S1.28 Mechanochemical coupling of F1-ATPase and intracellular ATP imaging

Editat anteriorment amb el títol: Guia de recurso

Biological Nanomotors with a Revolution, Linear, or Rotation Motion Mechanism

The ubiquitous biological nanomotors were classified into two categories in the past: linear and rotation motors. In 2013, a third type of biomotor, revolution without rotation (http://rnanano.osu.edu/movie.html), was discovered and found to be widespread among bacteria, eukaryotic viruses, and double-stranded DNA (dsDNA) bacteriophages. This review focuses on recent findings about various aspects of motors, including chirality, stoichiometry, channel size, entropy, conformational change, and energy usage rate, in a variety of well-studied motors, including FoF1 ATPase, helicases, viral dsDNA-packaging motors, bacterial chromosome translocases, myosin, kinesin, and dynein. In particular, dsDNA translocases are used to illustrate how these features relate to the motion mechanism and how nature elegantly evolved a revolution mechanism to avoid coiling and tangling during lengthy dsDNA genome transportation in cell division. Motor chirality and channel size are two factors that distinguish rotation motors from revolution motors. Rotation motors use right-handed channels to drive the right-handed dsDNA, similar to the way a nut drives the bolt with threads in same orientation; revolution motors use left-handed motor channels to revolve the right-handed dsDNA. Rotation motors use small channels (\u3c 2 nm in diameter) for the close contact of the channel wall with single-stranded DNA (ssDNA) or the 2-nm dsDNA bolt; revolution motors use larger channels (\u3e 3 nm) with room for the bolt to revolve. Binding and hydrolysis of ATP are linked to different conformational entropy changes in the motor that lead to altered affinity for the substrate and allow work to be done, for example, helicase unwinding of DNA or translocase directional movement of DNA

Temperature-sensitive reaction intermediate of F1-ATPase

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

Subunit rotation in a single FoF1-ATP synthase in a living bacterium monitored by FRET

FoF1-ATP synthase is the ubiquitous membrane-bound enzyme in mitochondria, chloroplasts and bacteria which provides the 'chemical energy currency' adenosine triphosphate (ATP) for cellular processes. In Escherichia coli ATP synthesis is driven by a proton motive force (PMF) comprising a proton concentration difference {\Delta}pH plus an electric potential {\Delta}{\Psi} across the lipid membrane. Single-molecule in vitro experiments have confirmed that proton-driven subunit rotation within FoF1-ATP synthase is associated with ATP synthesis. Based on intramolecular distance measurements by single-molecule fluorescence resonance energy transfer (FRET) the kinetics of subunit rotation and the step sizes of the different rotor parts have been unraveled. However, these experiments were accomplished in the presence of a PMF consisting of a maximum {\Delta}pH ~ 4 and an unknown {\Delta}{\Psi}. In contrast, in living bacteria the maximum {\Delta}pH across the plasma membrane is likely 0.75, and {\Delta}{\Psi} has been measured between -80 and -140 mV. Thus the problem of in vivo catalytic turnover rates, or the in vivo rotational speed in single FoF1-ATP synthases, respectively, has to be solved. In addition, the absolute number of functional enzymes in a single bacterium required to maintain the high ATP levels has to be determined. We report our progress of measuring subunit rotation in single FoF1-ATP synthases in vitro and in vivo, which was enabled by a new labeling approach for single-molecule FRET measurements.Comment: 9 pages, 6 figure

High-throughput formation of lipid bilayer membrane arrays with an asymmetric lipid composition

We present a micro-device in which more than 10,000 asymmetric lipid bilayer membranes are formed at a time on micro-chamber arrays. The arrayed asymmetric lipid bilayers, where lipid compositions are different between the inner and outer leaflets, are formed with high efficiency of over 97% by injecting several types of liquids into a micro-device that has hydrophilic-in-hydrophobic surfaces. The lipid compositional asymmetry is an intrinsic property of bio-membranes, and therefore, this micro-device extends the versatility of artificial lipid-bilayer systems, which were previously limited to symmetric bilayer formation, and could contribute to the understanding of the role of lipid compositional asymmetry in cell physiology and also to further analytical and pharmacological applications.UTokyo Research掲載「脂質組成の非対称性をもつ人工生体膜の新しい量産技術」 URI: http://www.u-tokyo.ac.jp/ja/utokyo-research/research-news/novel-high-throughput-micro-device-to-form-asymmetric-bio-membranes/UTokyo Research "Novel high-throughput micro-device to form asymmetric bio-membranes" URI: http://www.u-tokyo.ac.jp/en/utokyo-research/research-news/novel-high-throughput-micro-device-to-form-asymmetric-bio-membranes

High-speed atomic force microscopy reveals rotary catalysis of rotor-less F1-ATPase

F1 is an adenosine triphosphate (ATP)–driven motor in which three torque-generating β subunits in the α3β3 stator ring sequentially undergo conformational changes upon ATP hydrolysis to rotate the central shaft γ unidirectionally. Although extensive experimental and theoretical work has been done, the structural basis of cooperative torque generation to realize the unidirectional rotation remains elusive. We used high-speed atomic force microscopy to show that the rotorless F1 still “rotates”; in the isolated α3β3 stator ring, the three β subunits cyclically propagate conformational states in the counterclockwise direction, similar to the rotary shaft rotation in F1. The structural basis of unidirectionality is programmed in the stator ring. These findings have implications for cooperative interplay between subunits in other hexameric ATPases