Eads T M, Thomas D D, Austin R H
J Mol Biol. 1984 Oct 15;179(1):55-81. doi: 10.1016/0022-2836(84)90306-1.
We have studied submicrosecond and microsecond rotational motions within the contractile protein myosin by observing the time-resolved anisotropy of both absorption and emission from the long-lived triplet state of eosin-5-iodoacetamide covalently bound to a specific site on the myosin head. These results, reporting anisotropy data up to 50 microseconds after excitation, extend by two orders of magnitude the time range of data on time-resolved site-specific probe motion in myosin. Optical and enzymatic analyses of the labeled myosin and its chymotryptic digests show that more than 95% of the probe is specifically attached to sulfhydryl-1 (SH1) on the myosin head. In a solution of labeled subfragment-1 (S-1) at 4 degrees C, absorption anisotropy at 0.1 microseconds after a laser pulse is about 0.27. This anisotropy decays exponentially with a rotational correlation time of 210 ns, in good agreement with the theoretical prediction for end-over-end tumbling of S-1, and with times determined previously by fluorescence and electron paramagnetic resonance. In aqueous glycerol solutions, this correlation time is proportional to viscosity/temperature in the microsecond time range. Furthermore, binding to actin greatly restricts probe motion. Thus the bound eosin is a reliable probe of myosin-head rotational motion in the submicrosecond and microsecond time ranges. Our submicrosecond data for myosin monomers (correlation time 400 ns) also agree with previous results using other techniques, but we also detect a previously unresolvable slower decay component (correlation time 2.6 microseconds), indicating that the faster motions are restricted in amplitude. This restriction is not consistent with the commonly accepted free-swivel model of S-1 attachment in myosin. In synthetic thick filaments of myosin, both fast (700 ns) and slow (5 microseconds) components of anisotropy decay are observed. In contrast to the data for monomers, the anisotropy of filaments has a substantial residual component (26% of the initial anisotropy) that does not decay to zero even at times as long as 50 microseconds, implying significant restriction in overall rotational amplitude. This result is consistent with motion restricted to a cone half-angle of about 50 degrees. The combined results are consistent with a model in which myosin has two principal sites of segmental flexibility, one giving rise to submicrosecond motions (possibly corresponding to the junction between S-1 and S-2) and the other giving rise to microsecond motions (possibly corresponding to the junction between S-2 and light meromyosin).(ABSTRACT TRUNCATED AT 400 WORDS)
我们通过观察与肌球蛋白头部特定位点共价结合的5-碘乙酰胺曙红长寿命三重态吸收和发射的时间分辨各向异性，研究了收缩蛋白肌球蛋白内的亚微秒和微秒级旋转运动。这些结果报告了激发后长达50微秒的各向异性数据，将肌球蛋白中时间分辨位点特异性探针运动的数据时间范围扩展了两个数量级。对标记的肌球蛋白及其胰凝乳蛋白酶消化产物的光学和酶学分析表明，超过95%的探针特异性附着在肌球蛋白头部的巯基-1（SH1）上。在4℃的标记亚片段-1（S-1）溶液中，激光脉冲后0.1微秒的吸收各向异性约为0.27。这种各向异性以210纳秒的旋转相关时间呈指数衰减，与S-1端到端翻滚的理论预测以及先前通过荧光和电子顺磁共振测定的时间非常吻合。在水性甘油溶液中，该相关时间在微秒时间范围内与粘度/温度成正比。此外，与肌动蛋白结合极大地限制了探针运动。因此，结合的曙红是亚微秒和微秒时间范围内肌球蛋白头部旋转运动的可靠探针。我们关于肌球蛋白单体的亚微秒数据（相关时间400纳秒）也与使用其他技术的先前结果一致，但我们还检测到一个先前无法分辨的较慢衰减成分（相关时间2.6微秒），表明较快运动的幅度受到限制。这种限制与肌球蛋白中S-1附着的普遍接受的自由旋转模型不一致。在肌球蛋白的合成粗丝中，观察到各向异性衰减的快速（700纳秒）和慢速（5微秒）成分。与单体数据相反，细丝的各向异性有一个相当大的残余成分（初始各向异性的26%），即使在长达50微秒的时间也不会衰减到零，这意味着整体旋转幅度受到显著限制。这一结果与限制在约50度锥半角内的运动一致。综合结果与一个模型一致，在该模型中，肌球蛋白有两个主要的节段灵活性位点，一个导致亚微秒级运动（可能对应于S-1和S-2之间的连接处），另一个导致微秒级运动（可能对应于S-2和轻酶解肌球蛋白之间的连接处）。（摘要截短于400字）
