【作者】 张俊萍；

【导师】 郭维生；

【作者基本信息】 内蒙古大学 ， 生物物理学， 2017， 博士

【摘要】 肌球蛋白V作为最常见的线性分子马达之一,是将ATP水解过程中释放的化学能转化为机械功的纳米机器,参与细胞器和mRNA等的输运过程,其沿着肌动蛋白丝所作的定向和持续运动,是实现这些功能的基本条件。驻留时间、平均速度及运行长度是反映肌球蛋白V持续运动特性的重要参数。近年来的单分子实验结果表明,肌球蛋白V驻留时间的分布呈双指数曲线的形式,指数的系数与ATP和ADP的浓度及负载的大小密切相关,而平均速度与运行长度的分布也同样受到ATP和ADP的浓度及负载的影响。现有的理论研究尚难以完整地解释上述实验现象。肌球蛋白V的运动蕴含着深刻的化学动力学机制,因此建立力学化学动力学模型,是研究其运动特性的有效方法。基于以下实验结果:ATP浓度越高,肌球蛋白持续运动的驻留时间越短,平均速度越大;ADP的释放限制肌球蛋白力学化学循环速率,本文将ADP的释放及其逆过程和ATP与肌球蛋白V的结合这两个因素,作为决定肌球蛋白V运动特性的主要矛盾,对传统的多态模型进行了简化,建立了一种三态力学化学耦合模型。相对于传统的力学化学耦合模型,本模型在以下三个方面做了简化:(1)从统计意义上,认为肌球蛋白两个头部的力学和化学循环相对独立,从而在计算中只需考虑单头的循环过程;(2)将肌球蛋白V的化学循环过程,,简化为肌球蛋白V结合ATP、结合ADP和释放ADP几个步骤,忽略了 Pi的释放等细节。(3)在力学和化学耦合机制方面,不考虑肌球蛋白运动过程中复杂的构象变化及构象变化对双头化学反应速率的影响,仅考虑负载对化学反应速率的影响。相比之下,传统的多态模型考虑了较多的状态转化及力学化学耦合过程,虽然能够计算出更为精确的结果,但难以给出分子马达持续运动参数的解析解。与最简单的二态模型相比,本文的三态模型能够将ATP水解循环中ATP与ADP所起的作用区分开,从而能体现ATP与ADP对分子马达持续运动特性的影响,弥补二态模型的不足。依据简化的三态力学化学模型,采用化学动力学方法,本文定量研究了肌球蛋白V的持续运动特性,重点考察ATP与ADP的浓度以及负载对驻留时间、平均速度以及运行长度的影响,得到以下结果:(1)得出了驻留时间分布的双指数解析解和平均速度的数学表达式,明确了它们与ATP浓度、ADP浓度以及负载的定量关系;(2)通过肌球蛋白V双头驻留时间的联合概率分布,得出了运行长度分布的解析解。理论计算的结果与实验数据基本一致,证明本文提出的简化模型合理可行,为研究肌球蛋白V提供了新的理论工具。本文的研究还发现,ATP和ADP的浓度比对于驻留时间、平均速度的值具有决定性的作用,ATP和ADP之间的竞争是决定肌球蛋白V的运动特性的重要因素。更多还原

【Abstract】 Myosin V,as one of the most common linear molecular motor,is a nanometer-sized machine that transforms chemical energy liberated by ATP hydrolysis into mechanical work.It involves in many intracellular transport processses such as organelle and mRNA and so on.Directional and processive movement of myosin V along actin filaments is essential for the fulfillment of its functions.Dwell-time distributuions are widely reported for myosin V based on the single-molecule biophysical experiments which showed that the dwell time distribution is nearly dual exponential form with coefficients closely related to the ATP and ADP concentration and the load.As well as they have significant effects on mean velocity and run length distributuion of myosin V.The existing theoretical research is still difficult to fully explain the experimental phenomena.Myosin V movement contains a profound chemical kinetics mechanism,so mechanochemical model is an effective method for studying the characteristics of its movement.Single-molecule biophysical experiments show that increasing the concentration of ATP decreases the dwell time and increases the mean velocity.Meanwhile the ADP release is the rate-limiting transition in the myosin-V mechanochemical cycle.Based on the above facts,the traditional polymorphic models are simplified and a three-state mechanochemical coupling model is developed in which the processes of ADP release and its inverse and ATP binding are regarded as the decisive factors.Compared with the traditional mechanical chemical coupling model,the model is simplified in the following aspects.1.In the statistical meaning,it is considered that the mechanical and chemical cycles of the two heads of myosin are relatively independent.2.The chemical cycle of myosin V is simplified to several steps including ATP binding and ADP binding and ADP release with ignoring Pi release.3.In the aspect of mechanics and chemical coupling mechanism,the complex conformational changes and their effects on the rate of chemical reaction aren’t taken into account in myosin motion.The influence of external force on chemical reaction rate are considered.The traditional polymorphic model can calculate more accurate results.But due to considering so many the state transsitions and the chemical mechanical coupling,it is difficult to give an analytical solution for the processive motion of molecular motor.Compared with the most simple two-state model,the three-state model in this paper can separate the role of ATP and ADP in the ATP hydrolysis and then reflect the effects of ATP and ADP on the processivity of molecular motors.The three-state model makes up for the deficiency of the two state model.In this paper,according to a simplified mechanochemical coupling model,the processivity of myosin V is studied quantitatively.The effects of the ATP and ADP concentration and the load on the dwell time and the average velocity and the run length are investigated.The results are as follows:1.The analytical solutions of the dwell time distribution and the average velocity are obtained,and the quantitative relations between them and the ATP and ADP concentration and the load are defined.2.The joint probability distribution of the dwell time of myosin V is given and the analytical solution of the run length distribution is obtained.The theoretical results are in agreement with the experimental data,which proves that the simplified model proposed in this paper is reasonable and feasible.Our model may provide a new theoretical method for the study of myosin V.In this study it is also found that the ATP and ADP concentration ratio plays a decisive role for the dwell time and the average velocity,so the competition between ATP and ADP is an important factor in regulating movement characteristics of myosin V.更多还原