Analysis of the Open and Closed Conformations of the beta Subunits in Thermophilic F-1-ATPase by Solution NMR

作者：Kobayashi Masumi; Akutsu Hideo; Suzuki Toshiharu; Yoshida Masasuke; Yagi Hiromasa^*

来源：Journal of Molecular Biology, 2010, 398(2): 189-199.

DOI：10.1016/j.jmb.2010.03.013

• H+-ATPase
• NMR
• segmental isotope labeling
• CRINEPT
• F-1 rotation

F-1-ATPase, composed of alpha, beta, gamma, delta, and epsilon subunits, is a unique enzyme in terms of its rotational catalytic activity. The smallest unit showing this function is the alpha(3)beta(3)gamma complex. We have investigated the alpha(3)beta(3)gamma epsilon(Delta C) (epsilon(Delta C), truncated epsilon) complex from thermophilic Bacillus PS3 (TF1', 360 kDa) in the solution state by using the combination of extensive deuteration, segmental-labeling, and CRINEPT (cross-correlated relaxation-enhanced polarization transfer) NMR. Well-resolved CRINEPT-HMQC (heteronuclear multiple-quantum correlation) spectra of partially N-15-labeled TF1' were obtained for this huge and asymmetric protein complex. The spectrum of the C-terminal domain of the beta subunit revealed that the open form of the beta subunit in the TF1' complex is similar to that of the free beta monomer. The open beta subunit in the TF1' complex does not exhibit high affinity for nucleotides unlike the monomer, but this is in agreement with the results of single-molecule analysis of TF1 alpha(3)beta(3)gamma. On the other hand, the closed form of the beta subunit in the TF1' complex was shown to be distinct from that of the nucleotide-bound beta monomer. This is consistent with a previous report that the closed form of the TF1 beta monomer could be a catalytically activated state. The loop between the N-terminal beta-barrel and the central domain is highly flexible in the TF1' complex, in contrast to that in the alpha(3)beta(3) hexamer, suggesting that it is affected by the presence of the gamma subunit in this area.

• 出版日期2010-4-30