1P174 Direct observation of the rotation of V_1-ATPase from Enterococcus hirae and its torque(11.Molecular motor,Poster,The 51st Annual Meeting of the Biophysical Society of Japan)

Hiroshi Ueno, Yoshihiro Minagawa, Ichiro Yamato, Takeshi Murata, Ryota Iino, Eiro Muneyki
2013 Seibutsu Butsuri  
F 1 -ATPase (F 1 ) is an ATP-driven rotary motor. Average output of the torque during ATP hydrolysis reaction is reported to be 40-50 pNnm. However, the detailed torque profile as a function of rotor angle remains unknown. To know it, we have measured the torque by using magnetic tweezers. One of primary findings is that F 1 torque has a "microstructure", that is, a gradual decrease/increase followed by a steep jump/fall in torque repeats at least three times per 120 degree rotation during ATP
more » ... ydrolysis/synthesis, respectively. In addition, the efficiency of mechanochemical energy conversion is almost 100% in both ATP hydrolysis/synthesis reactions. Finally, the torque profiles provide a simple model that explains how F 1 attains 100% energy conversion and how F 1 rotates. A rotary motor enzyme F1-ATPase (F1) is a catalytic domain of FoF1-ATP synthase that produces the majority of ATP in respiring cells. Here, we visualize rotation of human mitochondrial F1, and present how each catalytic event drives rotation. Starting from rotor angle 0° of the γ-subunit in the stator α3β3 ring, ATP binding to one β-subunit in the ring drives a 0°>65° rotation, the Pi release from another β drives the next 65°>90°r otation, ATP hydrolysis on the third β occurs at 90°, and gamma turns to 120° to complete one cycle reaction. Rotation is often interrupted by persistent ADP binding and is stalled by azide or IF1. Based on this rotation scheme, the previous crystal structures of mitochondrial F1s are successfully assigned to each of the rotational isoforms. 1P172 腸内連鎖球菌 V-ATPase の大腸菌発現系 Expression of Enterococcus hirae V-ATPase in E. coli BL21 (DE3) There are several ion-pumping ATPases in cell membranes, working to maintain the homeostasis of a cell. V-ATPases function as ATP-dependent proton pumps in the membranes of acidic organelles of eukaryotic cells. V-ATPase is also expressed in a prokaryote, E. hirae, which transports Na + . We have already established expression and purification of V-ATPase in E. hirae. We are now trying to express this heterologous genes of V-ATPase whole complex in E. coli. In this poster, we report expression and purification of V-ATPase in E. coli along with its activity measurement. The vacuolar ATPases (V-ATPases) function as ATP-dependent proton pumps in the membranes of acidic organelles of eukaryotic cells. The V 1 domain is composed of A 3 B 3 DF complex which hydrolyzes ATP. To understand details of the assembly mechanism of V 1 domain, it is necessary to obtain the structures of every subunit and partial complex. We have already reported structures of A 3 B 3 and A 3 B 3 DF. A subunit was expressed in E. coli with His tag, purified with Ni 2+ affinity and gel filtration chromatography. In this poster, we will report the results of crystallization of purified A subunit with and without nucleotides. 1P174 Direct observation of the rotation of V 1 -ATPase from Enterococcus hirae and its torque We constructed an experimental setup to observe the rotation of Enterococcus hirae V 1 -ATPase (EhV 1 ). Proteins were prepared by Cellfree or E. coli expression systems that we developed recently. Rotation mutant was reconstituted from His 6 -A 3 B 3 and biotinylated-DF complexes, because mutation of endogenous cysteine residues in A-subunit decreased the ATPase activity. We have succeeded in observing the rotation of EhV 1 and the basic properties were determined by using a 40-nm gold colloid as a load-free probe (Minagawa, Y. et al., this meeting). Furthermore, we estimated the torque of EhV 1 from continuous rotation of large beads at 4 mM ATP using fluctuation theorem (Hayashi, K. et al., PRL 2010). Interestingly, torque of EhV 1 was ~3 times lower than that of F 1 -ATPase. 1P175 F 1 -ATPase の P-loop 変異体とリン酸解離の関係
doi:10.2142/biophys.53.s134_5 fatcat:4nkg7u4ps5cfxjqezz5ocxheji