Hydrophobic core malleability of a de novo designed three-helix bundle protein

De novo protein design provides a tool for testing the principles that stab ilize the structures of proteins. Recently, we described the design and str ucture determination of alpha D-3, a three-helix bundle protein with a well -packed hydrophobic core. Here, we test the malleability and adaptability o f this protein's structure by mutating a small, Ala residue (A60) in its co re to larger, hydrophobic side-chains, Leu and lie. Such changes introduce strain into the structures of natural proteins, and therefore generally des tabilize the native state. By contrast, these mutations were slightly stabi lizing (similar to1.5 kcal mol(-1)) to the tertiary structure of alpha D-3. The value of DeltaC(p) for unfolding of these mutants was not greatly affe cted relative to wild-type, indicating that the change in solvent accessibi lity for unfolding was similar. However, two-dimensional heteronuclear sing le quantum coherence spectra indicate that the protein adjusts to the intro duction of steric bulk in different ways. AGOL-alpha D-3 showed serious ero sion in the dispersion of both the amide backbone as well as the side-chain methyl chemical shifts. By contrast, A60I-alpha D-3 showed excellent dispe rsion of the backbone resonances, and selective changes in dispersion of th e aliphatic side-chains proximal to the site of mutation. Together, these d ata suggest that alpha D-3, although folded into a unique three-dimensional structure, is nevertheless more malleable and flexible than most natural, native proteins. (C) 2001 Academic Press.