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Random single amino acid deletion sampling unveils structural tolerance and the benefits of helical registry shift on GFP folding and structure

Arpino, James Alexander Joseph, Reddington, Samuel C., Halliwell, Lisa Marie, Rizkallah, Pierre and Jones, Darran Dafydd 2014. Random single amino acid deletion sampling unveils structural tolerance and the benefits of helical registry shift on GFP folding and structure. Structure 22 (6) , pp. 889-898. 10.1016/j.str.2014.03.014

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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 (EGFPG4Δ) conferred significantly higher cellular fluorescence. Folding analysis revealed that EGFPG4Δ retained more structure upon unfolding and refolded with almost 100% efficiency but at the expense of thermodynamic stability. The EGFPG4Δ 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.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Biosciences
Medicine
Subjects: Q Science > Q Science (General)
Publisher: Elsevier
ISSN: 0969-2126
Funders: BBSRC
Date of First Compliant Deposit: 30 March 2016
Date of Acceptance: 10 March 2014
Last Modified: 15 Oct 2019 12:47
URI: http://orca.cf.ac.uk/id/eprint/60136

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