Effective temperature of proteins in the active states

许多蛋白质利用ATP水解为ADP和P1过程中产生的化学能工作。一个基本的问题，蛋白质如何将这种化学能转化为机械功仍然没有解决。本研究的目的是检查由ATP的化学能激发的蛋白质分子的有效温度是否真的增加，如果是这样的话，那么我们就可以构建一个模型来解释蛋白质分子是如何转换化学能和机械能的。我们选择了肌动球蛋白作为实验体系，在本基金资助期间，取得了以下成果。(1)我们已经开发了一个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：肌动蛋白丝水平地保持在溶液中，用双光束光镊捕获两个连接在两端的微珠。测量了两个微珠的相对转动布朗运动，并由两个微珠之间的角度的均方和松弛时间计算了有效温度。(2)我们已经测量了在ATP存在下肌动蛋白丝与肌球蛋白相互作用的旋转波动：肌球蛋白VI，其每一个ATP水解具有36nm的步长，在五个36nm的步长期间旋转45度。这种运动不能用简单的沿着卷曲的肌动蛋白丝沿着行走的模型来解释。(3)肌球蛋白分子沿着肌动蛋白丝的热棘轮扩散。用单分子技术测量了前后步的比值。计算结果表明，前后向的势垒差为3kBT。该值与另一种蛋白质运动驱动蛋白几乎相同。

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.