Technical Highlight - February 2013
Short description: Protein redesign via backbone perturbations enables efficient expansion of sequence diversity while maintaining fold and thermostability.
Most protein design algorithms are based on sidechain optimization rather than backbone changes, and often return sequences that are highly similar to that of the parent protein, limiting the diversity of sequence space explored. Kuhlman and colleagues now demonstrate that through flexible backbone protein design, the entire hydrophobic core of the CheA phosphotransferase — consisting of 38 residues — can be redesigned while maintaining its four-helix bundle fold. There was a considerable increase in thermostability, as reflected by the Tm of 142°C for the redesigned protein, compared with 91°C for wild-type CheA. The redesigned sequence shared no sequence identity with the core of the parent protein.
In defining the optimal sequence, the authors compared fixed backbone protocols with flexible backbone designs using Rosetta software. Sequences were scored based on the total Rosetta energy score, quality of packing, correct prediction of secondary structure, performance in ab initio folding experiments and deviation from wild-type sequence. The flexible backbone algorithms returned the most divergent sequences and outperformed fixed backbone procedures.
The authors then experimentally validated the top-scoring sequence by structure determination via NMR and X-ray crystallography. The structures were in close agreement with the in silico model (with an all-atom root mean squared deviation of 1.3 Å), confirming that only small-scale backbone perturbations (1–2 Å) were needed for the redesign. There was no simple correlation between the number of buried hydrophobic residues and changes in protein stability, and thus the remarkable thermostability of the redesigned CheA core remains unexplained. Previous studies have observed an increase in side-chain motion in redesigned proteins, presumably due to imperfect packing. These studies have not yet been performed for redesigned CheA. It will be interesting to see if it is more rigid or flexible than the wild-type protein.
Overall, flexible backbone redesign appears promising for enzyme stabilization and the redesign of ligand-binding proteins and protein-protein interfaces, during which certain residues can be constrained to specific conformations. The remaining protein can sample a wide sequence space without altering the desired three-dimensional fold or decreasing thermostability.
G.S. Murphy et al. Increasing sequence diversity with flexible backbone protein design: the complete redesign of a protein hydrophobic core.
Structure. 20, 1086-96 (2012). doi:10.1016/j.str.2012.03.026