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Hiroyuki Noji

Publications and source records attributed to Hiroyuki Noji.

At least 19 recordsLinked to original sources
PubMed2024

In vitro one-pot construction of influenza viral genomes for virus particle synthesis based on reverse genetics system.

The reverse genetics system, which allows the generation of influenza viruses from plasmids encoding viral genome, is a powerful tool for basic research on viral infection mechanisms and application research such as vaccine development. However, conventional plasmid construction using Escherichia coli (E.coli) cloning is time-consuming and has difficulties handling DNA encoding genes toxic for E.coli or highly repeated sequences. These limitations hamper rapid virus synthesis. In this study, we establish a very rapid in vitro one-pot plasmid construction (IVOC) based virus synthesis. This method dramatically reduced the time for genome plasmid construction, which was used for virus synthesis, from several days or more to about 8 hours. Moreover, infectious viruses could be synthesized with a similar yield to the conventional E.coli cloning-based method with high accuracy. The applicability of this method was also demonstrated by the generation of recombinant viruses carrying reporter genes from the IVOC products. This method enables the pathogenicity analysis and vaccine development using genetically modified viruses, and it is expected to allow for faster analysis of newly emerging variants than ever before. Furthermore, its application to other RNA viruses is also expected.

Genome, Viral↗
PubMed2007

Visualization of RecA filaments and DNA by fluorescence microscopy.

We have developed two experimental methods for observing Escherichia coli RecA-DNA filament under a fluorescence microscope. First, RecA-DNA filaments were visualized by immunofluorescence staining with anti-RecA monoclonal antibody. Although the detailed filament structures below submicron scale were unable to be measured accurately due to optical resolution limit, this method has an advantage to analyse a large number of RecA-DNA filaments in a single experiment. Thus, it provides a reliable statistical distribution of the filament morphology. Moreover, not only RecA filament, but also naked DNA region was visualized separately in combination with immunofluorescence staining using anti-DNA monoclonal antibody. Second, by using cysteine derivative RecA protein, RecA-DNA filament was directly labelled by fluorescent reagent, and was able to observe directly under a fluorescence microscope with its enzymatic activity maintained. We showed that the RecA-DNA filament disassembled in the direction from 5' to 3' of ssDNA as dATP hydrolysis proceeded.

DNA↗
PubMed2006

Single molecule energetics of F1-ATPase motor.

Motor proteins are essential in life processes because they convert the free energy of ATP hydrolysis to mechanical work. However, the fundamental question on how they work when different amounts of free energy are released after ATP hydrolysis remains unanswered. To answer this question, it is essential to clarify how the stepping motion of a motor protein reflects the concentrations of ATP, ADP, and P(i) in its individual actions at a single molecule level. The F(1) portion of ATP synthase, also called F(1)-ATPase, is a rotary molecular motor in which the central gamma-subunit rotates against the alpha(3)beta(3) cylinder. The motor exhibits clear step motion at low ATP concentrations. The rotary action of this motor is processive and generates a high torque. These features are ideal for exploring the relationship between free energy input and mechanical work output, but there is a serious problem in that this motor is severely inhibited by ADP. In this study, we overcame this problem of ADP inhibition by introducing several mutations while retaining high enzymatic activity. Using a probe of attached beads, stepping rotation against viscous load was examined at a wide range of free energy values by changing the ADP concentration. The results showed that the apparent work of each individual step motion was not affected by the free energy of ATP hydrolysis, but the frequency of each individual step motion depended on the free energy. This is the first study that examined the stepping motion of a molecular motor at a single molecule level with simultaneous systematic control of DeltaG(ATP). The results imply that microscopically defined work at a single molecule level cannot be directly compared with macroscopically defined free energy input.

Adenosine Triphosphate↗
PubMed2006

Electrophysiological recordings of single ion channels in planar lipid bilayers using a polymethyl methacrylate microfluidic chip.

Planar lipid bilayers are used for functional studies of ion channel proteins using electrophysiological techniques. We have been developing a plastic micro-fluidic device for the reconstitution of planar lipid bilayers and electrophysiological recordings toward a "membrane protein chip" for high-throughput screening. In the previous report [Suzuki, H., Tabata, K.V., Noji, H., Takeuchi, S., 2006. Highly reproducible method of planar lipid bilayer reconstitution in polymethyl methacrylate microfluidic chip. Langmuir 22 (4), 1937-1942], we presented the method and device in which the reproducibility of planar lipid bilayers reached 90%, and multiple bilayers were formed simultaneously. In this communication, we show that our device has excellent electric properties suitable for ion channel analysis down to single molecular level. Additional aspects on the optical accessibility and controllability on lipid bilayer formation are also presented.

Electrochemistry↗
PubMed2006

Highly reproducible method of planar lipid bilayer reconstitution in polymethyl methacrylate microfluidic chip.

We developed a highly reproducible method for planar lipid bilayer reconstitution using a microfluidic system made of a polymethyl methacrylate (PMMA) plastic substrate. Planar lipid bilayers are formed at apertures, 100 microm in diameter, by flowing lipid solution and buffer alternately into an integrated microfluidic channel. Since the amount and distribution of the lipid solution at the aperture determines the state of the lipid bilayer, controlling them precisely is crucial. We designed the geometry of the fluidic system so that a constant amount of lipid solution is distributed at the aperture. Then, the layer of lipid solution was thinned by applying an external pressure and finally became a bilayer when a pressure of 200-400 Pa was applied. The formation process can be simultaneously monitored with optical and electrical recordings. The maximum yield for bilayer formation was 90%. Using this technique, four lipid bilayers are formed simultaneously in a single chip. Finally, a channel current through gramicidin peptide ion channels was recorded to prove the compatibility of the chip with single molecule electrophysiology.

Lipid Bilayers↗
PubMed2005

Temperature alternation by an on-chip microheater to reveal enzymatic activity of beta-galactosidase at high temperatures.

A method to measure enzymatic activity at high temperatures by rapid temperature alternation of a microreactor with a microheater is proposed. On-chip microreactor and microheater were integrated on a glass plate by MEMS technology; this microheater can control the temperature of the microreactor with a response speed of 34.2 and 31.5 K/s for temperature rise and fall, respectively, with an accuracy of 3 degrees C. The enzyme, beta-galactosidase, was revealed to survive short exposure (4-s pulses) to temperatures above that which would "normally" denature them. Its activity at 60 degrees C was revealed to be approximately 4 times greater than that at room temperature. This method not only gives new kinetic information in biochemistry but also enables application in highly sensitive biosensors.

Hot Temperature↗
PubMed2005

Activation of pausing F1 motor by external force.

A rotary motor F(1), a catalytic part of ATP synthase, makes a 120 degrees step rotation driven by hydrolysis of one ATP, which consists of 80 degrees and 40 degrees substeps initiated by ATP binding and probably by ADP and/or P(i) dissociation, respectively. During active rotations, F(1) spontaneously fails in ADP release and pauses after a 80 degrees substep, which is called the ADP-inhibited form. In the present work, we found that, when pushed >+40 degrees with magnetic tweezers, the pausing F(1) resumes its active rotation after releasing inhibitory ADP. The rate constant of the mechanical activation exponentially increased with the pushed angle, implying that F(1) weakens the affinity of its catalytic site for ADP as the angle goes forward. This finding explains not only its unidirectional nature of rotation, but also its physiological function in ATP synthesis; it would readily bind ADP from solution when rotated backward by an F(o) motor in the ATP synthase. Furthermore, the mechanical work for the forced rotation was efficiently converted into work for expelling ADP from the catalytic site, supporting the tight coupling between the rotation and catalytic event.

Adenosine Diphosphate↗
PubMed2005

Microfabricated arrays of femtoliter chambers allow single molecule enzymology.

Precise understanding of biological functions requires tools comparable in size to the basic components of life. Single molecule studies have revealed molecular behaviors usually hidden in the ensemble- and time-averaging of bulk experiments. Although most such approaches rely on sophisticated optical strategies to limit the detection volume, another attractive approach is to perform the assay inside very small containers. We have developed a silicone device presenting a large array of micrometer-sized cavities. We used it to tightly enclose volumes of solution, as low as femtoliters, over long periods of time. The microchip insures that the chambers are uniform and precisely positioned. We demonstrated the feasibility of our approach by measuring the activity of single molecules of beta-galactosidase and horseradish peroxidase. The approach should be of interest for many ultrasensitive bioassays at the single-molecule level.

Biological Assay↗
PubMed2005

Highly coupled ATP synthesis by F1-ATPase single molecules.

F1-ATPase is the smallest known rotary motor, and it rotates in an anticlockwise direction as it hydrolyses ATP. Single-molecule experiments point towards three catalytic events per turn, in agreement with the molecular structure of the complex. The physiological function of F1 is ATP synthesis. In the ubiquitous F0F1 complex, this energetically uphill reaction is driven by F0, the partner motor of F1, which forces the backward (clockwise) rotation of F1, leading to ATP synthesis. Here, we have devised an experiment combining single-molecule manipulation and microfabrication techniques to measure the yield of this mechanochemical transformation. Single F1 molecules were enclosed in femtolitre-sized hermetic chambers and rotated in a clockwise direction using magnetic tweezers. When the magnetic field was switched off, the F1 molecule underwent anticlockwise rotation at a speed proportional to the amount of synthesized ATP. At 10 Hz, the mechanochemical coupling efficiency was low for the alpha3beta3gamma subcomplex (F1-epsilon)), but reached up to 77% after reconstitution with the epsilon-subunit (F1+epsilon)). We provide here direct evidence that F1 is designed to tightly couple its catalytic reactions with the mechanical rotation. Our results suggest that the epsilon-subunit has an essential function during ATP synthesis.

Adenosine Triphosphate↗
PubMed2005

Chemomechanical coupling in single-molecule F-type ATP synthase.

An extremely small reaction chamber with a volume of a few femtoliters was developed for a highly sensitive detection of biological reaction. By encapsulating a single F(1)-ATPase (F(1)) molecule with ADP and an inorganic phosphate in the chamber, the chemomechanical coupling efficiency of ATP synthesis catalyzed by reversely rotated F(1) was successfully determined (Rondelez et al., 2005a, Nature, 444, 773-777). While the alpha3beta3gamma subcomplex of F(1) generated ATP with a low efficiency (approximately 10%), inclusion of the epsilon subunit into the subcomplex enhanced the efficiency up to 77%. This raises a new question about the mechanism of F(0)F(1)-ATP synthase (F(0)F(1)): How does the epsilon subunit support the highly coupled ATP synthesis of F(1)? To address this question, we measured the conformational dynamics of the epsilon subunit using fluorescence resonance energy transfer (FRET) at the single-molecule level. The experimental data revealed epsilon changes the conformation of its C-terminus helices in a nucleotide-dependent manner. It is plausible that the conformational change of epsilon switches the catalytic mode of F(0)F(1) for highly coupled ATP synthesis.

Adenosine Triphosphate↗
PubMed2004

Planar lipid bilayer reconstitution with a micro-fluidic system.

A planar lipid bilayer which is widely used for the electrophysiological study of membrane proteins in laboratories is reconstituted using a micro-fluidic system, in a manner that is suitable for automated processing. We fabricated micro-channels on both sides of the substrate, which are connected through a 100-200 microm aperture, and showed that the bilayer can be formed at the aperture by flowing the lipid solution and buffer, alternately. Parylene coating is found to be suitable for both bilayer formation and electric noise reduction. Future applications include a high-sensitivity ion sensor chip and a high-throughput drug screening device.

Electrochemistry↗
PubMed2004

Micro patterning of active proteins with perforated PDMS sheets (PDMS sieve).

We propose a novel technique for patterning active proteins on a glass substrate using a perforated polydimethylsiloxane (PDMS) sheet-sieve. The sieve, which has tapering holes, is fabricated by spin-coating PDMS on a pyramidal-shaped mold. By means of this sieve, FITC (fluorescent isothiocyanate, bovine)-albumin was successfully spotted in a 5 x 5 microm(2) area in an array. The patterned spots were perfectly isolated, which eliminates the problem of non-specific binding of proteins to undesired areas. To show that proteins maintained their activity after the patterning, we used F(1)-ATPase biomolecular motors; their activity can easily be verified by observing their rotary motion after patterning. Selective patterning with three kinds of fluorescent micro beads indicated the possibility of patterning of different proteins on the same substrate by using the sieve.

Adenosine Triphosphatases↗
PubMed2004

Mechanically driven ATP synthesis by F1-ATPase.

ATP, the main biological energy currency, is synthesized from ADP and inorganic phosphate by ATP synthase in an energy-requiring reaction. The F1 portion of ATP synthase, also known as F1-ATPase, functions as a rotary molecular motor: in vitro its gamma-subunit rotates against the surrounding alpha3beta3 subunits, hydrolysing ATP in three separate catalytic sites on the beta-subunits. It is widely believed that reverse rotation of the gamma-subunit, driven by proton flow through the associated F(o) portion of ATP synthase, leads to ATP synthesis in biological systems. Here we present direct evidence for the chemical synthesis of ATP driven by mechanical energy. We attached a magnetic bead to the gamma-subunit of isolated F1 on a glass surface, and rotated the bead using electrical magnets. Rotation in the appropriate direction resulted in the appearance of ATP in the medium as detected by the luciferase-luciferin reaction. This shows that a vectorial force (torque) working at one particular point on a protein machine can influence a chemical reaction occurring in physically remote catalytic sites, driving the reaction far from equilibrium.

Adenosine Diphosphate↗
PubMed2004

Chemomechanical coupling in F1-ATPase revealed by simultaneous observation of nucleotide kinetics and rotation.

F(1)-ATPase is a rotary molecular motor in which unidirectional rotation of the central gamma subunit is powered by ATP hydrolysis in three catalytic sites arranged 120 degrees apart around gamma. To study how hydrolysis reactions produce mechanical rotation, we observed rotation under an optical microscope to see which of the three sites bound and released a fluorescent ATP analog. Assuming that the analog mimics authentic ATP, the following scheme emerges: (i) in the ATP-waiting state, one site, dictated by the orientation of gamma, is empty, whereas the other two bind a nucleotide; (ii) ATP binding to the empty site drives an approximately 80 degrees rotation of gamma; (iii) this triggers a reaction(s), hydrolysis and/or phosphate release, but not ADP release in the site that bound ATP one step earlier; (iv) completion of this reaction induces further approximately 40 degrees rotation.

Adenosine Triphosphate↗
PubMed2003

Origin of apparent negative cooperativity of F(1)-ATPase.

In order to get insight into the origin of apparent negative cooperativity observed for F(1)-ATPase, we compared ATPase activity and ATPMg binding of mutant subcomplexes of thermophilic F(1)-ATPase, alpha((W463F)3)beta((Y341W)3)gamma and alpha((K175A/T176A/W463F)3)beta((Y341W)3)gamma. For alpha((W463F)3)beta((Y341W)3)gamma, apparent K(m)'s of ATPase kinetics (4.0 and 233 microM) did not agree with apparent K(m)'s deduced from fluorescence quenching of the introduced tryptophan residue (on the order of nM, 0.016 and 13 microM). On the other hand, in case of alpha((K175A/T176A/W463F)3)beta((Y341W)3)gamma, which lacks noncatalytic nucleotide binding sites, the apparent K(m) of ATPase activity (10 microM) roughly agreed with the highest K(m) of fluorescence measurements (27 microM). The results indicate that in case of alpha((W463F)3)beta((Y341W)3)gamma, the activating effect of ATP binding to noncatalytic sites dominates overall ATPase kinetics and the highest apparent K(m) of ATPase activity does not represent the ATP binding to a catalytic site. In case of alpha((K175A/T176A/W463F)3)beta((Y341W)3)gamma, the K(m) of ATPase activity reflects the ATP binding to a catalytic site due to the lack of noncatalytic sites. The Eadie-Hofstee plot of ATPase reaction by alpha((K175A/T176A/W463F)3)beta((Y341W)3)gamma was rather linear compared with that of alpha((W463F)3)beta((Y341W)3)gamma, if not perfectly straight, indicating that the apparent negative cooperativity observed for wild-type F(1)-ATPase is due to the ATP binding to catalytic sites and noncatalytic sites. Thus, the frequently observed K(m)'s of 100-300 microM and 1-30 microM range for wild-type F(1)-ATPase correspond to ATP binding to a noncatalytic site and catalytic site, respectively.

Adenosine Triphosphate↗
PubMed2003

The ATP-waiting conformation of rotating F1-ATPase revealed by single-pair fluorescence resonance energy transfer.

F1-ATPase is an ATP-driven rotary motor in which a rod-shaped gamma subunit rotates inside a cylinder made of alpha3beta3 subunits. To elucidate the conformations of rotating F1, we measured fluorescence resonance energy transfer (FRET) between a donor on one of the three betas and an acceptor on gamma in single F1 molecules. The yield of FRET changed stepwise at low ATP concentrations, reflecting the stepwise rotation of gamma. In the ATP-waiting state, the FRET yields indicated a gamma position approximately 40 degrees counterclockwise (= direction of rotation) from that in the crystal structures of mitochondrial F1, suggesting that the crystal structures mimic a metastable state before product release.

Adenosine Triphosphatases↗
PubMed2003

Evidence for rotation of V1-ATPase.

V(o)V(1)-ATPase is responsible for acidification of eukaryotic intracellular compartments and ATP synthesis of Archaea and some eubacteria. From the similarity to F(o)F(1)-ATP synthase, V(o)V(1)-ATPase has been assumed to be a rotary motor, but to date there are no experimental data to support this. Here we visualized the rotation of single molecules of V(1)-ATPase, a catalytic subcomplex of V(o)V(1)-ATPase. V(1)-ATPase from Thermus thermophilus was immobilized onto a glass surface, and a bead was attached to the D or F subunit through the biotin-streptavidin linkage. In both cases we observed ATP-dependent rotations of beads, the direction of which was always counterclockwise viewed from the membrane side. Given that three ATP molecules are hydrolyzed per one revolution, rates of rotation agree consistently with rates of ATP hydrolysis at saturating ATP concentrations. This study provides experimental evidence that V(o)V(1)-ATPase is a rotary motor and that both D and F subunits constitute a rotor shaft.

Adenosine Triphosphate↗