COMPARISONS OF THE ABILITY OF HUMAN IGG3 HINGE MUTANTS, IGM, IGE, ANDIGA2, TO FORM SMALL IMMUNE-COMPLEXES - A ROLE FOR FLEXIBILITY AND GEOMETRY

Various native and hinge-modified forms of Ig with identical Ids were reacted with an anti-Id mAb, and the resultant immune complexes were a nalyzed by negative stain immunoelectron microscopy. Complexes were sc ored 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 constrain ts. We found ring dimerization to correlate with the length of the upp er, but not middle or lower, hinge, In contrast, the geometry and size of complexes of those molecules lacking formal hinges were unpredicta ble, A hingeless IgG mutant and native IgE readily formed ring dimers, Remarkably, monomeric IgM formed more ring dimers than any of the oth er Igs tested, including IgG3, We also tagged the Fab arms and measure d 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 hi nge and Fab-Fab flexibility, In contrast, we found no correlation betw een the mean Fab-Fab angle in uncomplexed Igs and their ability to dim erize with anti-Id mAb, These data suggest that the physicochemical me thods typically used to evaluate molecular flexibility are often of lo w predictive value when tested in a functional assay.