Time-resolved rotational dynamics of phosphorescent-labeled myosin heads in contracting muscle fibers.
Time-resolved rotational dynamics of phosphorescent-labeled myosin heads in contracting muscle fibers.
复制标题
收缩肌纤维中磷光标记的肌球蛋白头的时间分辨旋转动力学。
DOI：
10.1021/bi00495a003
复制
发表时间：
1990
期刊：
影响因子：
2.9
通讯作者：
Thomas,DD
中科院分区：
文献类型：
--
作者：
Stein,RA;Ludescher,RD;Dahlberg,PS;Fajer,PG;Bennett,RL;Thomas,DD
Department of Biochemistry, University of Minnesota Medical School, Minneapolis, Minnesota 55455 Received March 22, 1990; Revised Manuscript Received July 17, 1990 abstract: We have measured the microsecond rotational motions of myosin heads in contracting rabbit psoas muscle fibers by detecting the transient phosphorescence anisotropy of eosin-5-maleimide attached specifically to the myosin head. Experiments were performed on small bundles (10-20 fibers) of glycerinated rabbit psoas muscle fibers at 4 C. The isometric tension and physiological ATPase activity of activated fibers were unaffected by labeling 60-80% of the heads. Following excitation of the probes by a 10-ns laser pulse polarized parallel to the fiber axis, the time-resolved emission anisotropy of muscle fibers in rigor (no ATP) showed no decay from 1 ps to 1 ms (r „=0.095), indicating that all heads are rigidly attached to actin on this time scale. In relaxation (5 mM MgATP but no Ca2+), theanisotropy decayed substantially over the microsecond time range, from an initial anisotropy (r0) of 0.066 to a final anisotropy (/·„) of 0.034, indicating large-amplitude rotational motions with correlation times of about 10 and 150 ps and an overall angular range of 40-50. In isometric contraction (MgATP plus saturating Ca2+), the amplitude of the anisotropy decay (and thus the amplitude of the microsecond motion) is slightly less than in relaxation, and the rotational correlation times are about twice as long, indicating slower motions than those observed in relaxation. While the residual anisotropy (at 1 ms) in contraction is much closer to that in relaxation than in rigor, the initial anisotropy (at 1 ps) is approximately equidistant betweenthose of rigor and relaxation. Therefore, the anisotropy decay in contraction is not a simple linear combination of those in rigor and relaxation, implying that there are myosin head rotations in contractionthat are distinct from those in both rigor and relaxation. Fiber stiffness in isometric contraction is about 70% of the rigor value, suggesting that a majority of cross-bridges are attached to actin. Therefore, much of the rotational motion observed in contraction probably occurs in the attached phase of the cross-bridge cycle.IN^ uscle contraction requires movement of the myosin cross-bridge, which consists of two globular actin-binding myosin “heads”(SI) connected to the thick filament by a single rodlike domain (S2). Motions involving molecular rotation of or within SI, elastic deformation of S2, or helix-coil tran-sitions within S2 have all been postulated [review by Cooke (1986)]. Much structural, biochemical, and mechanical ev-idence supports such motions, but most of this evidence is indirect, serving only to establish the plausibility of flexible cross-bridge mechanisms. Some of the most direct information about cross-bridge orientation and rotational motion, and their role in force generation, has come from spectroscopic molecular probes. These probes have three properties that no other method combines [review by Thomas (1987)]:(1) They can be attached covalently and specifically to myosin heads, so that the information comes directly and exclusively from heads.
