Stabilization of GroEL minichaperones by core and surface mutations.

We report the crystal structures of two hexa-substituted mutants of a GroEL minichaperone that are more stable than wild-type by 7.0 and 6.1 kcal mol(-1). Their structures imply that the increased stability results from multiple factors including improved hydrophobic packing, optimised hydrogen bonding and favourable structural rearrangements. It is commonly believed that protein core residues are immutable and generally optimized for energy, while on the contrary, surface residues are variable and hence unimportant for stability. But, it is now becoming clear that mutations of both core and surface residues can increase protein stability, and that protein cores are more flexible and thus more tolerant to mutation than expected. Sequence comparison of homologous proteins has provided a way to pinpoint the residues that contribute constructively to stability and to guide the engineering of protein stability. Stabilizing mutations identified by this approach are most frequently located at protein surfaces but with a few found in protein cores. In the latter case, local flexibility in the hydrophobic core is the key factor that allows the energetically favourable burial of larger hydrophobic side-chains without undue energetic penalties from steric clashes.