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Abstract

Comprehensive understanding of a protein fold is intertwined with successful design. Recent advances in designing de novo structures have shown that proteins can be designed for a few globular and heli- cal folds. However, designing all-b structures and barrels remains challenging because loops and intri- cate long range interactions that are important in these topologies are difficult to control. For designing novel catalysts, the (a/b)8 -barrel (or TIM-barrel) fold is one of the most important examples, for it is the most common topology for enzymes. For almost 30 year, attempts in designing de novo TIM barrel structures have all resulted in poorly folded proteins. Here we describe the successful design of a 4-fold symmetrical (a/b)8 barrel directly from geometrical and chemical principles. 22 designed variants with a wide range of stabilities from being molten globules to cooperatively folded proteins were experimentally characterized, and the results revealed the importance of sidechain-backbone hydrogen bonding for defining the characteristic a/b-barrel. The 184 residue TIM barrel structure is among the smallest TIM-barrels and has a fully-reversible melting temperature of 888C. The X-ray crystal structure shows atomic-level agreement with the design model. Despite this structural similarity, PSI- BLAST searches do not identify sequence similarities to known TIM-barrel proteins. More sensitive profile-profile searches suggest that the design is sufficiently distant from other native TIM-barrel superfamilies to be in a superfamily of its own, further implying that Nature has only sampled a subset of the sequence space available to the TIM-barrel fold. The ability to de novo design TIM-barrels opens new possibilities for custom-made enzymes.