Computational Design and Biophysical Characterization of Aggregation-Resistant Point Mutations for γD Crystallin Illustrate a Balance of Conformational Stability and Intrinsic Aggregation Propensity

gamma D 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 infomatic sequence-based predictors of peptide aggregation propensity and (2) how the mutations affected relative unfolding free energies (Delta Delta G(un)) and intrinsic aggregation propensity (IAP). M69Q was predicted to have Delta Delta G(un) >> 0, without significantly affecting IAP. S130P was predicted to have Delta Delta G(un) similar to 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 Delta Delta G(un) > 0 for M69Q and Delta Delta G(un) < 0 for S130P. Surprisingly, despite being the least conformationally stable, S130P was the most resistant to aggregation, indicating a significant decrease of its TAP compared to WT and M69Q.