Class I major histocompatibility complex proteins play a pivotal role in adaptive immunity by displaying epitopic peptides to CD8+ T cells. Dedicated molecular chaperones of MHC-I, tapasin and TAP-binding protein-related (TAPBPR), promote the selection of immunogenic antigens from a large pool of intracellular peptides. These chaperones stabilize nascent MHC-I, allowing the loading of the peptides to the empty peptide binding groove, the exchange of low-to-moderate affinity for high-affinity peptides (editing), and the momentary capture and release of peptides from a large peptide pool (proofreading). Interactions of the chaperoned MHC-I molecules with incoming peptides are transient in nature, and as a result, the precise antigen proofreading mechanism remains elusive. Therefore, this thesis work aims to understand the peptide proofreading mechanism of MHC-I by capturing and examining the biologically relevant MHC-I/TAPBPR peptide proofreading complex. Here, I demonstrate and characterize the direct interactions between TAPBPR and a repertoire of human MHC-I allotypes, revealing the polymorphic residues on the MHC-I/TAPBPR interacting surfaces can be engineered for altered chaperone recognition. Using deep mutational scanning of TAPBPR expressed at the plasma membrane, several gain-of-function TAPBPR mutants were identified that could significantly enhance the peptide exchange function of multiple disease-relevant MHC-I allotypes. I also adapt a structure-guided approach to engineering conformationally stable MHC-I molecules, named “open MHC-I,” by introducing a disulfide bond bridging conserved sites across the polymorphic MHC-I heavy chain and invariable light chain β2m interface (G120C and H31C). I show that the interchain disulfide bond increases the thermostability of molecules loaded with low- and moderate-affinity peptide cargos. Finally, we leverage one of the engineered high-fidelity TAPBPR variants, TAPBPRHiFi, containing three mutations (S104F, K211L, and R270Q) at the MHC-I binding site and conformationally stabilized open MHC-I to determine the solution structure of the human antigen proofreading complex of MHC-I/TAPBPR bound to a peptide decoy by cryogenic electron microscopy at an average resolution of 3.0 Å. Antigen proofreading is mediated by transient interactions formed between the nascent peptide binding groove with the N-terminal P2/P3 peptide anchors. Altogether, this work has important ramifications for understanding the selection of immunogenic epitopes for T cell screening and vaccine design applications.