Analysis of antibody folding and assembly

• Applicant: Professor Dr. Johannes Buchner
• Subject Area: Biochemistry
• Term: from 2011 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 204410927

To study protein folding, dynamics and association, antibodies are ideal model systems. The most common class of antibodies, IgGs, are comprised of two light chains and two heavy chains. In comparison to the classical IgG molecule, the IgM polymer, composed of “IgG-like” H2L2 subunits, is present in solution either as a pentamer (µ2L2)5-J in the presence of the protein J-chain, or as a hexamer (µ2L2)6 in the absence of J-chain. IgA, another immunoglobulin subtype, assembles to dimers in the presence of J-chain. During B-cell development, the quality control of the heavy chains relies on the interaction with the surrogate light chain (SLC) which consists of two proteins. The endoplasmic chaperone BiP plays an important role in the structure formation of antibodies. However, its precise role in IgM and IgA assembly as well as SLC function is still unclear.The project builds up on the advances achieved during the current funding period. The following key issues will be addressed:-Analysis of the mechanism of the oligomerization of IgM and IgA including the function of J-chain in this process: we want to understand how a short C-terminal peptide sequence (tail piece) in IgM can induce a precise geometry leading to the formation of dimers which finally assemble to hexameric protein complexes and how the J-chain can be covalently incorporated in the IgM assembly.-Structure, assembly and function of the SLC: our aim is analyze the complex assembly of this hetero-dimer and specifically determine the role of the unstructured extensions of the two proteins called unique regions in the assembly and function of the SLC.-The BiP-substrate interaction and its role in the assembly of Ig complexes: to define BiP’s role in these processes we want to map its binding sites in the tail piece and in SLC. This will allow us to make use of variants lacking BiP interaction sites using in vivo assembly experiments in comparison to the wildtype proteins.The project combines biochemical and biophysical in vitro approaches with cell-based experiments to obtain a comprehensive picture of the underlying mechanisms.

• DFG Programme: Research Grants