title: Mutation choice to eliminate buried free cysteines in protein therapeutics.
abstract: Buried free-cysteine (Cys) residues can contribute to an irreversible unfolding pathway that promotes protein aggregation, increases immunogenic potential, and significantly reduces protein functional half-life. Consequently, mutation of buried free-Cys residues can result in significant improvement in the storage, reconstitution, and pharmacokinetic properties of protein-based therapeutics. Mutational design to eliminate buried free-Cys residues typically follows one of two common heuristics: either substitution by Ser (polar and isosteric), or substitution by Ala or Val (hydrophobic); however, a detailed structural and thermodynamic understanding of Cys mutations is lacking. We report a comprehensive structure and stability study of Ala, Ser, Thr, and Val mutations at each of the three buried free-Cys positions (Cys16, Cys83, and Cys117) in fibroblast growth factor-1. Mutation was almost universally destabilizing, indicating a general optimization for the wild-type Cys, including van der Waals and H-bond interactions. Structural response to Cys mutation characteristically involved changes to maintain, or effectively substitute, local H-bond interactions-by either structural collapse to accommodate the smaller oxygen radius of Ser/Thr, or conversely, expansion to enable inclusion of novel H-bonding solvent. Despite the diverse structural effects, the least destabilizing average substitution at each position was Ala, and not isosteric Ser.
authors.affiliation: Department of Biomedical Sciences, Florida State University, Tallahassee, Florida, 32306-4300.
authors.forename: Xue
authors.initials: X
authors.lastname: Xia
authors.forename: Liam M
authors.initials: LM
authors.lastname: Longo
authors.forename: Michael
authors.initials: M
authors.lastname: Blaber
mesh_terms: D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry / Q000235:genetics*; D016220:Fibroblast Growth Factor 1 / Q000737:chemistry / Q000235:genetics* / Q000627:therapeutic use*; D006801:Humans; D009154:Mutation / Q000235:genetics*; D017433:Protein Structure, Secondary
chemical_list: D016220:Fibroblast Growth Factor 1; D003545:Cysteine
keywords: crystal structure; fibroblast growth factor-1; hydrogen bond; protein aggregation; protein engineering; protein half-life; protein structure; stability; thermodynamics