Position-dependent effects of loop proline residues on Fab stability and aggregation

Proline residues play an important role in the structural stability of proteins because their cyclic side chains impose strong conformational constraints on the peptide backbone. However, the contribution of proline residues to the stability and physicochemical properties of antibody fragments remains unclear. In this study, we investigated the structural role of proline residues in the loop regions of the H-chain of the antigen-binding fragment (Fab) of adalimumab. To evaluate the contribution of native proline residues, three loop-region prolines (Pro14, Pro41, and Pro210) were individually substituted with glycine. Circular dichroism spectroscopy and antigen binding analysis showed that these substitutions did not significantly affect the secondary structure or antigen binding activity of Fab. In contrast, differential scanning calorimetry revealed position-dependent effects on thermal stability. H:P41G exhibited a pronounced decrease in melting temperature, whereas H:P14G showed a biphasic thermal transition, suggesting disruption of the cooperative unfolding of the variable and constant domains. Eight proline-introduced variants in the constant domain were designed based on sequence conservation among human antibody isotypes. Among them, H:K209P exhibited partial stabilization and thermal transition at a higher temperature than that exhibited by wild-type Fab. Notably, the K209P substitution also reduced aggregation under pH-shifted stress conditions. These findings demonstrate that loop-region proline residues contribute to Fab stability and interdomain cooperation, highlighting the importance of considering the surrounding structural features when introducing proline residues in antibody engineering.