Hydrophobic Core Malleability of a De Novo Designed Three-helix Bundle Protein

Abstract

De novo protein design provides a tool for testing the principles that stabilize the structures of proteins. Recently, we described the design and structure determination of α 3D, a three-helix bundle protein with a well-packed hydrophobic core. Here, we test the malleability and adaptability of this protein's structure by mutating a small, Ala residue (A60) in its core to larger, hydrophobic side-chains, Leu and Ile. Such changes introduce strain into the structures of natural proteins, and therefore generally destabilize the native state. By contrast, these mutations were slightly stabilizing (∼1.5 kcal mol −1) to the tertiary structure of α 3D. The value of Δ C p for unfolding of these mutants was not greatly affected relative to wild-type, indicating that the change in solvent accessibility for unfolding was similar. However, two-dimensional heteronuclear single quantum coherence spectra indicate that the protein adjusts to the introduction of steric bulk in different ways. A60L-α 3D showed serious erosion in the dispersion of both the amide backbone as well as the side-chain methyl chemical shifts. By contrast, A60I-α 3D showed excellent dispersion of the backbone resonances, and selective changes in dispersion of the aliphatic side-chains proximal to the site of mutation. Together, these data suggest that α 3D, although folded into a unique three-dimensional structure, is nevertheless more malleable and flexible than most natural, native proteins.