Probe studies of the MgADP state of muscle cross-bridges: microscopic and wavelength-dependent fluorescence polarization from 1,5-IAEDANS-labeled myosin subfragment 1 decorating muscle fibers.
Probe studies of the MgADP state of muscle cross-bridges: microscopic and wavelength-dependent fluorescence polarization from 1,5-IAEDANS-labeled myosin subfragment 1 decorating muscle fibers.
复制标题
肌肉横桥 MgADP 状态的探针研究：来自 1,5-IAEDANS 标记的肌球蛋白亚片段 1 装饰肌纤维的微观和波长依赖性荧光偏振。
DOI：
10.1021/bi00388a052
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发表时间：
1987
期刊：
影响因子：
2.9
通讯作者：
Burghardt,TP
中科院分区：
文献类型：
--
作者：
Ajtai,K;Burghardt,TP
加利福尼亚大学心血管研究所，旧金山，旧金山，加利福尼亚州 94143-0524 收稿日期：1986 年 10 月 10 日；修订稿于 1987 年 1 月 14 日收到摘要：测量了在严格条件下和存在 MgADP 的情况下，来自 5-[[[(碘乙酰基)氨基]乙基]氨基]萘-1-磺酸 (1, 5-IAEDANS) 标记的肌球蛋白 (Sl) 装饰肌纤维亚片段 1 的微观和波长依赖性荧光偏振信号。使用显微荧光偏振，我们从肌纤维中选择一个小的均匀体积 (~ 0.1/um3) 并检测高度的角序。从这些数据中，我们表明，严密纤维的探针角分布与 MgADP 与 Sl 结合时存在的定量不同。使用与波长相关的荧光偏振，我们改变激发光的波长，从而改变探针吸收偶极子在 Sl 中固定参考系的方向。根据这些数据，我们表明 MgADP 与 Sl 的结合导致 S-1 相对于肌动蛋白丝的角度重新定向。严格状态与 MgADP 状态的探针角度分布之间的差异不能通过添加随机探针来解释。显微荧光偏振实验表明，早期使用 1, 5-IAEDANS 探针区分严格性与 MgADP 探针角分布的尝试失败了，因为所采用的光学技术分辨率较低。波长相关的荧光偏振实验表明，在通常使用的激发和发射波长下，Sl 上的探针偶极子方向对于检测 MgADP 角跃迁的严格方向变化并不理想。
Cardiovascular Research Institute, University of California, San Francisco, San Francisco, California 94143-0524 Received October 10, 1986; Revised Manuscript Received January 14, 1987 abstract: The microscopic and wavelength-dependent fluorescence polarization signals from the 5-[[[(iodoacetyl) amino] ethyl] amino] naphthalene-l-sulfonic acid (1, 5-IAEDANS) labeled subfragment 1 of myosin (Sl) decorating muscle fibers in rigor and in the presence of MgADP are measured. Using microscopic fluorescence polarization, we select a small uniform volume (~ 0.1/um3) from the muscle fiber and detect a high degree of angular order. From these data we show that the probe angulardistribution from fibers in rigor is quantitatively different from that present when MgADP is bound to Sl. Using wavelength-dependent fluorescence polarization, we vary the wavelength of the excitation light and thereby change the direction that theprobe absorption dipole makes with a reference frame fixed in Sl. From these data we show that the binding of MgADP to Sl causes an angular reorientation ofS-l relative to the actin filament. The difference between the angular distribution of probes for the rigor vs. MgADP states cannot be accounted for by the addition of random probes. The microscopic fluorescence polarization experiments suggest that the earlierattempts to distinguish a rigor from a MgADP probe angular distribution by using the 1, 5-IAEDANS probe failed due to the lower resolution of the optical technique employed. The wavelength-dependent fluorescence polarization experiments indicatethat the probe dipole orientation on Sl, at the typically used excitation and emission wavelengths, is not ideal for detecting the orientation change in the rigor to MgADP angular transition.