Biophysical characterization and molecular simulation of electrostatically driven self-association of a single-chain...

Colloidal protein-protein interactions (PPI) are often expected to impact key behaviors of proteins in solution, such as aggregation rates and mechanisms, aggregate structure, protein solubility, and solution viscosity. PPI of an anti-fluorescein single chain antibody variable fragment (scFv) were characterized experimentally at low to intermediate ionic strength using a combination of static light scattering and sedimentation equilibrium ultracentrifugation. Surprisingly, the results indicated that interactions were strongly net-attractive and electrostatics promoted self-association. Only repulsive interactions were expected based on prior work and calculations based a homology model of a related scFv crystal structure. However, the crystal structure lacks the charged, net-neutral linker sequence. PyRosetta was used to generate a set of scFv structures with different linker conformations, and coarse-grained Monte Carlo simulations were used to evaluate the effect of different linker configurations via second osmotic virial coefficient (B₂₂ ) simulations. The results show that the configuration of the linker has a significant effect on the calculated B₂₂ values, and can result in strong electrostatic attractions between oppositely charged residues on the protein surface. This is particularly relevant for development of non-natural antibody products, where charged linkers and other loop regions may be prevalent. The results also provide a preliminary computational framework to evaluate the effect of unstructured linkers on experimental protein-protein interaction parameters such as B₂₂ .