The light chain binding domain of expressed smooth muscle heavy meromyosin acts as a mechanical lever
The light chain binding domain of expressed smooth muscle heavy meromyosin acts as a mechanical lever
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DOI：
10.1074/jbc.m006438200
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发表时间：
2000-11-24
影响因子：
4.8
通讯作者：
Trybus, KM
中科院分区：
文献类型：
--
作者：
Warshaw, DM;Guilford, WH;Trybus, KM
结构数据表明分子运动肌球蛋白通过轻链结合域或“颈部”的摆动来移动肌动蛋白。为了检验颈部作为机械杠杆的假设，通过删除或添加轻链结合位点，平滑肌重肌球蛋白（HMM）突变体被表达为具有更短或更长的颈部。对突变 HMM 进行了动力学和机械表征，重点是激光陷阱测定中单位位移和力的测量。在运动测定中，两个较短颈的构建体具有较小的单位步长，并且肌动蛋白的移动速度比 WT HMM 更慢。与对照相比，包含额外必需轻链结合位点的较长颈构建体的单一步长增加了 1.4 倍。还观察到几个构建体的动力学变化。缺乏颈部的突变体产生的力在某种程度上降低，而长颈鹿构建体施加的力高于对照。单分子位移和力数据支持这样的假设：颈部充当刚性杠杆，运动和力的支点位于运动域内的一点。
Structural data led to the proposal that the molecular motor myosin moves actin by a swinging of the light chain binding domain, or "neck" To test the hypothesis that the neck functions as a mechanical lever, smooth muscle heavy meromyosin (HMM) mutants were expressed with shorter or longer necks by either deleting or adding light chain binding sites. The mutant HMMs were characterized kinetically and mechanically, with emphasis on measurements of unitary displacements and forces in the laser trap assay, Two shorter necked constructs had smaller unitary step sizes and moved actin more slowly than WT HMM in the motility assay. A longer necked construct that contained an additional essential light chain binding site exhibited a 1.4-fold increase in the unitary step size compared with its control. Kinetic changes were also observed with several of the constructs, The mutant lacking a neck produced force at a somewhat reduced level, while the force exerted by the giraffe construct was higher than control. The single molecule displacement and force data support the hypothesis that the neck functions as a rigid lever, with the fulcrum for movement and force located at a point within the motor domain.