title: Random single amino acid deletion sampling unveils structural tolerance and the benefits of helical registry shift on GFP folding and structure.
abstract: Altering a protein's backbone through amino acid deletion is a common evolutionary mutational mechanism, but is generally ignored during protein engineering primarily because its effect on the folding-structure-function relationship is difficult to predict. Using directed evolution, enhanced green fluorescent protein (EGFP) was observed to tolerate residue deletion across the breadth of the protein, particularly within short and long loops, helical elements, and at the termini of strands. A variant with G4 removed from a helix (EGFP(G4Δ)) conferred significantly higher cellular fluorescence. Folding analysis revealed that EGFP(G4Δ) retained more structure upon unfolding and refolded with almost 100% efficiency but at the expense of thermodynamic stability. The EGFP(G4Δ) structure revealed that G4 deletion caused a beneficial helical registry shift resulting in a new polar interaction network, which potentially stabilizes a cis proline peptide bond and links secondary structure elements. Thus, deletion mutations and registry shifts can enhance proteins through structural rearrangements not possible by substitution mutations alone.
authors.affiliation: School of Biosciences, Main Building, Park Place, Cardiff University, Cardiff CF10 3AT, UK.
authors.forename: James A J
authors.initials: JA
authors.lastname: Arpino
authors.affiliation: School of Biosciences, Main Building, Park Place, Cardiff University, Cardiff CF10 3AT, UK.
authors.forename: Samuel C
authors.initials: SC
authors.lastname: Reddington
authors.affiliation: School of Biosciences, Main Building, Park Place, Cardiff University, Cardiff CF10 3AT, UK.
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Halliwell
authors.affiliation: School of Medicine, Cardiff University, WHRI, Main Building, Heath Park, Cardiff CF14 4XN, UK.
authors.forename: Pierre J
authors.initials: PJ
authors.lastname: Rizkallah
authors.affiliation: School of Biosciences, Main Building, Park Place, Cardiff University, Cardiff CF10 3AT, UK. Electronic address: jonesdd@cf.ac.uk.
authors.forename: D Dafydd
authors.initials: DD
authors.lastname: Jones
mesh_terms: D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D019020:Directed Molecular Evolution; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics*; D007700:Kinetics; D008958:Models, Molecular; D017510:Protein Folding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D017384:Sequence Deletion
chemical_list: D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins