Generation and crystallization of empty disulfide-stabilized MHC class I proteins

  • MHC (Major Histocompatibility complex) class I molecules are membrane proteins that bind intracellular peptides of eight to ten amino acids, bring them to the surface, and present them to cytotoxic T cells of the immune system. MHC class I antigen presentation is critical to protect vertebrates against pathogens. In vitro folding of MHC class I molecules, which is performed with the purpose of studying MHC class I structure and peptide binding as well as for the preparation of recombinant proteins for the detection of T cells, is assisted by adding a specific peptide to occupy the binding groove and form a stable peptide-MHC (pMHC) complex. This process is slow and must be started anew for a different peptide of interest. However, in vitro folding of class I molecules without peptide is difficult to impossible to achieve because empty class I molecules are conformationally unstable and prone to denaturation. This delays the parallel production of multiple recombinant pMHC class I molecules. Therefore, we have developed a novel method to produced empty MHC class I molecules that are stable for subsequent therapeutic applications. Previously, our group has shown that small molecules, such as the dipeptide GM, can be used to fold class I molecules into a peptide-receptive conformation. We use these dipeptides in conjunction with a stabilized HLA-A*02:01. The additional Y84C/A139C disulfide bond links the α1/α2 helices in the F pocket region of the binding groove. In silico, molecular dynamics simulations show that this new disulfide bond, once formed, stabilizes the peptide-binding groove just like a full-length peptide. Disulfide stabilized HLA-A*02:01 can be folded with dipeptides that are subsequently removed during the purification steps. This results in empty MHC class I molecules that are stable in solution, are freeze-thaw compatible, and bind full-length peptides with rapid kinetics. Thus, the disulfide-stabilized empty class I molecules can be loaded directly

Download full text

Cite this publication

  • Export Bibtex
  • Export RIS

Citable URL (?):

Search for this publication

Search Google Scholar Search Catalog of German National Library Search OCLC WorldCat Search Catalog of GBV Common Library Network Search Catalog of Jacobs University Library Search Bielefeld Academic Search Engine
Meta data
Publishing Institution:IRC-Library, Information Resource Center der Jacobs University Bremen
Granting Institution:Jacobs Univ.
Referee:Sebastian Springer, Charlotte Uetrecht, Hector Marcelo Fernández Lahore
Advisor:Sebastian Springer
Persistent Identifier (URN):urn:nbn:de:gbv:579-opus-1009277
Document Type:PhD Thesis
Date of Successful Oral Defense:2020/06/16
Date of First Publication:2020/07/28
Academic Department:Life Sciences & Chemistry
PhD Degree:Biochemistry
Focus Area:Health
Call No:2020/10

$Rev: 13581 $