Recurring sequence-structure motifs in (βα)8-barrel proteins and experimental optimization of a chimeric protein designed based on such motifs

•The βα and αβ modules of TIM barrel proteins were clustered based on structure and sequence.•A number of recurring motifs have been identified.•A chimeric protein A0 was created by using the recurring motifs as interfaces.•A0 was significantly improved by six rounds of directed evolution.

An interesting way of generating novel artificial proteins is to combine sequence motifs from natural proteins, mimicking the evolutionary path suggested by natural proteins comprising recurring motifs. We analyzed the βα and αβ modules of TIM barrel proteins by structure alignment-based sequence clustering. A number of preferred motifs were identified. A chimeric TIM was designed by using recurring elements as mutually compatible interfaces. The foldability of the designed TIM protein was then significantly improved by six rounds of directed evolution. The melting temperature has been improved by more than 20 °C. A variety of characteristics suggested that the resulting protein is well-folded. Our analysis provided a library of peptide motifs that is potentially useful for different protein engineering studies. The protein engineering strategy of using recurring motifs as interfaces to connect partial natural proteins may be applied to other protein folds.