Hanson D C
Mol Immunol. 1985 Mar;22(3):245-50. doi: 10.1016/0161-5890(85)90157-9.
Nanosecond fluorescence depolarization studies reported in the accompanying companion paper showed that the long rotational correlation time, phi L, increased somewhat when rabbit IgG anti-dansyl antibodies were anchored in staphylococcal protein A (SpA) soluble complexes. The increases in phi L upon anchoring IgG probably resulted from "global coupling" effects caused by: increased steric hindrance of the antibody segments in the SpA complexes and intrinsic structural constraints already present in the monomeric IgG. Global coupling results from a restriction in the angular range of a flexible segment and is manifest when flexible motions alone cannot depolarize all of the fluorescence, so that the slower global tumbling of the entire particle is also required. Such effects cannot be resolved directly from experimental anisotropy data, however, because only a single long correlation time, phi L, is well defined over the limited time range of most fluorophores. In this paper, estimates of the anisotropy contributions from flexible and global motions of the IgG-SpA complexes are determined by contrasting theoretical and measured decays. For this analysis it was assumed that each of the experimental phi L-values is a weighted composite of the rotational correlation time associated with the less restricted flexible motions of the Fab arms, phi F, and the correlation time associated with global tumbling of the entire particle, phi G. A general two-exponential expression was used to relate phi F and phi G to phi L. This approach was meaningful because phi G-values of the various SpA complexes had been calculated from hydrodynamic measurements. The theoretical decays clearly show that, even if phi G is much longer than phi F, these two rotational motions still cannot be resolved over the experimentally accessible time range. Families of emission anisotropy decay curves for IgG antibodies with different amounts of intrinsic global coupling and for anchored antibodies with different amounts of steric hindrance were simulated by varying the preexponential weighting factors of the flexible and global terms. By comparing the calculated curves with the measured decays, it is evident that the rabbit IgG anti-dansyl antibodies do not have much intrinsic global coupling, but rather they are highly flexible. The curves also indicate that even for the exceptionally compact IgG4-SpA2 17-S complex, which showed the most steric hindrance in electron micrographs, the appropriate phi G weighting factor is only 0.28. Thus, as supposed earlier, the anchored antibodies exhibit considerable segmental flexibility. In closing, the above concepts are used to examine the results of
随附论文中报道的纳秒荧光去极化研究表明，当兔抗丹磺酰IgG抗体锚定在葡萄球菌蛋白A（SpA）可溶性复合物中时，长旋转相关时间phi L有所增加。IgG锚定后phi L的增加可能是由以下“全局耦合”效应引起的：SpA复合物中抗体片段的空间位阻增加以及单体IgG中已经存在的内在结构限制。全局耦合是由柔性片段的角度范围受限导致的，当仅靠柔性运动无法使所有荧光去极化时就会表现出来，因此还需要整个颗粒较慢的全局翻滚。然而，这种效应无法直接从实验各向异性数据中分辨出来，因为在大多数荧光团的有限时间范围内，只有一个长相关时间phi L能被很好地定义。在本文中，通过对比理论和测量的衰减来确定IgG-SpA复合物柔性和全局运动对各向异性的贡献估计值。对于此分析，假设每个实验phi L值是与Fab臂较少受限的柔性运动相关的旋转相关时间phi F和与整个颗粒全局翻滚相关的相关时间phi G的加权组合。使用一般的双指数表达式将phi F和phi G与phi L联系起来。这种方法是有意义的，因为各种SpA复合物的phi G值已从流体动力学测量中计算得出。理论衰减清楚地表明，即使phi G比phi F长得多，在实验可及的时间范围内这两种旋转运动仍然无法分辨。通过改变柔性和全局项的指数前加权因子，模拟了具有不同内在全局耦合量的IgG抗体以及具有不同空间位阻量的锚定抗体的发射各向异性衰减曲线族。通过将计算曲线与测量衰减进行比较，很明显兔抗丹磺酰IgG抗体没有太多内在全局耦合，而是具有高度柔性。曲线还表明，即使对于在电子显微镜下显示出最大空间位阻的异常紧密的IgG4-SpA2 17-S复合物，合适的phi G加权因子也仅为0.28。因此，如先前推测的那样，锚定抗体表现出相当大的片段柔性。最后，上述概念用于检验……的结果
