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.

Increased protein solubility is known to correlate with an increase in the proportion of lysine over arginine residues. Previous work has shown that the aggregation propensity of a single-chain variable fragment (scFv) does not correlate with its conformational stability or native-state protein−protein interactions. Here, we test the hypothesis that aggregation is driven by the colloidal stability of partially unfolded states, studying the behavior of scFv mutants harboring single or multiple site-specific arginine to lysine mutations in denaturing buffers. In 6 M guanidine hydrochloride (GdmCl) or 8 M urea, repulsive protein−protein interactions were measured for the wild-type and lysineenriched (4RK) scFvs reflecting weakened short-range attractions and increased excluded volume. In contrast to the arginine-enriched mutant (7KR) scFv exhibited strong reversible association. In 3 M GdmCl, the minimum concentration at which the scFvs were unfolded, the hydrodynamic radius of 4RK remained constant but increased for the wild type and especially for 7KR. Studies of single-point arginine to lysine scFv mutants indicated that the observed aggregation propensity of arginine under denaturing conditions was nonspecific. Interestingly, one such swap generated a scFv with especially low aggregation rates under low/high ionic strengths and denaturing buffers; molecular modeling identified hydrogen bonding between the arginine side chain and main chain peptide groups, stabilizing the structure. The arginine/lysine ratio is not routinely considered in biopharmaceutical scaffold design or current amyloid prediction methods. This work therefore suggests a simple method for increasing the stability of a biopharmaceutical protein against aggregation.

The Tat system transports folded proteins across the bacterial plasma membrane, and in Escherichia coli preferentially transports correctly-folded proteins. Little is known of the mechanism by which Tat proofreads a substrate's conformational state, and in this study we have addressed this question using a heterologous single-chain variable fragment (scFv) with a defined structure. We introduced mutations to surface residues while leaving the folded structure intact, and also tested the importance of conformational flexibility. We show that while the scFv is stably folded and active in the reduced form, formation of the 2 intra-domain disulphide bonds enhances Tat-dependent export 10-fold, indicating Tat senses the conformational flexibility and preferentially exports the more rigid structure. We further show that a 26-residue unstructured tail at the C-terminus blocks export, suggesting that even this short sequence can be sensed by the proofreading system. In contrast, the Tat system can tolerate significant changes in charge or hydrophobicity on the scFv surface; substitution of uncharged residues by up to 3 Lys-Glu pairs has little effect, as has the introduction of up to 5 Lys or Glu residues in a confined domain, or the introduction of a patch of 4 to 6 Leu residues in a hydrophilic region. We propose that the proofreading system has evolved to sense conformational flexibility and detect even very transiently-exposed internal regions, or the presence of unfolded peptide sections. In contrast, it tolerates major changes in surface charge or hydrophobicity.

HighlightsAggregation of an exemplar therapeutic antibody fragment (scFv) enhances immunogenicity in vivo.Epitope mapping reveals immunogenicity is directed to a specific epitope in aggregate species.Molecular simulation demonstrates biophysical stress enhances epitope presentation.Protein aggregates have distinct immunological profiles to their native counterparts.