Crowder M S, Cooke R
Biophys J. 1987 Feb;51(2):323-33. doi: 10.1016/S0006-3495(87)83338-6.
Electron paramagnetic resonance (EPR) spectroscopy of paramagnetic derivatives of ATP has been used to probe the angular distribution of myosin in glycerinated muscle fibers. Three nucleotide spin labels have been prepared with the nitroxide free radical moiety attached, via an ester linkage to either: the 2' or 3' positions of the ribose unit of ATP (SL-ATP), the 2' position of 3' deoxy ATP (2'SL-dATP), or the 3' position of 2' deoxy ATP (3'SL-dATP). In muscle fibers, these nucleotides are quickly hydrolyzed to their diphosphate forms. All three diphosphate analogues bind to the nucleotide site of myosin with similar affinities: rabbit psoas fibers, 7 X 10(3)/M; insect flight muscle, 5 X 10(3)/M; and rabbit soleus muscle, 2 X 10(4)/M. Analysis of the spectra showed that the principal z-axis of the nitroxide attached to bound nucleotides was oriented with respect to the filament axis. The principal axes of 3'SL-dADP and 2'SL-dADP appeared to be preferentially aligned at mean angles of 67 degrees +/- 4 degrees and 55 degrees +/- 5 degrees, respectively. The distribution of probes about these angles can be described by Gaussians with widths of 16 degrees +/- 4 degrees and 13 degrees +/- 5 degrees, respectively. The spectrum of bound SL-ADP was a linear combination of the spectra of the two deoxy analogues. These orientations were the same in the three muscle types examined, indicating a high degree of homology in the nucleotide binding site. Applying static strains as high as 0.2 N/mm2 to muscle fibers caused no change in the orientation of myosin-bound, spin-labeled nucleotides. When muscle fibers were stretched to decrease actin and myosin filament overlap, bound SL-ADP produced EPR spectra indicative of probes with a highly disordered angular distribution. Sodium vanadate and SL-ATP caused fiber stiffness to decrease, and the EPR spectrum of the bound analogue indicated an increase in the fraction of disoriented probes with a concomitant decrease in the fraction of oriented probes. These findings indicate that when myosin is bound to actin its nucleotide site is highly oriented relative to the fiber axis, and when this interaction is removed the orientation of the nucleotide site becomes highly disordered.
三磷酸腺苷（ATP）顺磁性衍生物的电子顺磁共振（EPR）光谱已被用于探测甘油化肌纤维中肌球蛋白的角分布。制备了三种核苷酸自旋标记物，其氮氧自由基部分通过酯键连接到：ATP核糖单元的2'或3'位（SL-ATP）、3'-脱氧ATP的2'位（2'SL-dATP）或2'-脱氧ATP的3'位（3'SL-dATP）。在肌纤维中，这些核苷酸会迅速水解为其二磷酸形式。所有三种二磷酸类似物以相似的亲和力结合到肌球蛋白的核苷酸位点：兔腰大肌纤维为7×10³/M；昆虫飞行肌为5×10³/M；兔比目鱼肌为2×10⁴/M。光谱分析表明，连接到结合核苷酸上的氮氧自由基的主z轴相对于细丝轴定向。3'SL-dADP和2'SL-dADP的主轴似乎分别以67°±4°和55°±5°的平均角度优先排列。围绕这些角度的探针分布可以分别用宽度为16°±4°和13°±5°的高斯分布来描述。结合的SL-ADP的光谱是两种脱氧类似物光谱的线性组合。在所研究的三种肌肉类型中，这些取向是相同的，表明核苷酸结合位点具有高度同源性。对肌纤维施加高达0.2 N/mm²的静态应变不会导致与肌球蛋白结合的自旋标记核苷酸的取向发生变化。当肌纤维被拉伸以减少肌动蛋白和肌球蛋白细丝的重叠时，结合的SL-ADP产生的EPR光谱表明探针的角分布高度无序。钒酸钠和SL-ATP导致纤维硬度降低，结合类似物的EPR光谱表明无序探针的比例增加，同时取向探针的比例降低。这些发现表明，当肌球蛋白与肌动蛋白结合时，其核苷酸位点相对于纤维轴高度定向，而当这种相互作用被消除时，核苷酸位点的取向变得高度无序。
