title: Modulating non-native aggregation and electrostatic protein-protein interactions with computationally designed single-point mutations.
abstract: Non-native protein aggregation is a ubiquitous challenge in the production, storage and administration of protein-based biotherapeutics. This study focuses on altering electrostatic protein-protein interactions as a strategy to modulate aggregation propensity in terms of temperature-dependent aggregation rates, using single-charge variants of human γ-D crystallin. Molecular models were combined to predict amino acid substitutions that would modulate protein-protein interactions with minimal effects on conformational stability. Experimental protein-protein interactions were quantified by the Kirkwood-Buff integrals (G22) from laser scattering, and G22 showed semi-quantitative agreement with model predictions. Experimental initial-rates for aggregation showed that increased (decreased) repulsive interactions led to significantly increased (decreased) aggregation resistance, even based solely on single-point mutations. However, in the case of a particular amino acid (E17), the aggregation mechanism was altered by substitution with R or K, and this greatly mitigated improvements in aggregation resistance. The results illustrate that predictions based on native protein-protein interactions can provide a useful design target for engineering aggregation resistance; however, this approach needs to be balanced with consideration of how mutations can impact aggregation mechanisms.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE 19716, USA.
authors.forename: C J
authors.initials: CJ
authors.lastname: O'Brien
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE 19716, USA.
authors.forename: M A
authors.initials: MA
authors.lastname: Blanco
authors.affiliation: Department of Chemical Engineering, University of Virginia, Charlottesville, VA 22903, USA.
authors.forename: J A
authors.initials: JA
authors.lastname: Costanzo
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE 19716, USA.
authors.forename: M
authors.initials: M
authors.lastname: Enterline
authors.affiliation: Department of Chemical Engineering, University of Virginia, Charlottesville, VA 22903, USA.
authors.forename: E J
authors.initials: EJ
authors.lastname: Fernandez
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE 19716, USA.|Department of Chemical and Biomolecular Engineering, Tulane University, New Orleans, LA 70118, USA.
authors.forename: A S
authors.initials: AS
authors.lastname: Robinson
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE 19716, USA.
authors.forename: C J
authors.initials: CJ
authors.lastname: Roberts
mesh_terms: D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D066329:Protein Aggregates; D060066:Protein Interaction Maps; D055550:Protein Stability; D055672:Static Electricity; D013696:Temperature; D038222:gamma-Crystallins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: C504903:CRYGD protein, human; D066329:Protein Aggregates; D038222:gamma-Crystallins
keywords: computational design; protein aggregation; protein engineering; protein–protein interactions