Proliferating cell nuclear antigen (PCNA) is a key regulatory protein in human DNA metabolism. PCNA forms a ring-like structure around the DNA that serves as a binding platform for a multitude of other proteins involved in DNA metabolism. Ataxia-telangiectasia-like disorder type 2 (ATLD2) is a neurodegenerative disease associated with impaired PCNA function. Recently, a mutation in the PCNA gene of ATLD2 patients was identified that encodes for the NM_002592.2(PCNA): c.443G > C(p.C148S) variant. However, the molecular effect of this mutation was unclear. Here we used non-equilibrium free (NEQ) energy calculations to predict the effect of this single amino acid mutation on PCNA stability and on the interaction of PCNA with one of its binding partner’s, p15. No change in binding affinity was predicted, while a significant decrease in the folding free energy was predicted for the PCNA^C148S variant. These results were validated experimentally by differential scanning fluorimetry and a FRET-based PCNA-p15 interaction assay. The experiments confirmed a reduced folding free energy of the variant, while no direct influence of the mutation on the PCNA-p15 interaction is detected. However, the lower folding free energy of the variant caused a time-dependent denaturation of PCNA^C148S. This provides a possible molecular explanation for the association of the PCNA^C148S variant with ATLD2. This study therefore underlines the effectiveness of NEQ alchemical free energy calculations in the analysis of disease conferring mutations.