The molecular dynamics of actin measured by a spin probe attached to lysine.
The molecular dynamics of actin measured by a spin probe attached to lysine.
复制标题
通过连接赖氨酸的自旋探针测量肌动蛋白的分子动力学。
DOI：
10.1016/0003-9861(87)90024-5
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发表时间：
1987
影响因子：
3.9
通讯作者：
Cooke,R
中科院分区：
文献类型：
--
作者：
Waring,AJ;Cooke,R
用自旋探针3-（5-氟-2，4-二硝基苯胺基）乙酰基标记兔骨骼肌G-肌动蛋白。胰蛋白酶消化标记的肌动蛋白，然后通过超滤和离子交换柱层析表明，该标签连接到残留的赖氨酸-61。该残基存在于9-kDa的N末端片段中，该片段容易被蛋白水解酶降解。测量抗坏血酸盐还原氮氧键的速率，以确定探针对溶剂中小分子的可及性。这些实验表明，与G-肌动蛋白结合的标记物相对难以接近抗坏血酸，这表明它被埋在蛋白质结构中。聚合进一步降低了探针的可及性。用Mn 2+取代结合的Ca 2+降低了所观察到的电子顺磁共振信号的强度，表明自旋标记距离肌动蛋白分子上的金属结合位点约2 nm。附着在G-肌动蛋白上的标记物显示出旋转运动的吸收光谱特征，相关时间（τc）为7 × 10− 9 s，比整个分子的相关时间更快。用饱和转移电子顺磁共振测量，附着在F-肌动蛋白上的标记物的τc值为2 × 10−5s，这表明探针的迁移率比细丝的要高。重酶解肌球蛋白或肌钙蛋白原肌球蛋白与标记肌动蛋白结合导致旋转相关时间进一步增加，当两者结合时，观察到迁移率的最大下降（τc= 1 × 10−4s）。上述结果表明，9 kDa的肌动蛋白片段相对于分子的其余部分是移动的，并且这种移动性可以受到重肌球蛋白或肌钙蛋白-原肌球蛋白结合的影响。
Rabbit skeletal muscle G-actin was labeled with a spin probe, 3-(5-fluoro-2,4-dinitroanilino)proxyl. Tryptic digestion of the labeled actin followed by ultrafiltration and ion-exchange column chromatography indicated that the label was attached to residue Lys-61. This residue is found within a 9-kDa N-terminal segment that is easily degraded by proteolytic enzymes. The rate of reduction of the nitroxide bond by ascorbate was measured to determine the accessibility of the probe to small molecules in the solvent. These experiments showed that label bound to G-actin was relatively inaccessible to ascorbate, suggesting that it is buried within the protein structure. Polymerization further decreased the accessibility of the probe. Replacing bound Ca2+with Mn2+decreased the observed intensity of the electron paramagnetic resonance signal, indicating the spin label is about 2 nm distant from the metal binding site on the actin molecule. Labels attached to G-actin displayed an absorption spectrum characteristic of rotational motion with a correlation time (τc) of 7 × 10−9s, which is faster than that for the whole molecule. Labels attached to F-actin had a value ofτc, measured using saturation transfer electron paramagnetic resonance, of 2 × 10−5s, which shows that the probe has a greater degree of mobility than the filament. The binding of heavy meromyosin or troponintropomyosin to labeled actin resulted in a further increase in the rotational correlation times, with the greatest decrease in mobility (τc= 1 × 10−4s) observed when both were bound. Together the above results suggest that the 9-kDa segment of actin is mobile relative to the rest of the molecule and that this mobility can be influenced by the binding of heavymeromyosin or troponin-tropomyosin.