Effective temperature of proteins in the active states

许多蛋白质利用ATP水解成ADP和P1过程中产生的化学能工作。一个基本的问题，即蛋白质如何将化学能转化为机械能，仍未得到解决。本研究的目的是检验在ATP化学能的激发下，蛋白质分子的有效温度是否真的升高了。如果是这样，那就建立一个模型来解释化学和机械能是如何被蛋白质分子转化的。我们选择了肌动球蛋白作为实验系统，在本次资助期间，我们取得了以下成果。(1)我们开发了一种实验系统来测量肌动蛋白丝与肌球蛋白分子相互作用的有效温度：将肌动蛋白丝水平放置在溶液中，并用双光束光学镊子捕获两端连接的两个微珠。测量了两个磁珠的相对旋转布朗运动，并根据磁珠与松弛线夹角的均方计算了有效温度。(2)我们测量了在ATP存在下肌动蛋白丝与肌凝蛋白相互作用的旋转波动：肌凝蛋白VI，每一个ATP水解的步长为36nm，在5个36nm的步骤中旋转45度。这种运动不能用简单的沿着盘绕的肌动蛋白丝行走的模型来解释。肌凝蛋白分子沿着肌动蛋白丝的热棘轮扩散。用单分子技术测定了正反两步的比值。结果表明，正反向能垒差为3kBT。对于另一种蛋白质马达驱动蛋白，这个值几乎相同。

Many proteins work using chemical energy that generates during hydrolysis of ATP into ADP and P1. A basic question, how proteins converts this chemical energy to mechanical works remains unsolved. The aims of this study were to examine if the effective temperature of a protein molecule excited by chemical energy of ATP is really increased and. If so, to construct a model to explain how chemical and mechanical energies are converted by a protein molecule. We chose actomyosin as the experimental system, and during the period of this grant, we obtained the following achievements.(1) We have developed an experimental system to measure the effective temperature of an actin filament interacting with myosin molecules: an actin filament was horizontally held in solution being caught two microbeads attached to the both ends using double beam optical tweezers. Relative rotational Brownian movement of the two beads was measured and the effective temperature was calculated from the mean square of the angle between the two beads and the relaxation tirne.(2) We have measured the rotational fluctuation of an actin filament interacting with myosin in the presence of ATP: Myosin VI, which has a 36nm step length per one ATP hydrolysis, rotated 45 degree during five 36nm steps. This movement cannot be explained according to simple walking model along the coiled actin filament.(3) Myosin molecules are diffusing along the thermal ratchet of an actin filament. The ratio of forward and backward steps was measured using singlemolecule technique. The result indicates that difference of the energy barrier for forward and backward direction is 3kBT. This value is almost same for another protein motor kinesin.