DataSheet1_Angle-dependent rotation velocity consistent with ADP release in bacterial F1-ATPase.pdf

A model-based method is used to extract a short-lived state in the rotation kinetics of the F1-ATPase of a bacterial species, Paracoccus denitrificans (PdF1). Imaged as a single molecule, PdF1 takes large 120ø steps during it rotation. The apparent lack of further substeps in the trajectories not only renders the rotation of PdF1 unlike that of other F-ATPases, but also hinders the establishment of its mechano-chemical kinetic scheme. We addressed these challenges using the angular velocity extracted from the single-molecule trajectories and compare it with its theoretically calculated counterpart. The theory-experiment comparison indicate the presence of a 20μs lifetime state, 40o after ATP binding. We identify a kinetic cycle in which this state is a three-nucleotide occupancy state prior to ADP release from another site. A similar state was also reported in our earlier study of the Thermophilic bacillus F1-ATPase (lifetime ∼10μs), suggesting thereby a common mechanism for removing a nucleotide release bottleneck in the rotary mechanism.

本研究采用基于模型的方法，从脱氮副球菌（Paracoccus denitrificans）F₁-ATP合酶（F₁-ATPase，缩写PdF₁）的旋转动力学过程中提取一种瞬态状态。作为单分子成像的PdF₁在旋转过程中会产生较大的120°步幅，但其运动轨迹中似乎不存在额外的亚步幅，这不仅使得PdF₁的旋转机制与其他F₁-ATP合酶截然不同，同时也阻碍了其力化学动力学模型的构建。本研究借助从单分子轨迹中提取的角速度，并将其与理论计算得到的对应值进行对比，以此解决上述难题。理论与实验的对比结果表明，在ATP结合后的40°位置存在一种寿命为20微秒的瞬态状态。我们确定了一个动力学循环，该循环中此瞬态状态为另一位点释放ADP之前的三核苷酸占据状态。在本团队此前对嗜热芽孢杆菌F₁-ATP合酶的研究中，也曾报道过类似的状态（寿命约为10微秒），这表明旋转机制中存在一种可解决核苷酸释放瓶颈的通用机制。