RT Journal Article SR Electronic T1 Extension of a de novo TIM barrel with a rationally designed secondary structure element JF bioRxiv FD Cold Spring Harbor Laboratory SP 2020.10.16.342774 DO 10.1101/2020.10.16.342774 A1 Jonas Gregor Wiese A1 Sooruban Shanmugaratnam A1 Birte Höcker YR 2020 UL http://biorxiv.org/content/early/2020/10/16/2020.10.16.342774.abstract AB The ability to construct novel enzymes is a major aim in de novo protein design. A popular enzyme fold for design attempts is the TIM barrel. This fold is a common topology for enzymes and can harbor many diverse reactions. The recently published de novo design of a four-fold symmetric TIM barrel provides a well understood minimal scaffold for potential enzyme designs. Here we explore opportunities to extend and diversify this scaffold by adding a short de novo helix on top of the barrel. Due to the size of the protein we developed a design pipeline based on computational ab initio folding that solves a less complex sub-problem focused around the helix and its vicinity and adapt it to the entire protein. We provide biochemical characterization and a high-resolution X-ray structure for one variant and compare it to our design model. The successful extension of this robust TIM-barrel scaffold opens opportunities to diversify it towards more pocket like arrangements and as such can be considered a building block for future design of binding or catalytic sites.Competing Interest StatementThe authors have declared no competing interest.