Analysis of thermostability for seven Phe to Ala and six Pro to Gly mutants in the Fab constant region of adalimumab.
J Biochem
; 174(4): 345-353, 2023 Sep 29.
Article
en En
| MEDLINE
| ID: mdl-37390406
To identify amino acids that play important roles in the structural stability of Fab, seven phenylalanine residues in the Fab constant region of the therapeutic antibody adalimumab were subjected to alanine mutagenesis. Six Fab mutants, H:F130A, H:F154A, H:F174A, L:F118A, L:F139A and L:F209A, showed decreased thermostability compared with wild-type Fab. In contrast, the Tm for the L:F116A mutant was 1.7°C higher than that of wild-type Fab, indicating that the F116 residue was unfavorable for Fab thermostability. Six proline mutants, H:P131G, H:P155G, H:P175G, L:P119G, L:P120G and L:P141G, were also prepared to investigate the effect of proline residues adjacent to mutated phenylalanine residues. The thermostability of the H:P155G and L:P141G mutants in particular was significantly reduced, with decreases in Tm of 5.0 and 3.0°C, respectively, compared with wild-type Fab. The H:P155 and L:P141 residues have a cis conformation, whereas the other mutated proline residues have a trans conformation. H:P155 and L:P141 had stacking interactions with the H:F154 and L:Y140, respectively, at the interface between the variable and constant regions. It is suggested that the interactions of the aromatic ring with a cis-form proline at the interface between the variable and constant regions is important for stability of Fab.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Fenilalanina
/
Alanina
Tipo de estudio:
Prognostic_studies
Idioma:
En
Revista:
J Biochem
Año:
2023
Tipo del documento:
Article
País de afiliación:
Japón
Pais de publicación:
Reino Unido