Volkán-Kacsó Sándor, Marcus Rudolph A
Noyes Laboratory of Chemical Physics, California Institute of Technology, Pasadena, CA, United States.
Segerstrom Science Center, Azusa Pacific University, Azusa, CA, United States.
Front Microbiol. 2022 Apr 22;13:861855. doi: 10.3389/fmicb.2022.861855. eCollection 2022.
In this chapter, we review single-molecule observations of rotary motors, focusing on the general theme that their mechanical motion proceeds in substeps with each substep described by an angle-dependent rate constant. In the molecular machine F1-ATPase, the stepping rotation is described for individual steps by forward and back reaction rate constants, some of which depend strongly on the rotation angle. The rotation of a central shaft is typically monitored by an optical probe. We review our recent work on the theory for the angle-dependent rate constants built to treat a variety of single-molecule and ensemble experiments on the F-ATPase, and relating the free energy of activation of a step to the standard free energy of reaction for that step. This theory, an elastic molecular transfer theory, provides a framework for a multistate model and includes the probe used in single-molecule imaging and magnetic manipulation experiments. Several examples of its application are the following: (a) treatment of the angle-dependent rate constants in stalling experiments, (b) use of the model to enhance the time resolution of the single-molecule imaging apparatus and to detect short-lived states with a microsecond lifetime, states hidden by the fluctuations of the imaging probe, (c) treatment of out-of-equilibrium "controlled rotation" experiments, (d) use of the model to predict, without adjustable parameters, the angle-dependent rate constants of nucleotide binding and release, using data from other experiments, and (e) insights obtained from correlation of kinetic and cryo-EM structural data. It is also noted that in the case where the release of ADP would be a bottleneck process, the binding of ATP to another site acts to accelerate the release by 5-6 orders of magnitude. The relation of the present set of studies to previous and current theoretical work in the field is described. An overall goal is to gain mechanistic insight into the biological function in relation to structure.
在本章中，我们回顾了旋转马达的单分子观测结果，重点关注其机械运动以亚步进行的一般主题，每个亚步由一个与角度相关的速率常数描述。在分子机器F1 - ATP酶中，单个步骤的步进旋转由正向和反向反应速率常数描述，其中一些速率常数强烈依赖于旋转角度。中心轴的旋转通常由光学探针监测。我们回顾了我们最近关于构建与角度相关的速率常数理论的工作，该理论用于处理关于F - ATP酶的各种单分子和整体实验，并将一个步骤的活化自由能与该步骤的标准反应自由能联系起来。这个理论，即弹性分子转移理论，为多态模型提供了一个框架，并包括单分子成像和磁操纵实验中使用的探针。其应用的几个例子如下：(a) 在失速实验中处理与角度相关的速率常数；(b) 使用该模型提高单分子成像设备的时间分辨率，并检测具有微秒寿命的短寿命状态，这些状态被成像探针的波动所掩盖；(c) 处理非平衡“受控旋转”实验；(d) 使用该模型，在不使用可调参数的情况下，根据其他实验数据预测核苷酸结合和释放的与角度相关的速率常数；(e) 从动力学和低温电子显微镜结构数据的相关性中获得的见解。还需要注意的是，在ADP的释放可能是一个瓶颈过程的情况下，ATP与另一个位点的结合作用是将释放加速5 - 6个数量级。描述了本系列研究与该领域先前和当前理论工作的关系。一个总体目标是获得与结构相关的生物学功能的机制性见解。
