Structural dynamics of a single-chain Fv antibody against (4-hydroxy-3-nitrophenyl)acetyl.
Int J Biol Macromol
; 91: 151-7, 2016 Oct.
Article
em En
| MEDLINE
| ID: mdl-27222286
ABSTRACT
Protein structure dynamics are critical for understanding structure-function relationships. An antibody can recognize its antigen, and can evolve toward the immunogen to increase binding strength, in a process referred to as affinity maturation. In this study, a single-chain Fv (scFv) antibody against (4-hydroxy-3-nitrophenyl)acetyl, derived from affinity matured type, C6, was designed to comprise the variable regions of light and heavy chains connected by a (GGGGS)3 linker peptide. This scFv was expressed in Escherichia coli in the insoluble fraction, solubilized in the presence of urea, and refolded by stepwise dialysis. The correctly refolded scFv was purified, and its structural, physical, and functional properties were analyzed using analytical ultracentrifugation, circular dichroism spectrometry, differential scanning calorimetry, and surface plasmon resonance biosensor. Thermal stability of C6 scFv increased greatly upon antigen binding, due to favorable enthalpic contributions. Antigen binding kinetics were comparable to those of the intact C6 antibody. Structural dynamics were analyzed using the diffracted X-ray tracking method, showing that fluctuations were suppressed upon antigen binding. The antigen binding energy determined from the angular diffusion coefficients was in good agreement with that calculated from the kinetics analysis, indicating that the fluctuations detected at single-molecule level are well reflected by antigen binding events.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Fenilacetatos
/
Anticorpos de Cadeia Única
/
Nitrofenóis
Tipo de estudo:
Prognostic_studies
Idioma:
En
Revista:
Int J Biol Macromol
Ano de publicação:
2016
Tipo de documento:
Article