使用占空比优化的三重ALEX通过三色FRET同时监测单个FoF1-ATP合酶的两个耦合马达
Simultaneous monitoring of the two coupled motors of a single FoF1-ATP synthase by three-color FRET using duty cycle-optimized triple-ALEX
摘要：
FoF 1-ATP合成酶是为活细胞提供“化学能量货币”三磷酸腺苷（ATP）的酶。ATP的形成是通过酶内亚基的逐步内部旋转来完成的。简而言之，质子通过ATP合酶的膜结合Fo部分的移位驱动c亚基环相对于非旋转亚基a和b的10步旋转运动。这种旋转被传递到F1扇区的γ和γ子单元，导致120度的步长。为了解开这种对称性错配，我们通过单分子荧光共振能量转移（FRET）方法监测亚基旋转，该方法使用三个特异性连接到酶的荧光团：一个连接到F1马达，另一个连接到Fo马达，第三个连接到非旋转亚基。为了减少由于单个荧光团的光谱波动引起的物理伪影，已经开发了占空比优化的交替三激光方案（DCO-ALEX）。同时观察两个电机的步长可以检测Fo和F1转子部件之间的可逆弹性变形。
英文摘要：
FoF1-ATP synthase is the enzyme that provides the 'chemical energy currency' adenosine triphosphate, ATP, for living cells. The formation of ATP is accomplished by a stepwise internal rotation of subunits within the enzyme. Briefly, proton translocation through the membrane-bound Fo part of ATP synthase drives a 10-step rotary motion of the ring of c subunits with respect to the non-rotating subunits a and b. This rotation is transmitted to the gamma and epsilon subunits of the F1 sector resulting in 120 degree steps. In order to unravel this symmetry mismatch we monitor subunit rotation by a single-molecule fluorescence resonance energy transfer (FRET) approach using three fluorophores specifically attached to the enzyme: one attached to the F1 motor, another one to the Fo motor, and the third one to a non-rotating subunit. To reduce photophysical artifacts due to spectral fluctuations of the single fluorophores, a duty cycle-optimized alternating three-laser scheme (DCO-ALEX) has been developed. Simultaneous observation of the stepsizes for both motors allows the detection of reversible elastic deformations between the rotor parts of Fo and F1.
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