ABSTRACT
The C(H)3 domain of antibodies is characterized by two antiparallel beta-sheets forming a disulfide-linked sandwich-like structure. At acidic pH values and low ionic strength, C(H)3 becomes completely unfolded. The addition of salt transforms the acid-unfolded protein into an alternatively folded state exhibiting a characteristic secondary structure. The transition from native to alternatively folded C(H)3 is a fast reaction. Interestingly, this reaction involves the formation of a defined oligomer consisting of 12-14 subunits. Association is completely reversible and the native dimer is quantitatively reformed at neutral pH. This alternatively folded protein is remarkably stable against thermal and chemical denaturation and the unfolding transitions are highly cooperative. With a t(m) of 80 degrees C, the stability of the alternatively folded state is comparable to that of the native state of C(H)3. The defined oligomeric structure of C(H)3 at pH 2 seems to be a prerequisite for the cooperative unfolding transitions.
Subject(s)
Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/metabolism , Immunoglobulin Constant Regions/chemistry , Immunoglobulin Constant Regions/metabolism , Acids/pharmacology , Animals , Anions/pharmacology , Calorimetry, Differential Scanning , Chromatography, Gel , Circular Dichroism , Hydrogen-Ion Concentration , Kinetics , Light , Mice , Molecular Weight , Osmolar Concentration , Protein Denaturation/drug effects , Protein Folding , Protein Structure, Quaternary/drug effects , Protein Structure, Tertiary/drug effects , Protein Subunits , Salts/pharmacology , Scattering, Radiation , Solvents , Temperature , Thermodynamics , UltracentrifugationABSTRACT
Immunoglobulin heavy chain binding protein (BiP), a member of the Hsp70 chaperone family, and the oxidoreductase protein-disulfide isomerase (PDI) play an important role in the folding and oxidation of proteins in the endoplasmic reticulum. However, it was not clear whether both cooperate in this process. We show here that BiP and PDI act synergistically in the in vitro folding of the denatured and reduced Fab fragment. Several ATP-dependent cycles of binding, release, and rebinding of the unfolded antibody chains by BiP are required for efficient reactivation. Our data suggest that in the absence of BiP unfolded antibody chains collapse rapidly upon refolding, rendering cysteine side chains inaccessible for PDI. BiP binds the unfolded polypeptide chains and keeps them in a conformation in which the cysteine residues are accessible for PDI. These findings support the idea of a network of folding helper proteins in the endoplasmic reticulum, which makes this organelle a dedicated protein-processing compartment.
