Conformational changes accompanying the dissociation and association of immunoglobulin-G subunits

Abstract

The circular dichroism spectra, between 210 and 320 nm, of human pooled IgG, two IgG myeloma proteins and their isolated heavy and light chains have been determined. Below 250 nm all spectra are dominated by a negative band at 217 nm, probably due to β-structure. Above 250 nm the spectra are complex and indicate the presence of several overlapping side-chain transitions. Three different samples of pooled IgG give similar spectra as do their heavy chains and light chains. However, the heavy chains differ significantly from the light chains. The myeloma protein spectra differ from the pooled IgG and from each other, probably reflecting variable region conformational differences. Pooled IgG and one of the myeloma proteins shows a positive band at 235 nm, contributed by the light chain and shown to be probably due to a tyrosine transition on the basis of titration data. Algebraic addition of the subunit spectra did not yield the spectrum of the parent IgG indicating unique conformational features associated with the intact protein. The differences suggest a greater contribution of certain light chain transitions in native IgG than expected. The 235-nm tyrosine band is considerably enhanced as are other bands, probably due to tryptophan residues. There is evidence of other residues experiencing altered environments on subunit interaction but assignment of these changes to either subunit or to specific side-chains is difficult. The 217-nm band is reduced in intensity in native IgG when compared to the subunits suggesting secondary structure changes. Recombination of the myeloma subunits results in the complete recovery of the native conformation. This does not occur when pooled IgG subunits are recombined. A partial recovery takes place but differences between the native and recombined proteins are apparent throughout the wavelength range. Of particular interest is the failure to fully recover the 235-nm light chain tyrosine band.