title: Computational design and biophysical characterization of aggregation-resistant point mutations for γD crystallin illustrate a balance of conformational stability and intrinsic aggregation propensity.
abstract: γD crystallin is a natively monomeric eye-lens protein that is associated with hereditary juvenile cataract formation. It is an attractive model system as a multidomain Greek-key protein that aggregates through partially folded intermediates. Point mutations M69Q and S130P were used to test (1) whether the protein-design algorithm RosettaDesign would successfully predict mutants that are resistant to aggregation when combined with informatic sequence-based predictors of peptide aggregation propensity and (2) how the mutations affected relative unfolding free energies (ΔΔG(un)) and intrinsic aggregation propensity (IAP). M69Q was predicted to have ΔΔG(un) ≫ 0, without significantly affecting IAP. S130P was predicted to have ΔΔG(un) ∼ 0 but with reduced IAP. The stability, conformation, and aggregation kinetics in acidic solution were experimentally characterized and compared for the variants and wild-type (WT) protein using circular dichroism and intrinsic fluorescence spectroscopy, calorimetric and chemical unfolding, thioflavin-T binding, chromatography, static laser light scattering, and kinetic modeling. Monomer secondary and tertiary structures of both variants were indistinguishable from WT, while ΔΔG(un) > 0 for M69Q and ΔΔG(un) < 0 for S130P. Surprisingly, despite being the least conformationally stable, S130P was the most resistant to aggregation, indicating a significant decrease of its IAP compared to WT and M69Q.
authors.affiliation: Department of Chemical Engineering, University of Delaware, Newark, Delaware 19716, United States.
authors.forename: Erinc
authors.initials: E
authors.lastname: Sahin
authors.forename: Jacob L
authors.initials: JL
authors.lastname: Jordan
authors.forename: Michelle L
authors.initials: ML
authors.lastname: Spatara
authors.forename: Andrea
authors.initials: A
authors.lastname: Naranjo
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Costanzo
authors.forename: William F
authors.initials: WF
authors.lastname: Weiss
authors.forename: Anne Skaja
authors.initials: AS
authors.lastname: Robinson
authors.forename: Erik J
authors.initials: EJ
authors.lastname: Fernandez
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Roberts
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D017076:Computer-Aided Design; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D017354:Point Mutation; D011487:Protein Conformation; D017510:Protein Folding; D055550:Protein Stability; D013816:Thermodynamics; D038222:gamma-Crystallins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: C504903:CRYGD protein, human; D038222:gamma-Crystallins