The present work investigates the influence of electrostatic surface potential distribution of monoclonal antibodies (MAbs) on intermolecular interactions and viscosity. Electrostatic models suggest MAb-1 has a less uniform surface charge distribution than MAb-2. The patches of positive and negative potential on MAb-1 are predicted to favor intermolecular attraction, even in the presence of a small net positive charge. Consistent with this expectation, MAb-1 exhibits a negative second virial coefficient (B₂₂), an increase in static structure factor, S((q→0)), and a decrease in hydrodynamic interaction parameter, H((q→0)), with increase in MAb-1 concentration. Conversely, MAb-2 did not show such heterogeneous charge distribution as MAb-1 and hence favors intermolecular repulsion (positive B₂₂), lower static structure factor, S((q→0)), and repulsion induced increase in momentum transfer, H((q→0)), to result in lower viscosity of MAb-2. Charge swap mutants of MAb-1, M-5 and M-7, showed a decrease in charge asymmetry and concomitantly a loss in self-associating behavior and lower viscosity than MAb-1. However, replacement of charge residues in the sequence of MAb-2, M-10, did not invoke charge distribution to the same extent as MAb-1 and hence exhibited a similar viscosity and self-association profile as MAb-2.

The solution pH dependent measured dipole moments of MAb1 appears to be in line with the observed intermolecular interactions and viscosity behavior suggesting that dipole-dipole interaction plays an important role in governing the high concentration solution behavior of this MAb.

Internalization of G-protein coupled receptors is mediated by phosphorylation of the C-terminus, followed by binding with the cytosolic protein arrestin. To explore structural factors that may play a role in internalization of cannabinoid receptor 1 (CB1), we utilize a phosphorylated peptide derived from the distal C-terminus of CB1 (CB15P454-473). Complexes formed between the peptide and human arrestin-2 (wt-arr21-418) were compared to those formed with a truncated arrestin-2 mutant (tr-arr21-382) using isothermal titration calorimetry and nuclear magnetic resonance spectroscopy. The penta-phosphopeptide CB15P454-473 adopts a helix-loop conformation, whether binding to full-length arrestin-2 or its truncated mutant. This structure is similar to that of a hepta-phosphopeptide, mimicking the distal segment of the rhodopsin C-tail (Rh7P330-348), binding to visual arrestin, suggesting that this adopted structure bears functional significance. Isothermal titration calorimetry (ITC) experiments show that the CB15P454-473 peptide binds to tr-arr21-382 with higher affinity than to the full-length wt-arr21-418. As the observed structure of the bound peptides is similar in either case, we attribute the increased affinity to a more exposed binding site on the N-domain of the truncated arrestin construct. The transferred nOe data from the bound phosphopeptides are used to predict a model describing the interaction with arrestin, using the data driven HADDOCK docking program. The truncation of arrestin-2 provides scope for positively charged residues in the polar core of the protein to interact with phosphates present in the loop of the CB15P454-473 peptide.

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