Folding of an antibody variable domain in two functional conformations in vitro: calorimetric and spectroscopic study of the anti-ferritin antibody VL domain

Abstract

Understanding refolding pathways of recombinant antibody fragments is essential for efficient production of these proteins of high biomedical significance. The recombinant VL domain of mouse anti-human ferritin antibody F11 formed two distinct functional conformations obtained by refolding from bacterial inclusion bodies using two different procedures. Involvement of a dialysis step at pH 2-3 resulted in the VL-1 conformation with fluorescence of the highly conserved Trp-35 residue quenched by the spatially proximal disulfide bond. This conformation was identical to the 'native' VL domain folded in host cells and purified from the cytoplasm. In the absence of the acidic dialysis step, the VL domain adopted a previously unreported conformation, VL-2, that demonstrated prominent fluorescence due to a local structural disorder around Trp-35. Furthermore, VL-2 showed changes in secondary structure and significantly lower stability as determined by differential scanning calorimetry and denaturant-induced unfolding. While more flexible VL-2 binds human ferritin both in solution and after surface adsorption of the antibody domain, the VL-1 conformer needs an adsorption-induced conformational change to allow the access of ferritin to the antigen-binding site. Noteworthy, the two macroscopic conformations constitute kinetically trapped dimers and do not interconvert at elevated temperatures (3 weeks at 37 degrees C or 15 min at 60 degrees C), which indicates a high energetic barrier between them. As a major finding, this paper provides the first description for two stable and functional conformations of an antibody domain.

Elongation of the C-terminal domain of an anti-amyloid β single-chain variable fragment increases its thermodynamic stability and decreases its aggregation tendency

“…Also in consonance, the initial ellipticity minimum at 230 nm in the CD spectra, characteristic of some V L domains, 20,21 progressively disappears during the first transition. These observations pointed out the V L domain of scFv-h3D6 as being the preferential target for protein stability redesign.…”

“…13 These anomalies are contributions from the aromatic or cystinyl side-chains within the far-UV. 18 The minimum at 230 nm was also reported for an IgG1-Fc 19 and described for some V L domains, 20,21 the latter attributed to the interaction of the aromatic residues with the conserved Trp35.…”

Development of a high-performance immunolatex based on “soft landing” antibody immobilization mechanism

“…S2 in Supporting Information). The far-and near-ultraviolet CD are sensitive to variations in secondary and tertiary protein structures, respectively, and fluorescence intensity and maximum wavelength are also suitable indicators of changes in tertiary structure [31]. These results show that the anti-hFT structure did not change within 60 min at different pH levels.…”

Decreased Amyloidogenicity Caused by Mutational Modulation of Surface Properties of the Immunoglobulin Light Chain BRE Variable Domain

“…The peaks at 225 nm probably reflect the presence of aromatic clusters in the native protein structures. 34 These data indicate that all the proteins prepared in this study adopt conformations similar to those of the wild-type proteins at low temperatures. In contrast, at 60 °C, the proteins exhibited spectra typical of the unfolded state with minima around 205 nm.…”