Barnett V A, Thomas D D
Biochemistry. 1987 Jan 13;26(1):314-23. doi: 10.1021/bi00375a044.
We have measured the conventional electron paramagnetic resonance (EPR) spectrum of spin-labeled myosin filaments as a function of the nucleotide occupancy of the active site of the enzyme. The probe used was 4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidine-1-oxyl (IASL), which reacts specifically with sulfhydryl 1 of the myosin head. In the absence of nucleotide, the probe remains strongly immobilized (rigidly attached to the myosin head) so that no nanosecond rotational motions are detectable. When MgADP is added to IASL-labeled myosin filaments (T = 20 degrees C), the probe mobility increases slightly. During steady-state MgADP hydrolysis (T = 20 degrees C), the probe undergoes large-amplitude nanosecond rotational motion. These results are consistent with previous studies of myosin monomers, heavy meromyosin, and myosin subfragment 1. Isoclinic points observed in overlays of sequential EPR spectra recorded during ATP hydrolysis strongly suggest that the probes fall into two motional classes, separated by approximately an order of magnitude in effective rotational correlation time. Both of the observed states are distinct from the conformation of myosin in the absence of nucleotides, and the spectrum of the less mobile population is indistinguishable from that observed in the presence of MgADP. The addition of ADP and vanadate to IASL-myosin gives rise to two motional classes virtually identical with those observed in the presence of ATP, but the relative concentrations of the spin populations are significantly different. We have quantitated the percentage of myosin in each motional state during ATP hydrolysis. The result agrees well with the predicted percentages in the two predominant chemical states in the myosin ATPase cycle. Spectra obtained in the presence of nucleotide analogues permit us to assign the conformational states to specific chemical states. We propose that the two motional classes represent two distinct local conformations of myosin that are in exchange with one another during the ATP hydrolysis reaction cycle.
我们测量了自旋标记肌球蛋白丝的传统电子顺磁共振（EPR）光谱，该光谱是酶活性位点核苷酸占据情况的函数。所使用的探针是4-（2-碘乙酰胺基）-2,2,6,6-四甲基哌啶-1-氧基（IASL），它与肌球蛋白头部的巯基1特异性反应。在没有核苷酸的情况下，探针保持强烈固定（牢固地附着在肌球蛋白头部），因此无法检测到纳秒级的旋转运动。当将MgADP添加到IASL标记的肌球蛋白丝中（T = 20摄氏度）时，探针的流动性略有增加。在稳态MgADP水解过程中（T = 20摄氏度），探针经历大幅度的纳秒级旋转运动。这些结果与先前对肌球蛋白单体、重酶解肌球蛋白和肌球蛋白亚片段1的研究一致。在ATP水解过程中记录的连续EPR光谱叠加图中观察到的等倾点强烈表明，探针分为两个运动类别，有效旋转相关时间相差约一个数量级。观察到的两种状态均与不存在核苷酸时肌球蛋白的构象不同，且流动性较小的群体的光谱与在MgADP存在下观察到的光谱无法区分。向IASL-肌球蛋白中添加ADP和钒酸盐会产生两个与在ATP存在下观察到的运动类别几乎相同的运动类别，但自旋群体的相对浓度有显著差异。我们对ATP水解过程中每种运动状态下的肌球蛋白百分比进行了定量。结果与肌球蛋白ATP酶循环中两种主要化学状态的预测百分比非常吻合。在核苷酸类似物存在下获得的光谱使我们能够将构象状态与特定化学状态相关联。我们提出，这两个运动类别代表肌球蛋白的两种不同局部构象，它们在ATP水解反应循环中相互交换。
