Search

Single-molecule imaging and manipulation with optical microscopy have become essential methods for studying biomolecular machines; however, only few efforts have been directed towards synthetic molecular machines. Single-molecule optical microscopy was now applied to a synthetic molecular rotor, a double-decker porphyrin (DD). By attaching a magnetic bead (ca. 200 nm) to the DD, its rotational dynamics were captured with a time resolution of 0.5 ms. DD showed rotational diffusion with 90° steps, which is consistent with its four-fold structural symmetry. Kinetic analysis revealed the first-order kinetics of the 90° step with a rate constant of 2.8 s−1. The barrier height of the rotational potential was estimated to be greater than 7.4 kJ mol−1 at 298 K. The DD was also forcibly rotated with magnetic tweezers, and again, four stable pausing angles that are separated by 90° were observed. These results demonstrate the potency of single-molecule optical microscopy for the elucidation of elementary properties of synthetic molecular machines. [ABSTRACT FROM AUTHOR]

V1-ATPase is a rotary molecular motor in which the mechanical rotation of the rotor DF subunits against the stator A3B3 ring is driven by the chemical free energy of ATP hydrolysis. Recently, using X-ray crystallography, we solved the high-resolution molecular structure of Enterococcus hirae V1-ATPase (EhV1) and revealed how the three catalytic sites in the stator A3B3 ring change their structure on nucleotide binding and interaction with the rotor DF subunits. Furthermore, recently, we also demonstrated directly the rotary catalysis of EhV1 by using single-molecule high-speed imaging and analyzed the properties of the rotary motion in detail. In this critical review, we introduce the molecular structure and rotary dynamics of EhV1 and discuss a possible model of its chemomechanical coupling scheme. © 2014 IUBMB Life, 66(9):624-630, 2014 [ABSTRACT FROM AUTHOR]

FoF1-Adenosine triphosphate (ATP) synthase, a complex of two rotary motor proteins, reversibly converts the electrochemical potential of protons across the cell membrane into phosphate transfer potential of ATP to provide the energy currency of the cell. The water-soluble motor is F1-ATPase, which possesses ATP synthesis/hydrolysis catalytic sites. Isolated F1 hydrolyses ATP to rotate the rotary shaft against the stator ring. The membrane-embedded motor is Fo, which is driven by proton flow down the proton electrochemical potential. In the FoF1 complex, the direction of mechanical rotation, the chemical reaction, and the proton transport are determined by the relative amplitudes between the Gibbs free energy of the ATP hydrolysis reaction and the electrochemical potential of protons across the membrane. Therefore, FoF1-ATP synthase is a highly efficient molecular device in which the chemical, mechanical, and potential energies are tightly and reversibly converted. In this critical review, we summarize our latest knowledge about the operation mechanism of this sophisticated nanomachine, revealed by its structure and dynamics. © 2013 IUBMB Life, 65(3):238-246, 2013. [ABSTRACT FROM AUTHOR]

F1-ATPase is a rotary molecular motor that makes 120° stepping rotations, with each step being driven by a single-ATP hydrolysis. In this study, a new reaction intermediate of F1-ATPase was discovered at a temperature below 4°C, which makes a pause at the same angle in its rotation as when ATP binds. The rate constant of the intermediate reaction was strongly dependent on temperature with a Q10 factor of 19, implying that the intermediate reaction accompanies a large conformational change. Kinetic analyses showed that the intermediate state does not correspond to ATP binding or hydrolysis. The addition of ADP to the reaction mixture did not alter the angular position of the intermediate state, but specifically lengthened the time constant of this state. Conversely, the addition of inorganic phosphate caused a pause at an angle of +80° from that of the intermediate state. These observations strongly suggest that the newly found reaction intermediate is an ADP-releasing step. [ABSTRACT FROM AUTHOR]

The crystal structure of subunit F of vacuole-type ATPase/synthase (prokaryotic V-ATPase) was determined to of 2.2 Å resolution. The subunit reveals unexpected structural similarity to the response regulator proteins that include the Escherichia coli chemotaxis response regulator CheY. The structure was successfully placed into the low-resolution EM structure of the prokaryotic holo-V-ATPase at a location indicated by the results of crosslinking experiments. The crystal structure, together with the single-molecule analysis using fluorescence resonance energy transfer, showed that the subunit F exhibits two conformations, a ‘retracted’ form in the absence and an ‘extended’ form in the presence of ATP. Our results postulated that the subunit F is a regulatory subunit in the V-ATPase. [ABSTRACT FROM AUTHOR]

The cover page of the journal "Angewandte Chemie International Edition" is presented.