The design and structural characterization of a synthetic pentatricopeptide repeat protein

Benjamin Gully, Kunal Shah, Mihwa Lee, Kate Shearston, Nicole Smith, Agata Sadowska, Amanda Blythe, Kalia Bernath-Levin, William Stanley, Ian Small, Charlie Bond

Research output: Contribution to journalArticle

19 Citations (Scopus)
337 Downloads (Pure)

Abstract

Proteins of the pentatricopeptide repeat (PPR) superfamily are characterized by tandem arrays of a degenerate 35-amino-acid [alpha]-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 2.2 Å 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
JournalACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY
Volume71
Issue number2
DOIs
Publication statusPublished - Feb 2015

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Proteins
RNA-Binding Proteins
Consensus Sequence
RNA
RNA Editing
Organelle Biogenesis
Solubility
Sequence Analysis
Hot Temperature
Electrons
Amino Acids
Messenger RNA

Cite this

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title = "The design and structural characterization of a synthetic pentatricopeptide repeat protein",
abstract = "Proteins of the pentatricopeptide repeat (PPR) superfamily are characterized by tandem arrays of a degenerate 35-amino-acid [alpha]-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 2.2 {\AA} 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.",
author = "Benjamin Gully and Kunal Shah and Mihwa Lee and Kate Shearston and Nicole Smith and Agata Sadowska and Amanda Blythe and Kalia Bernath-Levin and William Stanley and Ian Small and Charlie Bond",
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The design and structural characterization of a synthetic pentatricopeptide repeat protein. / Gully, Benjamin; Shah, Kunal; Lee, Mihwa; Shearston, Kate; Smith, Nicole; Sadowska, Agata; Blythe, Amanda; Bernath-Levin, Kalia; Stanley, William; Small, Ian; Bond, Charlie.

In: ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY, Vol. 71, No. 2, 02.2015, p. 196-208.

Research output: Contribution to journalArticle

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AU - Gully, Benjamin

AU - Shah, Kunal

AU - Lee, Mihwa

AU - Shearston, Kate

AU - Smith, Nicole

AU - Sadowska, Agata

AU - Blythe, Amanda

AU - Bernath-Levin, Kalia

AU - Stanley, William

AU - Small, Ian

AU - Bond, Charlie

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AB - Proteins of the pentatricopeptide repeat (PPR) superfamily are characterized by tandem arrays of a degenerate 35-amino-acid [alpha]-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 2.2 Å 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.

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