Roux K H, Strelets L, Brekke O H, Sandlie I, Michaelsen T E
Department of Biological Science, Florida State University, Tallahassee 30306-4370, USA.
J Immunol. 1998 Oct 15;161(8):4083-90.
Various native and hinge-modified forms of Ig with identical Ids were reacted with an anti-Id mAb, and the resultant immune complexes were analyzed by negative stain immunoelectron microscopy. Complexes were scored for their geometry (linear versus ring complexes) and size (dimer, trimer, etc.). Ring dimers are the thermodynamically most favorable configuration, unless inhibited by steric and/or flexibility constraints. We found ring dimerization to correlate with the length of the upper, but not middle or lower, hinge. In contrast, the geometry and size of complexes of those molecules lacking formal hinges were unpredictable. A hingeless IgG mutant and native IgE readily formed ring dimers. Remarkably, monomeric IgM formed more ring dimers than any of the other Igs tested, including IgG3. We also tagged the Fab arms and measured the mean Fab-Fab angles and the degree of angular variation for each type of Ig. Surprisingly, IgM proved the most flexible by this assay. In hinged Igs, there was a correlation between length of the upper hinge and Fab-Fab flexibility. In contrast, we found no correlation between the mean Fab-Fab angle in uncomplexed Igs and their ability to dimerize with anti-Id mAb. These data suggest that the physicochemical methods typically used to evaluate molecular flexibility are often of low predictive value when tested in a functional assay.
将具有相同个体基因型的各种天然和铰链修饰形式的免疫球蛋白（Ig）与抗个体基因型单克隆抗体（mAb）反应，然后通过负染免疫电子显微镜分析所得的免疫复合物。根据复合物的几何形状（线性与环状复合物）和大小（二聚体、三聚体等）进行评分。环状二聚体是热力学上最有利的构型，除非受到空间位阻和/或柔韧性限制。我们发现环状二聚化与上铰链区的长度相关，而与中铰链区或下铰链区的长度无关。相比之下，那些缺乏正规铰链的分子形成的复合物的几何形状和大小是不可预测的。一种无铰链的IgG突变体和天然IgE很容易形成环状二聚体。值得注意的是，单体IgM形成的环状二聚体比任何其他测试的Ig都多，包括IgG3。我们还标记了Fab臂，并测量了每种Ig的平均Fab-Fab角度和角度变化程度。令人惊讶的是，通过该测定法证明IgM最具柔韧性。在有铰链的Ig中，上铰链区的长度与Fab-Fab柔韧性之间存在相关性。相比之下，我们发现未复合的Ig中的平均Fab-Fab角度与其与抗个体基因型mAb二聚化的能力之间没有相关性。这些数据表明，通常用于评估分子柔韧性的物理化学方法在功能测定中进行测试时，其预测价值往往较低。
