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 (β/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.

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Figure 1: Geometric constraints on the secondary structure arrangement in an ideal four-fold-symmetric TIM barrel.
Figure 2: Sequence determinants of de novo–designed TIM barrel.
Figure 3: Effect of two-fold- versus four-fold-symmetrical barrel interior on stability.
Figure 4: Stability and structure of sTIM-11.
Figure 5: Sequence relationships between natural (β/α)8-barrels and sTIM-11.

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We thank R. Krishnamurty and C. Tinberg for comments on the manuscript, as well as S. Ovchinnikov, A.C. Stiel and S. Schmidt for technical advice. D.A.F.V. thanks the Dirección General de Asuntos del Personal Académico UNAM for a sabbatical stay fellowship, and K.F. acknowledges a fellowship from the International Max Planck Research School (IMPRS) “From Molecules to Organisms” Tübingen. F.P. was supported by Human Frontier Science Program Long-term fellowship LT000070/2009-l. This work was facilitated through the use of advanced computational storage and networking infrastructure provided by the Hyak supercomputer system at the University of Washington. This research was also done using resources provided by the Open Science Grid (OSG), which is supported by the US National Science Foundation and the US Department of Energy's Office of Science. We would like to particularly thank M. Rynge and J. McGee of the OSG Engagement Team at the Renaissance Computing Institute (RENCI) and M. Livny and the HTCondor Team of the University of Wisconsin–Madison for their technical and logistical guidance in our use of OSG resources. This work was supported by grants from the Defense Threat Reduction Agency (DTRA) and the Howard Hughes Medical Institute to D.B., Deutsche Forschungsgemeinschaft grant HO4022/1-2 and Max Planck funds to B.H., and grants CONACYT 99857 and PAPIIT-UNAM IN219913 to D.A.F.V.

Author information




P.-S.H., K.F., D.A.F.V., B.H. and D.B. designed the research. P.-S.H. wrote program code and designed structures with help from B.H. and D.A.F.V. P.-S.H., F.P. and D.A.F.V. built the clones. K.F. and B.H. solved the crystal structure of sTIM-11 and collected thermodynamic data for sTIMs. F.P. characterized the first designs. P.-S.H., K.F., B.H. and D.B. collected and analyzed sequence and structure comparison data. K.F. and B.H. generated the cluster map. P.-S.H., K.F., B.H. and D.B. wrote the manuscript with help from all the authors. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Po-Ssu Huang or Birte Höcker or David Baker.

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The authors declare no competing financial interests.

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Supplementary Results, Supplementary Figures 1–9 and Supplementary Tables 1–5. (PDF 6188 kb)

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Huang, PS., Feldmeier, K., Parmeggiani, F. et al. De novo design of a four-fold symmetric TIM-barrel protein with atomic-level accuracy. Nat Chem Biol 12, 29–34 (2016).

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