ATP synthase

Scientists have created a working nanoscale electomotor. The science team designed a turbine engineered from DNA that is powered by hydrodynamic flow inside a nanopore, a nanometer-sized hole in a membrane of solid-state silicon nitride. The tiny motor could help spark research into future applications such as building molecular factories or even medical probes of molecules inside the bloodstream. Scientists have created the world's first working nanoscale electromotor, according to research published in the journal Nature Nanotechnology. The science team designed a turbine engineered from DNA that is powered by hydrodynamic flow inside a nanopore, a nanometer-sized hole in a membrane of solid-state silicon nitride. The tiny motor could help spark research into future applications such as building molecular factories for useful chemicals or medical probes of molecules inside the bloodstream to detect diseases such as cancer. "Common macroscopic machines become inefficient at the nanoscale," said study co-author professor Aleksei Aksimentiev, a professor of physics at the University of Illinois at Urbana-Champagne. "We have to develop new principles and physical mechanisms to realize electromotors at the very, very small scales." The experimental work on the tiny motor was conducted by Cees Dekker of the Delft University of Technology and Hendrik Dietz of the Technical University of Munich. Dietz is a world expert in DNA origami. His lab manipulated DNA molecules to make the tiny motor's turbine, which consisted of 30 double-stranded DNA helices engineered into an axle and three blades of about 72 base pair length. Decker's lab work demonstrated that the turbine can indeed rotate by applying an electric field. Aksimentiev's lab carried out all-atom molecular dynamics simulations on a system of five million atoms to characterize the physical phenomena of how the motor works. The system was the smallest representation that could yield meaningful results about the experiment; however, "it was one of the largest ever simulated from the DNA origami perspective," Aksimentiev said. Mission Impossible to Mission Possible The Texas Advanced Computing Center (TACC) awarded Aksimentiev a Leadership Resource Allocation to aid his study of mesoscale biological systems on the National Science Foundation (NSF)-funded Frontera, the top academic supercomputer in the U.S. "Frontera was instrumental in this DNA nanoturbine work," Aksimentiev said. "We obtained microsecond simulation trajectories in two to three weeks instead of waiting for a year or more on smaller computing systems. The big simulations were done on Frontera using about a quarter of the machine -- over 2,000 nodes," Aksimentiev said. "However, it's not just the hardware, but also the interaction with TACC staff. It's extremely important to make the best use of the resources once we have the opportunity." Aksimentiev was also awarded supercomputer allocations for this work by the NSF-funded Advanced Cyberinfrastructure Coordination Ecosystem: Services & Support (ACCESS) on Expanse of the San Diego Supercomputer Center and Anvil of Purdue University. "We had up to 100 different nanomotor systems to simulate. We had to run them for different conditions and in a speedy manner, which the ACCESS supercomputers assisted with perfectly," Aksimentiev said. "Many thanks to the NSF for their support -- we would not be able to do the science that we do without these systems." DNA as a Building Block The success with the working DNA nanoturbine builds on a previous study that also used Frontera and ACCESS supercomputers. The study showed that a single DNA helix is the tiniest electromotor that one can build -- it can rotate up to a billion revolutions per minute. DNA has emerged as a building material at the nanoscale, according to Aksimentiev. "The way DNA base pair is a very powerful programming tool. We can program geometrical, three-dimensional objects from DNA using the Cadnano software just by programming the sequence of letters that make up the rungs of the double helix," he explained. Another reason for using DNA as the building block is that it carries a negative charge, an essential characteristic to make the electromotor. "We wanted to reproduce one of the most spectacular biological machines -- ATP synthase, which is driven by electric field. We chose to do our motor with DNA," Aksimentiev said. "This new work is the first nanoscale motor where we can control the rotational speed and direction," he added. It's done by adjusting the electric field across the solid state nanopore membrane and the salt concentrations of the fluid that surrounds the rotor. "In the future, we might be able to synthetize a molecule using the new nanoscale electromotor, or we can use it to as an element of a bigger molecular factory, where things are moved around. Or we could imagine it as a vehicle for soft propulsion, where synthetic systems can go into a blood stream and probe molecules or cells one at a time," Aksimentiev said. If you think this sounds like something out of a 1960's sci-fi movie, you are right. In the movie Fantastic Voyage, a team of Americans in a nuclear submarine is shrunk and injected into a scientist's body to fix a blood clot and need to work quickly before the miniaturization wears off. As far-fetched as this might sound, Aksimentiev says that the concept and the elements of the machines we are developing today could enable something like this to happen. "We were able to accomplish this because of supercomputers," Aksimentiev said. "Supercomputers are becoming more and more indispensable as the complexity of the systems that we build increases. They're the computational microscopes, which at ultimate resolutions can see the motion of individual atoms and how that is coupled to a bigger system." Funding came from ERC Advanced Grant no. 883684 and the NanoFront and BaSyC programmes; ERC Consolidator Grant to H.D. (GA no. 724261), the Deutsche Forschungsgemeinschaft via the Gottfried-Wilhelm-Leibniz Programme (to H.D.) and the SFB863 Project ID 111166240 TPA9; National Science Foundation grant DMR-1827346; the Max Planck School Matter to Life and the MaxSynBio Consortium. Supercomputer time was provided through TACC Leadership Resource Allocation MCB20012 on Frontera and through ACCESS allocation MCA05S028.

F-Type ATP synthase, a catalytic complex of proteins, synthesizes adenosine triphosphate (ATP), the energy currency of living cells. A lot of ambiguity exists over the rotational mechanism of this spinning enzyme. Now, researchers from Japan have demonstrated how each chemical event of ATP metabolism is linked to the 'stepwise' rotational movement of the F1 component of ATPase. Especially, they clarified the angle of shaft rotation before ATP-cleavage, a long-standing enigma, to be 200°. F-Type ATP synthase, a catalytic complex of proteins, synthesizes adenosine triphosphate (ATP), the energy currency of living cells. A lot of ambiguity exists over the rotational mechanism of this spinning enzyme. Now, researchers from Japan have demonstrated how each chemical event of ATP metabolism is linked to the 'stepwise' rotational movement of the F1 component of ATPase. Especially, they clarified the angle of shaft rotation before ATP-cleavage, a long-standing enigma, to be 200°. Adenosine triphosphate (ATP) is the energy currency of cells. It powers various cellular processes that require energy, including enzymatic reactions. ATP is synthesized with the help of an enzyme complex called F-type ATP synthase. This enzyme complex has a bidirectional functionality, working to synthesize ATP as well as hydrolyzing it, depending on environmental and cellular cues. ATP synthase consists of two rotating motors―F1 and F0. The F1 subcomplex is mainly composed of α, β, and γ subunits. During the hydrolysis of ATP, the F1-ATPase show rotational motion. Therefore, F1-ATPase is also known as the world's smallest rotary biological molecule motor. However, the underlying mechanism of how ATP hydrolysis makes the molecule rotate remains unclear. To address this knowledge gap, a team of researchers from Japan, led by Associate Professor Tomoko Masaike from Tokyo University of Science, set out to investigate the events behind the rotational mechanism of F1-ATPase in the thermophilic bacterium, Bacillus PS3. Elaborating on the objectives of their study, Dr. Masaike explains, "We wanted to clarify the mechanism by which F1-ATPase rotates the central shaft during ATP hydrolysis. We focused on clarifying the angle of rotation of the central shaft between the binding of the substrate ATP to the enzyme and the cleavage of its high-energy phosphate bond." The study, which was undertaken in collaboration with Professor Takayuki Nishizaka from Gakushuin University and Yuh Hasimoto from Hamamatsu Photonics K.K., was made available online on 21 December 2022 and 7 February 2023 in print in Biophysical Journal. Previous investigations of the F1 subunits of Bacillus PS3 have established that ATP cleavage involves chemomechanical coupling, i.e., each rotational stepping motion is linked to a chemical reaction step. The angle of rotation between ATP binding and its cleavage at the same catalytic site has been previously estimated to be 200°. However, experimental evidence to substantiate this has so far been lacking. To address this, the researchers studied the rotation by creating a hybrid F1 using one mutant β and two wild type βs. Since the rate of both ATP cleavage and ATP binding was extremely slow in the mutant, the researchers could observe the pauses or dwells between rotational steps easily. Upon performing a single-molecule rotation assay with varying concentrations of ATP, they could observe three sets of short and long dwells associated alternately with 80° and 40° intervals per revolution. To investigate the events associated with the dwells, the authors performed dwell-time analyses. The long pause before the 40° sub-step was independent of ATP concentration and was confirmed as the 'catalytic dwell'―a pause in the rotation of the shaft due to ATP cleavage. Alternately, the short pause before the 80° step was clearly dependent on ATP concentration and thus identified as the 'ATP-waiting dwell' (pause to enable β subunit to bind ATP). "Upon investigating the rotation of the shaft, we could provide visible evidence through optical microscopy that the shaft angles at ATP-binding and cleaving events in Bacillus PS3 were 0° and 200°, respectively" says Hasimoto. With this study, the authors have resolved a long-term debate over the ATP-cleavage shaft angle and established the chemomechanical correlation of ATPase function. Talking about the future impacts of their novel study, Associate Prof. Masaike elaborates, "Since F1-ATPase is the world's smallest biological rotational motor protein, it can be used as a reference to understand the mechanism of energy transduction in living organisms. This knowledge can be revolutionary in developing efficient nanomachines. Moreover, ATP synthase from Mycobacterium tuberculosis has recently been identified as a potential target for drug discovery. Therefore, to stop its rotation using inhibitors, understanding the mechanism of rotation is quite important."

The redox regulation mechanism responsible for efficient production of ATP under varying light conditions in photosynthetic organisms has now been unveiled. Researchers investigated the enzyme responsible for this mechanism and uncovered how the amino acid sequences present in the enzyme regulate ATP production. Their findings provide valuable insights into the process of photosynthesis and the ability to adapt to changing metabolic conditions. The redox regulation mechanism responsible for efficient production of ATP under varying light conditions in photosynthetic organisms has now been unveiled by Tokyo Tech researchers. They investigated the enzyme responsible for this mechanism and uncovered how the amino acid sequences present in the enzyme regulate ATP production. Their findings provide valuable insights into the process of photosynthesis and the ability to adapt to changing metabolic conditions. ATP, the compound essential for the functioning of photosynthetic organisms such as plants and algae, is produced by an enzyme called "chloroplast ATP synthase" (CFoCF1). To control ATP production under varying light conditions, the enzyme uses a redox regulatory mechanism that modifies the ATP synthesis activity in response to changes in the redox state of cysteine (Cys) residues, which exist as dithiols under reducing (light) conditions, but forms a disulfide bond under oxidizing (dark) conditions. However, this mechanism has not been fully understood so far. Now, in a study published in the Proceedings of the National Academy of Sciences, a team of researchers from Japan, led by Prof. Toru Hisabori from Tokyo Institute of Technology (Tokyo Tech), has uncovered the role of the amino acid sequences present in CFoCF1, revealing how the enzyme regulates ATP production in photosynthetic organisms. To understand how the conformation of the amino acids present in CFoCF1 contributes to the redox regulation mechanism, the researchers used the unicellular green alga, Chlamydomonas reinhardtii, to produce the enzyme. "By leveraging the powerful genetics of Chlamydomonas reinhardtii as a model organism for photosynthesis, we conducted a comprehensive biochemical analysis of the CFoCF1 molecule," explains Prof. Hisabori. With the alga as the host organism, the team introduced plasmids (extrachromosomal DNA molecule that can replicate independently) that encoded the F1 component of the CFoCF1 protein, namely the part of the enzyme containing catalytic sites for ATP synthesis. They additionally introduced mutated versions of the gene to change the amino acid sequences of the protein, specifically targeting the DDE motif (a cluster of negatively charged amino acids), the redox loop, and the β-hairpin domain. They then purified CFoCF1, generating five different variations of it that included a wild-type strain with no changes to the amino acid sequence and four mutant strains: one with the DDE motif replaced with neutral amino acids, Asn-Asn-Gln, one without the β-hairpin domain, one without the redox loop, and one lacking both the redox loop and the β-hairpin domain. Upon testing the ATP synthesis activity of these mutants under reducing (mimicking the light conditions) and oxidizing (mimicking the dark conditions) conditions, the researchers found that the wild-type enzyme and the mutant enzyme with changes to the DDE motif functioned normally (showed high activity when reduced and low activity when oxidized). However, the enzyme complexes without the redox loop or the β-hairpin domain did not show the redox-response, indicating that both regions were involved in the redox regulation mechanism. The researchers suggested that under dark conditions, the disulfide bond between the Cys residues makes the redox loop rigid and weakens the interaction between the redox loop and the β-hairpin. This causes the β-hairpin to remain stuck within a cavity in the protein. However, when the disulfide bond is reduced in presence of light, the redox loop regains its flexibility and pulls the β-hairpin out of the cavity, enabling it to partake in the ATP synthesis activity. "The redox regulation of ATP synthesis is accomplished by a cooperative interaction between two γ subunit domains of CFoCF1 unique to photosynthetic organisms," says Prof. Hisabori. "We propose that it results from the interaction of the β-hairpin and the redox loop with the catalytic site." The results are an important step towards understanding the photosynthesis process better, with the potential for significant implications in the fields of agriculture and bioenergy.