The molecular basis of frictional loads in the in vitro motility assay with applications to the study of the loaded mechanochemistry of molecular motors.
The molecular basis of frictional loads in the in vitro motility assay with applications to the study of the loaded mechanochemistry of molecular motors.
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DOI：
10.1002/cm.20441
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发表时间：
2010-05
期刊：
影响因子：
2.9
通讯作者：
Moore, Jeffrey R.
中科院分区：
文献类型：
--
作者：
Greenberg, Michael J.;Moore, Jeffrey R.
分子马达将化学能转化为机械运动，产生完成一系列细胞功能所需的力。由于分子马达产生力，它们通常在负载条件下工作，其中马达的机械力化学会因负载的存在而改变。已经开发了几种生物物理技术来研究分子马达的负载行为和力产生能力，但这些技术中的大多数需要专门的设备。摩擦加载分析是对体外运动性分析的改进，可以在标准的荧光显微镜上进行，从而允许高通量地测量分子马达的加载的机械力化学。在这里，我们通过将载荷建模为一系列弹性或粘弹性元件来描述摩擦载荷分析的分子基础的模型。该模型计算不同结合蛋白施加的摩擦负荷，允许在运动性分析中测量等渗动力学、力-速度关系和功率曲线。我们通过计算和实验证明，摩擦载荷实验中最广泛使用的肌动蛋白结合蛋白α-肌动蛋白施加的摩擦载荷表现为粘弹性载荷，而不是纯弹性载荷。作为模型的测试，我们以α-肌动蛋白为负载，考察了正常和类疲劳条件下兔骨骼肌肌球蛋白的摩擦负载行为。我们发现，与纤维研究一致，类疲劳状态会导致肌球蛋白等长力、空载滑动速度、最大功率输出减少，并使峰值功率输出的负载发生移动。
Molecular motors convert chemical energy into mechanical movement, generating forces necessary to accomplish an array of cellular functions. Since molecular motors generate force, they typically work under loaded conditions where the motor mechanochemistry is altered by the presence of a load. Several biophysical techniques have been developed to study the loaded behavior and force generating capabilities of molecular motors yet most of these techniques require specialized equipment. The frictional loading assay is a modification to the in vitro motility assay that can be performed on a standard epifluorescence microscope, permitting the high-throughput measurement of the loaded mechanochemistry of molecular motors. Here, we describe a model for the molecular basis of the frictional loading assay by modeling the load as a series of either elastic or viscoelastic elements. The model, which calculates the frictional loads imposed by different binding proteins, permits the measurement of isotonic kinetics, force-velocity relationships, and power curves in the motility assay. We show computationally and experimentally that the frictional load imposed by alpha-actinin, the most widely employed actin binding protein in frictional loading experiments, behaves as a viscoelastic rather than purely elastic load. As a test of the model, we examined the frictional loading behavior of rabbit skeletal muscle myosin under normal and fatigue-like conditions using alpha-actinin as a load. We found that, consistent with fiber studies, fatigue-like conditions cause reductions in myosin isometric force, unloaded sliding velocity, maximal power output, and shift the load at which peak power output occurs.
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DOI：
10.1073/pnas.0706124105
发表时间：
2008-07-08
影响因子：
11.1
作者：
Ferrer, Jorge M.;Lee, Hyungsuk;Lang, Matthew J.
通讯作者：
Lang, Matthew J.
影响因子：
64.8
作者：
FINER, JT;SIMMONS, RM;SPUDICH, JA
通讯作者：
SPUDICH, JA
影响因子：
3.4
作者：
Evans, E;Ritchie, K
通讯作者：
Ritchie, K
DOI：
10.1098/rspb.1938.0050
发表时间：
1938-10-01
期刊：
PROCEEDINGS OF THE ROYAL SOCIETY SERIES B-BIOLOGICAL SCIENCES
影响因子：
--
作者：
Hill, AV
通讯作者：
Hill, AV
影响因子：
5.5
作者：
CADY, EB;ELSHOVE, H;MOLL, A
通讯作者：
MOLL, A