利用DCO-ALEX FRET研究嗜热碱细菌FoF1-ATP合酶中旋转质子马达的步长
Step size of the rotary proton motor in single FoF1-ATP synthase from a thermoalkaliphilic bacterium by DCO-ALEX FRET
摘要：
嗜热酶可以在较高温度下工作，但在室温下显示出降低的活性。它们通常在制备过程中更稳定，因此被认为结构更刚性。与在环境温度下生长的细菌的蛋白质相比，结晶通常更容易，特别是对于膜蛋白。产ATP酶FoF 1-ATP合酶由嗜热碱Caldalkalibacillus thermarum菌株TA2.A1的Fo马达驱动，该马达由13个C-亚基组成的环组成。我们应用了单分子福斯特共振能量转移（FRET）方法，使用占空比优化的交替激光激发（DCO-ALEX）来监测预期的13步旋转Fo电机工作。开发了新的FRET转换直方图，以识别与大肠杆菌酶的10步Fo马达相比更小的步长。停留时间分析揭示了温度和LDAO依赖性的Fo电机活动的单分子水平上。Fo马达的来回步进发生得很快，表明这种嗜热酶的膜部分具有高柔性。
英文摘要：
Thermophilic enzymes can operate at higher temperatures but show reduced activities at room temperature. They are in general more stable during preparation and, accordingly, are considered to be more rigid in structure. Crystallization is often easier compared to proteins from bacteria growing at ambient temperatures, especially for membrane proteins. The ATP-producing enzyme FoF1-ATP synthase from thermoalkaliphilic Caldalkalibacillus thermarum strain TA2.A1 is driven by a Fo motor consisting of a ring of 13 c-subunits. We applied a single-molecule Förster resonance energy transfer (FRET) approach using duty cycle-optimized alternating laser excitation (DCO-ALEX) to monitor the expected 13-stepped rotary Fo motor at work. New FRET transition histograms were developed to identify the smaller step sizes compared to the 10-stepped Fo motor of the Escherichia coli enzyme. Dwell time analysis revealed the temperature and the LDAO dependence of the Fo motor activity on the single molecule level. Back-and-forth stepping of the Fo motor occurs fast indicating a high flexibility in the membrane part of this thermophilic enzyme.
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