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

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Publishing Institution:IRC-Library, Information Resource Center der Jacobs University Bremen
Granting Institution:Jacobs Univ.
Author:RAGHAVENDRA ANJANAPPA
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
Language:English
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

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