The design and structural characterization of a synthetic pentatricopeptide repeat protein

Benjamin S. Gully, Kunal R. Shah, Mihwa Lee, Kate Shearston, Nicole M. Smith, Agata Sadowska, Amanda J. Blythe, Kalia Bernath-Levin, Will A. Stanley, Ian D. Small, Charles S. Bond

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20 Citations (Scopus)

Abstract

Proteins of the pentatricopeptide repeat (PPR) superfamily are characterized by tandem arrays of a degenerate 35-amino-acid -hairpin motif. PPR proteins are typically single-stranded RNA-binding proteins with essential roles in organelle biogenesis, RNA editing and mRNA maturation. A modular, predictable code for sequence-specific binding of RNA by PPR proteins has recently been revealed, which opens the door to the de novo design of bespoke proteins with specific RNA targets, with widespread biotechnological potential. Here, the design and production of a synthetic PPR protein based on a consensus sequence and the determination of its crystal structure to 2.2Å resolution are described. The crystal structure displays helical disorder, resulting in electron density representing an infinite superhelical PPR protein. A structural comparison with related tetratricopeptide repeat (TPR) proteins, and with native PPR proteins, reveals key roles for conserved residues in directing the structure and function of PPR proteins. The designed proteins have high solubility and thermal stability, and can form long tracts of PPR repeats. Thus, consensus-sequence synthetic PPR proteins could provide a suitable backbone for the design of bespoke RNA-binding proteins with the potential for high specificity.

Original languageEnglish
Pages (from-to)196-208
Number of pages13
JournalActa Crystallographica Section D: Biological Crystallography
Volume71
DOIs
Publication statusPublished - 1 Jan 2015
Externally publishedYes

Keywords

  • synthetic pentatricopeptide repeat protein

Cite this

Gully, B. S., Shah, K. R., Lee, M., Shearston, K., Smith, N. M., Sadowska, A., Blythe, A. J., Bernath-Levin, K., Stanley, W. A., Small, I. D., & Bond, C. S. (2015). The design and structural characterization of a synthetic pentatricopeptide repeat protein. Acta Crystallographica Section D: Biological Crystallography, 71, 196-208. https://doi.org/10.1107/S1399004714024869