De novo design of a four-fold symmetric TIM-barrel protein with atomic-level accuracy.

Despite efforts for over 25 years, de novo protein design has not succeeded in achieving the TIM-barrel fold. Here we describe the computational design of four-fold symmetrical (ß/α)8 barrels guided by geometrical and chemical principles. Experimental characterization of 33 designs revealed the importance of side chain-backbone hydrogen bonds for defining the strand register between repeat units. The X-ray crystal structure of a designed thermostable 184-residue protein is nearly identical to that of the designed TIM-barrel model. PSI-BLAST searches do not identify sequence similarities to known TIM-barrel proteins, and sensitive profile-profile searches indicate that the design sequence is distant from other naturally occurring TIM-barrel superfamilies, suggesting that Nature has sampled only a subset of the sequence space available to the TIM-barrel fold. The ability to design TIM barrels de novo opens new possibilities for custom-made enzymes.

Identification of protein scaffolds for enzyme design using scaffold selection.

The identification of suitable protein structures that can serve as scaffolds for the introduction of catalytic residues is crucial for the design of new enzymes. Here we describe how the automated and rapid scaffold search program ScaffoldSelection can be used to find the best starting points, namely protein structures that are most likely to tolerate the introduction and promote the proper formation of a specific catalytic motif.

Computational protein design of ligand binding and catalysis.

The vision of custom-made proteins by computation appears closer than ever. Computational methods have advanced rapidly in recent years and proteins have been designed to catalyze new reactions. A number of second-generation enzyme designs analyzed possible bottlenecks and started tackling emergent problems. Detailed experimental analysis combined with structure determination and molecular dynamics simulations as well as design optimization with directed evolution techniques have led to important insights. While ligand recognition seems to be particularly problematic, new approaches focus on this design aspect and promising improvements have been made.