FRET标记的FoF1-ATP合酶β亚基的构象变化
Regulatory conformational changes of the epsilon subunit in single FRET-labeled FoF1-ATP synthase
摘要：
FoF 1-ATP合酶是细菌FoF 1-ATP合酶的内在调节因子，FoF 1-ATP合酶是一种普遍存在的膜包埋酶，在大多数生物体中利用质子动力合成三磷酸腺苷（ATP）。大肠杆菌酶F1部分的X射线结构显示，β-内酰胺酶的C-末端结构域可以延伸到α和β亚基形成的中央空腔中。这种插入阻止了中心γ亚基的旋转，从而防止了浪费的ATP水解。在这里，我们的目标是开发一个实验系统，可以揭示条件下，在这种条件下，在体外抑制全酶FoF 1-ATP合酶。标记的C-末端结构域的的α和γ亚基特异性与两个不同的荧光团的单分子Foerster共振能量转移（smFRET）允许监测构象的α在重构的酶在实时。为未来的三色smFRET实验制作了新的突变体，以揭示p53中调控构象变化的细节。
英文摘要：
Subunit epsilon is an intrinsic regulator of the bacterial FoF1-ATP synthase, the ubiquitous membrane-embedded enzyme that utilizes a proton motive force in most organisms to synthesize adenosine triphosphate (ATP). The C-terminal domain of epsilon can extend into the central cavity formed by the alpha and beta subunits, as revealed by the recent X-ray structure of the F1 portion of the Escherichia coli enzyme. This insertion blocks the rotation of the central gamma subunit and, thereby, prevents wasteful ATP hydrolysis. Here we aim to develop an experimental system that can reveal conditions under which epsilon inhibits the holoenzyme FoF1-ATP synthase in vitro. Labeling the C-terminal domain of epsilon and the gamma subunit specifically with two different fluorophores for single-molecule Foerster resonance energy transfer (smFRET) allowed monitoring of the conformation of epsilon in the reconstituted enzyme in real time. New mutants were made for future three-color smFRET experiments to unravel the details of regulatory conformational changes in epsilon.
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