Major histocompatibility complex class I (MHC I) reports a cell's health status by presenting antigenic peptides inside its binding groove. However, MHC I binding grooves can differ largely in their plasticity, from binding grooves that are conformationally stable by themselves to those that require a high-affinity peptide to be bound to attain conformational stability. These latter MHC I alleles are dependent on the C-terminus of the peptide that stabilizes the F-pocket region of their binding grooves. It has remained unclear to what extent a peptide-MHC I complex (pMHC I) can tolerate the (intermittent) partial dissociation of high-affinity peptides, especially of the peptide's N-terminus. Using bias exchange umbrella sampling (BEUS), a technique to achieve enhanced sampling in molecular dynamics (MD) simulations, we obtained the free-energy profiles of the N-terminal dissociation of a respective high-affinity peptide from HLA-B*35:01 and HLA-B*44:02, two alleles on opposite ends of the scale regarding binding groove plasticity. The potential of mean force (PMF) for HLA-B*35:01 was calculated for two different sets of starting structures and is compared with a PMF obtained previously with a different force field to disentangle allele differences from force field and sampling effects. For both alleles, the free-energy profiles indicate that the peptide N-terminus dissociates in a substantial fraction of the pMHC I, suggesting that their crystal structures with fully bound peptides only partially capture the dynamic conformational ensemble of pMHC I in solution, and thus in the cell.