Adhikari B, Hideg K, Fajer P G
Institute of Molecular Biophysics, Department of Biological Science and National High Magnetic Field Laboratory, Florida State University, Tallahassee, FL 32310, USA.
Proc Natl Acad Sci U S A. 1997 Sep 2;94(18):9643-7. doi: 10.1073/pnas.94.18.9643.
The recent determination of the myosin head atomic structure has led to a new model of muscle contraction, according to which mechanical torque is generated in the catalytic domain and amplified by the lever arm made of the regulatory domain [Fisher, A. J., Smith, C. A., Thoden, J., Smith, R., Sutoh, K., Holden, H. M. & Rayment, I. (1995) Biochemistry 34, 8960-8972]. A crucial aspect of this model is the ability of the regulatory domain to move independently of the catalytic domain. Saturation transfer-EPR measurements of mobility of these two domains in myosin filaments give strong support for this notion. The catalytic domain of the myosin head was labeled at Cys-707 with indane dione spin label; the regulatory domain was labeled at the single cysteine residue of the essential light chain and exchanged into myosin. The mobility of the regulatory domain in myosin filaments was characterized by an effective rotational correlation time (tauR) between 24 and 48 micros. In contrast, the mobility of the catalytic domain was found to be tauR = 5-9 micros. This difference in mobility between the two domains existed only in the filament form of myosin. In the monomeric form, or when bound to actin, the mobility of the two domains in myosin was indistinguishable, with tauR = 1-4 micros and >1,000 micros, respectively. Therefore, the observed difference in filaments cannot be ascribed to differences in local conformations of the spin-labeled sites. The most straightforward interpretation suggests a flexible hinge between the two domains, which would have to stiffen before force could be generated.
最近对肌球蛋白头部原子结构的测定产生了一种新的肌肉收缩模型，根据该模型，机械扭矩在催化结构域中产生，并由调节结构域构成的杠杆臂进行放大[Fisher, A. J., Smith, C. A., Thoden, J., Smith, R., Sutoh, K., Holden, H. M. & Rayment, I. (1995) Biochemistry 34, 8960 - 8972]。该模型的一个关键方面是调节结构域能够独立于催化结构域移动。对肌球蛋白丝中这两个结构域的流动性进行的饱和转移 - 电子顺磁共振测量有力地支持了这一观点。肌球蛋白头部的催化结构域在Cys - 707处用茚满二酮自旋标记进行标记；调节结构域在必需轻链的单个半胱氨酸残基处进行标记，并交换到肌球蛋白中。肌球蛋白丝中调节结构域的流动性通过24至48微秒之间的有效旋转相关时间（tauR）来表征。相比之下，发现催化结构域的值为tauR = 5 - 9微秒。这两个结构域之间流动性的差异仅存在于肌球蛋白的丝状体形式中。在单体形式或与肌动蛋白结合时，肌球蛋白中两个结构域的流动性无法区分，分别为tauR = 1 - 4微秒和>1000微秒。因此，在丝状体中观察到的差异不能归因于自旋标记位点局部构象的差异。最直接的解释表明两个结构域之间存在一个灵活的铰链，在产生力之前这个铰链必须变硬。
