Fig. 2: Role of the disulfide bonds in the knob.
From: Mechanistic principles of an ultra-long bovine CDR reveal strategies for antibody design
a Cysteines and disulfide bond pattern in the knob NC-Cow1 (PDB:6OO0). b Immunoprecipitation of Expi293 supernatants after transient expression of Fab fragments followed by SDS-PAGE. Two independent experiments. c FUV CD spectra of the NC-Cow1 Fab wt compared to C → S mutants. d Binding of 100 nM NC-Cow1 Fab variants to immobilized HIV-1 Env protein in SPR. e Thermal unfolding of the NC-Cow1 Fab wt and mutants measured with DSC. f Molecular mass and eluting peaks of NC-Cow1 Fab wt and C → S mutants in SEC MALS. In c, d, e and f, black is the wildtype (wt) NC-Cow1 Fab, the remaining colors represent different NC-Cow1 Fab mutants with cysteine residues replaced by serine residues in the knob. Refer to a for the exact position of the mutations. g, h and i Comparative MD simulations of the Fv of NC-Cow1 wt (black) and variants with two (orange), one (magenta) or no (yellow) disulfide bonds in the knob. RMSD for the g framework, h the whole structure (knob plus the framework) and i the knob itself. A snapshot of the finally sampled knob structure is indicated.
