Fung L W, Kalaw B O, Hatfield R M, Dias M N
Department of Chemistry, Loyola University of Chicago, Illinois 60626, USA.
Biophys J. 1996 Feb;70(2):841-51. doi: 10.1016/S0006-3495(96)79626-1.
The segmental motions of cross-linked erythrocyte skeletal protein (spectrin-actin-protein 4.1) samples, labeled with nitroxide spin labels, were monitored by conventional first-harmonic and saturation transfer second-harmonic electron paramagnetic resonance methods. Skeletal proteins were extracted from human red blood cells and treated with three oxidative reagents (diamide, hydrogen peroxide, and phenylhydrazine) to cross-link sulfhydryl groups and with one fixative reagent (glutaraldehyde) to cross-link lysine residues. The treatments provided extensive cross-linking between spectrin-actin-protein 4.1 molecules, as determined by gel electrophoresis, and surface charge modification, as determined by pl measurements. However, segmental motions of the cross-linked skeletal proteins remained generally similar to those in normal skeletal proteins. Both the weakly immobilized and the strongly immobilized motions were similar in cross-linked and control samples. Small differences in some motional components were detected. In some cases, faster mobilities were observed, with approximately 5% of the strongly immobilized motions converted to the weakly immobilized motions in the cross-linked samples. It is often believed that the consequence of membrane protein oxidation is restricted protein dynamics, giving membrane rigidity. However, our studies provide needed experimental evidence to indicate that segmental motions are maintained with very little modification even in the presence of extensive cross-linking. Thus cross-linking does not restrict the internal molecular flexibility that gives rise to segmental motions.
用氮氧化物自旋标记物标记的交联红细胞骨架蛋白（血影蛋白-肌动蛋白-蛋白4.1）样品的片段运动，通过传统的一次谐波和饱和转移二次谐波电子顺磁共振方法进行监测。从人红细胞中提取骨架蛋白，并用三种氧化试剂（二酰胺、过氧化氢和苯肼）处理以交联巯基，并用一种固定剂（戊二醛）处理以交联赖氨酸残基。如通过凝胶电泳所确定的，这些处理在血影蛋白-肌动蛋白-蛋白4.1分子之间提供了广泛的交联，并且如通过pl测量所确定的，实现了表面电荷修饰。然而，交联骨架蛋白的片段运动总体上仍与正常骨架蛋白中的片段运动相似。交联样品和对照样品中弱固定和强固定运动均相似。检测到一些运动成分存在微小差异。在某些情况下，观察到更快的迁移率，在交联样品中约5%的强固定运动转变为弱固定运动。人们通常认为膜蛋白氧化的结果是蛋白质动力学受限，导致膜刚性增加。然而，我们的研究提供了必要的实验证据，表明即使在存在广泛交联的情况下，片段运动也能在几乎没有改变的情况下得以维持。因此，交联并不限制产生片段运动的分子内部灵活性。
