Luminescence resonance energy transfer measurements in myosin
Luminescence resonance energy transfer measurements in myosin
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DOI：
10.1016/s0006-3495(98)77953-6
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发表时间：
1998-05-01
影响因子：
3.4
通讯作者：
Selvin, PR
中科院分区：
文献类型：
--
作者：
Getz, EB;Cooke, R;Selvin, PR
肌球蛋白被认为通过两个结构域之间的相对方向旋转产生力。直接测量结构域之间的距离可能会确认和量化这些构象变化，但由于所涉及的距离很大，这些努力受到了阻碍。在这里，我们表明，发光共振能量转移（LRET），它使用发光镧系元素作为能量转移供体，是能够测量这些长距离。具体而言，我们测量了肌球蛋白头部的催化结构域（Cys（707））和调节轻链结构域（Cys（108））之间的距离。在没有核苷酸或肌动蛋白的肌球蛋白复合物中测量到21.2 +/-1.9%的能量转移效率，对应于73埃的距离，与Rayment等人的晶体结构一致。在与肌动蛋白结合时，能量转移效率降低4.5 +/-1.0%，表明肌球蛋白的构象变化，其涉及Cys（707）相对于轻链结构域的相对旋转和/或平移。ADP的加入也改变了能量转移效率，可能是通过连接到Cys的探针的旋转（707）。这些结果表明，LRET能够对相对较大的肌动球蛋白复合物进行准确测量，并且能够检测肌球蛋白的催化和轻链结构域之间的构象变化。
Myosin is thought to generate force by a rotation between the relative orientations of two domains. Direct measurements of distances between the domains could potentially confirm and quantify these conformational changes, but efforts have been hampered by the large distances involved. Here we show that luminescence resonance energy transfer (LRET), which uses a luminescent lanthanide as the energy-transfer donor, is capable of measuring these long distances. Specifically, we measure distances between the catalytic domain (Cys(707)) and regulatory light chain domain (Cys(108)) of the myosin head. An energy transfer efficiency of 21.2 +/- 1.9% is measured in the myosin complex without nucleotide or actin, corresponding to a distance of 73 Angstrom, consistent with the crystal structure of Rayment et al. Upon binding to actin, the energy transfer efficiency decreases by 4.5 +/- 1.0%, indicating a conformational change in myosin that involves a relative rotation and/or translation of Cys(707) relative to the light chain domain. Addition of ADP also alters the energy transfer efficiency, likely through a rotation of the probe attached to Cys(707). These results demonstrate that LRET is capable of making accurate measurements on the relatively large actomyosin complex, and is capable of detecting conformational changes between the catalytic and light chain domains of myosin.