based on image defocus.
Molecular motor is a nano-sized chemical engine that converts chemical free energy to mechanical motions. Hence, the energetics is as important as kinetics in order to understand its operation principle. We review experiments to evaluate the thermodynamic properties of a rotational F1-ATPase motor (F1-motor) at a single-molecule level. We show that the F1-motor achieves 100% thermodynamic efficiency at the stalled state. Furthermore, the motor reduces the internal irreversible heat inside the motor to almost zero and achieves a highly-efficient free energy transduction close to 100% during rotations far from quasistatic process. We discuss the mechanism of how the F1-motor achieves such a high efficiency, which highlights the remarkable property of the nano-sized engine F1-motor.
Hydration structures around F-actin and myosin subfragment-1 (S1), which play central roles as counterparts in muscle contraction, were investigated by small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS). The radius of gyration of chymotryptic S1 was evaluated to be 41.3±1.1 Å for SAXS, 40.1±3.0 Å for SANS in H2O, and 37.8±0.8 Å for SANS in D2O, respectively. The values of the cross-sectional radius of gyration of F-actin were 25.4±0.03 Å for SAXS, 23.4±2.4 Å for SANS in H2O, and 22.6±0.6 Å for SANS in D2O, respectively. These differences arise from different contributions of the hydration shell to the scattering curves. Analysis by model calculations showed that the hydration shell of S1 has the average density 10-15% higher than bulk water, being the typical hydration shell. On the other hand, the hydration shell of F-actin has the average density more than 19% higher than bulk water, indicating that F-actin has a denser, unusual hydration structure. The results indicate a difference in the hydration structures around F-actin and S1. The unusual hydration structure around F-actin may have the structural property of so-called “hyper-mobile water” around F-actin.
Maxwell's demon is a hypothetical creature that can convert information to free energy. A debate that has lasted for more than 100 years has revealed that the demon's operation does not contradict the laws of thermodynamics; hence, the demon can be realized physically. We briefly review the first experimental demonstration of Maxwell's demon of Szilard's engine type that converts information to free energy. We pump heat from an isothermal environment by using the information about the thermal fluctuations of a Brownian particle and increase the particle's free energy.
A database for the 3D structures of available compounds is essential for the virtual screening by molecular docking. We have developed the LigandBox database (http://ligandbox.protein.osaka-u.ac.jp/ligandbox/) containing four million available compounds, collected from the catalogues of 37 commercial suppliers, and approved drugs and biochemical compounds taken from KEGG_DRUG, KEGG_COMPOUND and PDB databases. Each chemical compound in the database has several 3D conformers with hydrogen atoms and atomic charges, which are ready to be docked into receptors using docking programs. The 3D conformations were generated using our molecular simulation program package, myPresto. Various physical properties, such as aqueous solubility (LogS) and carcinogenicity have also been calculated to characterize the ADME-Tox properties of the compounds. The Web database provides two services for compound searches: a property/chemical ID search and a chemical structure search. The chemical structure search is performed by a descriptor search and a maximum common substructure (MCS) search combination, using our program kcombu. By specifying a query chemical structure, users can find similar compounds among the millions of compounds in the database within a few minutes. Our database is expected to assist a wide range of researchers, in the fields of medical science, chemical biology, and biochemistry, who are seeking to discover active chemical compounds by the virtual screening.
Attenuated total reflectance (ATR)-FTIR spectroscopy has been widely used to probe protein structural changes under various stimuli, such as light absorption, voltage change, and ligand binding, in aqueous conditions. Time-resolved measurements require a trigger, which can be controlled electronically; therefore, light and voltage changes are suitable. Here we developed a novel, rapid buffer-exchange system for time-resolved ATR-FTIR spectroscopy to monitor the ligand- or ion-binding reaction of a protein. By using the step-scan mode (time resolution; 2.5 ms), we confirmed the completion of the buffer-exchange reaction within ~25 ms; the process was monitored by the infrared absorption change of a nitrate band at 1,350 cm-1. We also demonstrated the anion-binding reaction of a membrane protein, Natronomonas pharaonis halorhodopsin (pHR), which binds a chloride ion in the initial anion-binding site near the retinal chromophore. The formation of chloride- or nitrate-bound pHR was confirmed by an increase of the retinal absorption band at 1,528 cm-1. It also should be noted that low sample consumption (~1 μg of protein) makes this new method a powerful technique to understand ligand-protein and ion-protein interactions, particularly for membrane proteins.
In the translation process, translating ribosomes usually move on an mRNA until they reach the stop codon. However, when ribosomes translate an aberrant mRNA, they stall. Then, ribosomes are rescued from the aberrant mRNA, and the aberrant mRNA is subsequently degraded. In eukaryotes, Pelota (Dom34 in yeast) and Hbs1 are responsible for solving general problems of ribosomal stall in translation. In archaea, aPelota and aEF1α, homologous to Pelota and Hbs1, respectively, are considered to be involved in that process. In recent years, great progress has been made in determining structures of Dom34/aPelota and Hbs1/aEF1α. In this review, we focus on the functional roles of Dom34/aPelota and Hbs1/aEF1α in ribosome rescue, based on recent structural studies of them. We will also present questions to be answered by future work.
Flagella/cilia are motile organelles with more than 400 proteins. To understand the mechanism of such complex systems, we need methods to describe molecular arrangements and conformations three-dimensionally in vivo. Cryo-electron tomography enabled us such a 3D structural analysis. Our group has been working on 3D structure of flagella/cilia using this method and revealed highly ordered and beautifully organized molecular arrangement. 3D structure gave us insights into the mechanism to generate bending motion with well defined waveforms. In this review, I summarize our recent structural studies on flagella/cilia by cryo-electron tomography, mainly focusing on dynein microtubule-based ATPase motor proteins and the radial spoke, a regulatory protein complex.
Kinesin-1 (conventional kinesin) is a molecular motor that transports various cargo such as endoplasmic reticulum and mitochondria in cells. Its two head domains walk along microtubule by hydrolyzing ATP, while the tail domains at the end of the long stalk bind to the cargo. When a kinesin is not carrying cargo, its motility and ATPase activity is inhibited by direct interactions between the tail and head. However, the mechanism of this tail regulation is not well understood. Here, we apply single molecule fluorescence resonance energy transfer (smFRET) to observe this interaction in stalk-truncated kinesin. We found that kinesin with two tails forms a folding conformation and dissociates from microtubules, whereas kinesin with one tail remains bound to the microtubule and is immobile even in the presence of ATP. We further investigated the head-tail interaction as well as head-head coordination on the microtubule at various nucleotide conditions. From these results, we propose a two-step inhibition model for kinesin motility.
Conditioned taste aversion (CTA) can be classically conditioned in the pond snail Lymnaea stagnalis and subsequently be consolidated into long-term memory (LTM). The neural trace that subserves CTA-LTM can be summarized as follows: A polysynaptic inhibitory postsynaptic potential recorded in the neuron 1 medial (N1M) cell in the conditioned snails as a result of activation of the cerebral giant cell (CGC) is larger and lasts longer than that in control snails. The N1M cell is ultimately activated by the CGC via the neuron 3 tonic (N3t) cell. That is, the inhibitory monosynaptic inputs from the N3t cell to the N1M cell are facilitated. The N1M and N3t cells are the members of feeding central pattern generator, whereas the CGC is a multimodal interneuron thought to play a key role in feeding behavior. Here we examined the involvement of a second messenger, cAMP, in the establishment of the memory trace. We injected cAMP into the CGC and monitored the potentials of the B3 motor neuron activated by the CGC. B3 activity is used as an index for the synaptic inputs from the N3t cell to the N1M cell. We found that the B3 potentials were transiently enlarged. Thus, when the cAMP concentration is increased in the CGC by taste aversion training, cAMP-induced changes may play a key role in the establishment of a memory trace in the N3t cell.
Protein-bound water molecules play crucial roles in their structure and function, but their detection is an experimental challenge, particularly in aqueous solution at room temperature. By applying attenuated total reflection (ATR) Fourier-transform infrared (FTIR) spectroscopy to a light-driven Cl- pump pharaonis halorhodopsin (pHR), here we detected an O-H stretching vibration of protein-bound water molecules in the active center. The pHR(Cl-) minus pHR(Br-) ATR-FTIR spectra show random fluctuation at 3600-3000 cm-1, frequency window of water vibration, which can be interpreted in terms of dynamical fluctuation of aqueous water at room temperature. On the other hand, we observed a reproducible spectral feature at 3617 (+)/3630 (-) cm-1 in the pHR(Cl-) minus pHR(Br-) spectrum, which is absent in the pHR(Cl-) minus pHR(Cl-) and in the pHR(Br-) minus pHR(Br-) spectra. The water O-H stretching vibrations of pHR(Cl-) and pHR(Br-) at 3617 and 3630 cm-1, respectively, are confirmed by light-induced difference FTIR spectra in isotope water (H218O) at 77 K. The observed water molecule presumably binds to the active center of pHR, and alter its hydrogen bond during the Cl- pumping photocycle.
The bacterial flagellar motor is made of a rotor and stators. In Salmonella it is thought that about a dozen MotA/B complexes are anchored to the peptidoglycan layer around the motor through the C-terminal peptidoglycan-binding domain of MotB to become active stators as well as proton channels. MotB consists of 309 residues, forming a single transmembrane helix (30-50), a stalk (51-100) and a C-terminal peptidoglycan-binding domain (101-309). Although the stalk is dispensable for torque generation by the motor, it is required for efficient motor performance. Residues 51 to 72 prevent premature proton leakage through the proton channel prior to stator assembly into the motor. However, the role of residues 72-100 remains unknown. Here, we analyzed the torque-speed relationship of the MotB(Δ72-100) motor. At a low speed near stall, this mutant motor produced torque at the wild-type level. Unlike the wild-type motor, however, torque dropped off drastically by slight decrease in external load and then showed a slow exponential decay over a wide range of load by its further reduction. Since it is known that the stator is a mechanosensor and that the number of active stators changes in a load-dependent manner, we interpreted this unusual torque-speed relationship as anomaly in load-dependent control of the number of active stators. The results suggest that residues 72-100 of MotB is required for proper load-dependent control of the number of active stators around the rotor.
What is the general principle of sensory transduction? Sensory transduction is defined as energy transformation from the external world to the internal world. The energy of the external world, such as thermal energy (heat), electro-magnetic energy (light), mechanical energy (sound) and the energy from molecules (chemicals), is converted into electrochemical events in the animal nervous system. The following five classes of special sense receptors are utilized for energy conversion: vision (photo); audition (sound); taste and smell (chemo); and tactile (mechano). There are also other special sense receptors, including thermo and noxious receptors. The focus of this study is on photoreceptors, sound-receptors and odorant-receptors because the transduction mechanisms of these receptors are explained biochemically and understood by a common physical principle; these biochemical models are well known in neuroscience. The following notable problems are inherent in these biochemical models: the cGMP ionophore model of the vertebrate photoreceptor cannot explain the fast photoresponse (~msec); the tip links connection model of stereocilia in the basilar membrane for opening the K+ channel on the tip of a hair has difficulty explaining the high frequency vibration of hair cells without a damping of the oscillation, and the odorant shape-specific receptor model for olfactory transduction has difficulty in discriminating the minute differences among similar fragrant smells of essential oils with different molecular shapes. These difficulties might arise from a lack of the physical sense when the transduction models were proposed. This article will reconsider these problems and propose rational models for visual, olfactory and auditory transduction.