Dynamic inter-subunit interactions in thermophilic F(1)-ATPase subcomplexes studied by cross-correlated relaxation-enhanced polarization transfer NMR.
F(1)-ATPase is a unique enzyme in terms of its rotational catalytic activity. The smallest unit showing this property is the alpha(3)beta(3)gamma complex (351 kDa). For investigation of such a huge system by means of solution NMR, we have explored a suitable NMR method using F(1)-ATPase subcomplexes from a thermophilic Bacillus PS3 including an alpha(3)beta(3) hexamer (319 kDa). Pulse sequences for large molecules, effects of deuteration and simplification of the spectra were examined in this work. Since the beta subunit includes the catalytic site, this was the target of the analysis in this work. The combination of [(15)N,(1)H]-CRINEPT-HMQC-[(1)H]-TROSY, deuteration of both alpha and beta subunits, and segmental isotope-labeling was found essential to analyze such a huge and complex molecular system. Utilizing this method, subcomplexes composed of alpha and beta subunits were investigated in terms of inter-subunit interactions. It turned out that there is equilibrium among monomers, heterodimers and the alpha(3)beta(3) hexamers in solution. The rate of exchange between the dimer and hexamer is in the slow regime on the NMR time scale. In chemical shift perturbation experiments, the N-terminal domain was found to be involved in strong inter-subunit interactions. In contrast, the C-terminal domain was found to be mobile even in the hexamer.
Kobayashi
Masumi
M
Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka, 565-0871, Japan.
Yagi
Hiromasa
H
Yamazaki
Toshio
T
Yoshida
Masasuke
M
Akutsu
Hideo
H
D001407:Bacillus / Q000201:enzymology; D019281:Dimerization; D008958:Models, Molecular; D008962:Models, Theoretical; D046912:Multiprotein Complexes / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods; D021122:Protein Subunits / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism*; D013696:Temperature; D013816:Thermodynamics
D046912:Multiprotein Complexes; D021122:Protein Subunits; D006180:Proton-Translocating ATPases