Antiferritin single-chain Fv fragment is a functional protein with properties of a partially structured state: Comparison with the completely folded V-L domain

Differential scanning calorimetry and spectroscopic probes were applied to study folding and stability of the single-chain Fv fragment (scFv) of the a nti-human ferritin antibody F11 and its isolated variable light-chain (VL) domain. The scFv fragment followed variable heavy-chain domain (VH)-linker- V-L orientation and contained (Gly(4)Ser)(3) linker peptide. The two protei ns were produced in Escherichia coli and refolded from denaturant-solubiliz ed inclusion bodies. The isolated VL domain demonstrated a typical immunogl obulin fold with well-defined secondary and tertiary structure and was capa ble of binding human ferritin with K-a = 1.8 x 10(7) M-1, similar to 1/30 O f the affinity of the parent F11 antibody. Involvement of this VL domain in to the two-domain scFv fragment yielded a distorted secondary and significa ntly destabilized tertiary structure in which neither of the two constituen t domains attained complete folding. The thermal unfolding enthalpy of scFv F11 at pH 7.0 was as low as 5.0 J . g(-1) versus 16.3 J . g(-1) obtained f or the VL domain and 24.7 J . g(-1) for the parent F11 antibody (mouse IgG2 a subclass). Intrinsic fluorescence and near-ultraviolet circular dichroic (CD) spectra, and binding of the hydrophobic probe 8-anilino-1-naphthalene sulfonate, confirmed partial loss of tertiary interactions in scFv. The spe ctroscopic and calorimetric properties of scFv F11 under physiological cond itions are consistent with a model of a partially structured state with a d istorted beta-sheet as a secondary structure and partial loss of tertiary i nteractions, which closely resembles the alternatively folded A-state adopt ed by an immunoglobulin at pH 2-3 [Buchner, J., Renner, M., Lilie, H,, Hint , H.-J,, Jaenicke, R., Kiefhaber, T., and Rudolph, R. (1991) Biochemistry 3 0, 6922-6929]. However, scFv F11 demonstrated only an similar to 4-fold dec rease in the antigen-binding affinity (K-a = 1.3 x 10(8) M-1) versus the pa rent F11 antibody. The scFv fragment F11 provides the first description of a functional protein trapped under physiological conditions in a partially structured state. This state is either close to the native one in the antig en-binding affinity or, alternatively, initial weak binding of the antigeni c epitope induces folding of scFv F11 into a more structured conformation t hat generates relatively high affinity.