Effective temperature of proteins in the active states

许多蛋白质使用的化学能是在将ATP水解成ADP和P1的过程中产生的。一个基本的问题，即蛋白质如何将这种化学能转化为机械功，仍然没有解决。这项研究的目的是检验由三磷酸腺苷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，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。我们选择肌动球蛋白作为实验系统，在此资助期间，我们取得了以下成果：(1)我们开发了一套测量肌动蛋白细丝与肌球蛋白分子相互作用的有效温度的实验系统：将肌动蛋白细丝水平固定在溶液中，用双光束光钳捕获两端附着的两个微球。测量了两珠的相对旋转布朗运动，并根据两珠夹角的均方和松弛时间计算了有效温度。(2)我们测量了在ATP存在下肌球蛋白与肌球蛋白相互作用的旋转涨落：肌球蛋白VI在5个36 nm的步骤中旋转了45度，每一次ATP水解的步长为36 nm。这种运动不能用简单的肌动蛋白细丝行走模型来解释。(3)肌球蛋白分子沿着肌动蛋白细丝的热棘轮扩散。用单分子技术测量正反向步数之比。结果表明，正反向势垒之差为3kBT。对于另一种蛋白质运动蛋白来说，这个值几乎是相同的。

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.