title: Structural dynamics of a single-chain Fv antibody against (4-hydroxy-3-nitrophenyl)acetyl.
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.
authors.affiliation: Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, 1-5 Hangi-cho, Shimogamo, Sakyo-ku, Kyoto 606-8522, Japan.
authors.forename: Yusui
authors.initials: Y
authors.lastname: Sato
authors.affiliation: Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, 1-5 Hangi-cho, Shimogamo, Sakyo-ku, Kyoto 606-8522, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Tanaka
authors.affiliation: Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, 1-5 Hangi-cho, Shimogamo, Sakyo-ku, Kyoto 606-8522, Japan.
authors.forename: Satomi
authors.initials: S
authors.lastname: Inaba
authors.affiliation: Japan Synchrotron Radiation Research Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5198, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Sekiguchi
authors.affiliation: Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Maruno
authors.affiliation: Graduate School of Frontier Sciences, The University of Tokyo, Kashiwanoha, Kashiwa, Chiba 277-8561, Japan.
authors.forename: Yuji C
authors.initials: YC
authors.lastname: Sasaki
authors.affiliation: Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan.
authors.forename: Harumi
authors.initials: H
authors.lastname: Fukada
authors.affiliation: Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Kobayashi
authors.affiliation: Research Institute for Biological Sciences (RIBS), Tokyo University of Science, 2669 Yamazaki, Noda, Chiba 278-0022, Japan.
authors.forename: Takachika
authors.initials: T
authors.lastname: Azuma
authors.affiliation: Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, 1-5 Hangi-cho, Shimogamo, Sakyo-ku, Kyoto 606-8522, Japan. Electronic address: oda@kpu.ac.jp.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Oda
mesh_terms: D008958:Models, Molecular; D009596:Nitrophenols / Q000276:immunology*; D010648:Phenylacetates / Q000276:immunology*; D000072417:Protein Domains; D055550:Protein Stability; D017433:Protein Structure, Secondary; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000276:immunology* / Q000302:isolation & purification; D013696:Temperature
chemical_list: D009596:Nitrophenols; D010648:Phenylacetates; D057127:Single-Chain Antibodies; C026242:4-hydroxy-5-nitrophenyl acetic acid
keywords: Antigen binding; Single-molecule analysis; Thermal stability