The effect of charge mutations on the stability and aggregation of a human single chain Fv fragment

Abstract

a b s t r a c tThe aggregation propensities for a series of single-chain variable fragment (scFv) mutant proteins containing supercharged sequences, salt bridges and lysine/arginine-enriched motifs were characterised as a function of pH and ionic strength to isolate the electrostatic contributions. Recent improvements in aggregation predictors rely on using knowledge of native-state protein-protein interactions. Consistent with previous findings, electrostatic contributions to native protein-protein interactions correlate with aggregate growth pathway and rates. However, strong reversible self-association observed for selected mutants under native conditions did not correlate with aggregate growth, indicating 'sticky' surfaces that are exposed in the native monomeric state are inaccessible when aggregates grow. We find that even though similar native-state protein-protein interactions occur for the arginine and lysine-enriched mutants, aggregation propensity is increased for the former and decreased for the latter, providing evidence that lysine suppresses interactions between partially folded states under these conditions. The supercharged mutants follow the behaviour observed for basic proteins under acidic conditions; where excess net charge decreases conformational stability and increases nucleation rates, but conversely reduces aggregate growth rates due to increased intermolecular electrostatic repulsion. The results highlight the limitations of using conformational stability and native-state protein-protein interactions as predictors for aggregation propensity and provide guidance on how to engineer stabilizing charged mutations.

Electrostatic interactions drive native‐like aggregation of human alanine:glyoxylate aminostransferase

“…Most studies on the pathogenesis of protein deficits interpret the aggregation propensity of disease-causing variants as being due to the population of folding intermediates showing sticky hydrophobic patches (misfolding-based aggregation) [55,56]. Our results, along with those on other proteins of pharmacological interest [2,17,57], highlight that a deep analysis of the electrostatic surface should be also carried out to provide insights into possible alterations of the surface charge anisotropy that could promote electrostatic aggregation of a protein once folded (native-like aggregation). In particular, by combining the data reported here with those of previous reports [26,28,[58][59][60], we delineated a more comprehensive picture describing AGT aggregation.…”

“…Among them, the association of protein folding intermediates exposing hydrophobic patches is a well-known mechanism [13]. However, theoretical [14] and experimental [15][16][17] evidences suggest that electrostatic forces can play an important role in inducing or enhancing protein aggregation as well as in stabilizing protein-protein complexes, by influencing binding rate and/or equilibrium [18]. The process of electrostatic aggregation is based on the attraction between complementary charged domains of a protein surface, and it usually decreases at high ionic strength, due to screening effects [15].…”

Charge and hydrophobicity are key features in sequence-trained machine learning models for predicting the biophysical properties of clinical-stage antibodies

“…Using the Goyon et al (2017) dataset we studied the importance of CDR (complementaritydetermining regions) length and aromatic content for predicting behaviour on HIC (hydrophobic interaction columns) (Hebditch et al, 2018). Lastly, we have developed tools for predicting the presence of hydrophobic and charged patches as well as fold state stability (Hebditch and Warwicker, 2019) from crystal structures available in the PDB (Berman et al, 2007) and applied these observations to experimental work (Austerberry et al, 2017). After the release of the Jain et al…”

Computational investigation of protein surface polarity and pH-dependence: application to antibodies shows that antibody-antigen interfaces tend to be relatively polar

“…Much research has focused on the role of anisotropic surface patches of charge and hydrophobicity in causing reversible and irreversible protein association (Yadav et al, 2012;Perchiacca et al, 2012;Chow et al, 2016;Li et al, 2016;Roberts et al, 2014b;Austerberry et al, 2017). This experimental work has led to efforts in predicting protein surface patches in silico.…”