通过DCO-ALEX FRET监测单个FoF1-ATP合成酶的旋转电机内的瞬时弹性能量存储
Monitoring transient elastic energy storage within the rotary motors of single FoF1-ATP synthase by DCO-ALEX FRET
摘要：
FoF 1-ATP合成酶为活细胞提供“化学能量货币”三磷酸腺苷（ATP）。催化作用是由酶内亚基旋转与三个ATP结合位点构象变化的机械化学耦合驱动的。通过ATP合酶的膜结合Fo部分的质子转运为c亚基环的10步旋转运动提供动力。这种旋转传递到F1部分的γ和γ亚基。由于γ和γ亚基旋转120度的步骤，我们的目标是解开这种对称性不匹配的实时监测亚基旋转使用单分子福斯特共振能量转移（FRET）。一个荧光团特异性地连接到F1马达，另一个连接到脂质体重构酶的Fo马达。由于单一荧光团的光谱波动的光物理伪影最小化由先前开发的占空比优化的交替激光激发方案（DCO-ALEX）。我们报告了Fo和F1转子部件之间的可逆弹性变形的检测，并使用Monte Carlo模拟估计了无负载旋转期间的最大角位移
英文摘要：
The enzyme FoF1-ATP synthase provides the 'chemical energy currency' adenosine triphosphate (ATP) for living cells. Catalysis is driven by mechanochemical coupling of subunit rotation within the enzyme with conformational changes in the three ATP binding sites. Proton translocation through the membrane-bound Fo part of ATP synthase powers a 10-step rotary motion of the ring of c subunits. This rotation is transmitted to the gamma and epsilon subunits of the F1 part. Because gamma and epsilon subunits rotate in 120 deg steps, we aim to unravel this symmetry mismatch by real time monitoring subunit rotation using single-molecule Forster resonance energy transfer (FRET). One fluorophore is attached specifically to the F1 motor, another one to the Fo motor of the liposome-reconstituted enzyme. Photophysical artifacts due to spectral fluctuations of the single fluorophores are minimized by a previously developed duty cycle-optimized alternating laser excitation scheme (DCO-ALEX). We report the detection of reversible elastic deformations between the rotor parts of Fo and F1 and estimate the maximum angular displacement during the load-free rotation using Monte Carlo simulations
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