Antibodies are essential in the human adaptive immune response against infection and disease, with immunoglobulin gamma (IgG) being the most abundant in human serum and widely used as therapeutic molecules. The IgG structure is comprised of multiple domains that make up the antigen-binding region (Fab), the immune cell binding region (Fc), and a linker region (hinge). Aside from the upper-most domains that interact directly with antigens, the backbone of the antibody structure is considered to be genetically constant, owing to a finite number of immune receptor targets. Despite this, over 250 alleles of IgG constant domains have been identified across the four IgG subclasses, many of which encode amino acid polymorphisms and, in some cases, whole exon deletions.
The effect of naturally occurring polymorphisms on stability was evaluated for 35 genetically distinct IgG antibodies. A fluorescent based technique called red edge excitation shift (REES) was used to compare changes to the structural integrity of each antibody after heating as a measure of thermal stability. REES relies on the fluorescent excitation properties of tryptophan which are influenced by solvation state. We were able to resolve stability differences between antibodies within each subclass which shared high structural homology but harboured amino acid polymorphisms. Of note, polymorphisms located at the interface of individual domains tended to have greater implications on the molecular stability.
Structural effects of exon deletions in the hinge sequence of IgG3 alleles were investigated using small angle x-ray scattering (SAXS). The extended hinge of IgG3 is understood to increase protease susceptibility and flexibility of IgG3 subclass of antibodies compared to the other subclasses. The structures of full-length IgG3 antibodies with three different hinge lengths were characterised using SAXS to provide insights into the motility of the Fc and Fab regions as governed by hinges of varying lengths. These findings begin to unravel the interplay between genetic variation and antibody dynamics, aiding our understanding of antibody evolution.