Real-time monitoring of conformational dynamics of the epsilon subunit in F1-ATPase.
It has been proposed that C-terminal two alpha-helices of the epsilon subunit of F1-ATPase can undergo conformational transition between retracted folded-hairpin form and extended form. Here, using F(1) from thermophilic Bacillus PS3, we monitored this transition in real time by fluorescence resonance energy transfer (FRET) between a donor dye and an acceptor dye attached to N terminus of the gamma subunit and C terminus of the epsilon subunit, respectively. High FRET (extended form) of F1 turned to low FRET (retracted form) by ATP, which then reverted as ATP was hydrolyzed to ADP. 5'-Adenyl-beta,gamma-imidodiphosphate, ADP + AlF4-, ADP + NaN3, and GTP also caused the retracted form, indicating that ATP binding to the catalytic beta subunits induces the transition. The ATP-induced transition from high FRET to low FRET occurred in a similar time scale to the ATP-induced activation of ATPase from inhibition by the epsilon subunit, although detailed kinetics were not the same. The transition became faster as temperature increased, but the extrapolated rate at 65 degrees C (physiological temperature of Bacillus PS3) was still too slow to assign the transition as an obligate step in the catalytic turnover. Furthermore, binding affinity of ATP to the isolated epsilon subunit was weakened as temperature increased, and the dissociation constant extrapolated to 65 degrees C reached to 0.67 mm, a consistent value to assume that the epsilon subunit acts as a sensor of ATP concentration in the cell.
Iino
Ryota
R
ATP System Project, ERATO, Japan Science and Technology Agency, Nagatsuta 5800-3, Yokohama 226-0026, Japan.
Murakami
Tomoe
T
Iizuka
Satoshi
S
Kato-Yamada
Yasuyuki
Y
Suzuki
Toshiharu
T
Yoshida
Masasuke
M
D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D001407:Bacillus / Q000378:metabolism; D001412:Bacillus subtilis; D002384:Catalysis; D002417:Cattle; D004396:Coloring Agents / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer; D006868:Hydrolysis; D007700:Kinetics; D009154:Mutation; D009711:Nucleotides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013997:Time Factors; D000097795:ATPase Inhibitory Protein
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D004396:Coloring Agents; D009711:Nucleotides; D011506:Proteins; D000097795:ATPase Inhibitory Protein