These conformational PLX-4720 differences are probably a result of small changes in the electrostatic interactions between amino acid residues rather than by ��gross�� conformational changes caused by hinge flexibility or by structural constrains imposed by the oligosaccharide moieties. Identifying functional residues is a complex issue. Activity can be modulated by residues that are distant from the binding site and these interactions within and between functional sites are crucial for protein activity. The study of closeness values in protein structure has proved useful in characterizing functional sites. Such studies are also useful for describing the Life Science Reagents interaction networks within protein structures that are connected to active site residues, as those found in enzymes where effectors bind to residues distant from the active sites. Central residues have high closeness values and they are assumed to transmit and efficiently integrate in formation to the rest of the protein. Our earlier studies suggested that highly connected residues may be involved in the differences in fine specificity in four murine mAbs expressing different CH regions and identical V regions. As observed in the homology models, an electrostatic network between the tyrosines residues interacting at the interface of the V region and the CH1 domain of murine mAb 18B7 could be more unfavorable for the net electrostatic forces of binding. These interaction networks formed at the CH1 domain appear to be directly responsible for the differences in affinity and specificity between these mAbs. The differences observed in the specificity of these mAbs also affected the characteristics of binding to a panel of self-antigens, indicating that polyreactivity is not only preserved after Ig class switch, but it also depends on the contribution of structural changes caused by the CH domains. This finding implies that polyreactivity is influenced by the surrounding CH structures, possibly making the Ab-combining site more plastic, thus allowing these Abs to recognize a variety of Ags. Consequently, the structural heterogeneity conferred by the different CH regions may result in the production of polyreactive Ab by changing charge and/or hydrophobicity of the V region. This implies that mousehuman chAb construction may yield Igs with binding characteristics that are different from the parental murine mAb, including the possibility for unexpected self-reactivity. In summary CH region glycosylation does not affect Ag binding, but exchanging murine and human CH domains can have profound effects on affinity and specificity. Although the generalizability of the observations with mAb 18b7 and chAb 18B7 to other chimeric Abs is unknown, our findings suggest caution with assuming that simply replacing the C region domain maintains the specificity and affinity of the V region.