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Structural and dynamic changes associated with beneficial engineered single-amino-acid deletion mutations in enhanced green fluorescent protein

Arpino, James Alexander Joseph, Rizkallah, Pierre and Jones, Darran Dafydd 2014. Structural and dynamic changes associated with beneficial engineered single-amino-acid deletion mutations in enhanced green fluorescent protein. Acta Crystallographica Section D Biological Crystallography 70 (8) , pp. 2152-2162. 10.1107/S139900471401267X

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Abstract

Single-amino-acid deletions are a common part of the natural evolutionary landscape but are rarely sampled during protein engineering owing to limited and prejudiced molecular understanding of mutations that shorten the protein backbone. Single-amino-acid deletion variants of enhanced green fluorescent protein (EGFP) have been identified by directed evolution with the beneficial effect of imparting increased cellular fluorescence. Biophysical characterization revealed that increased functional protein production and not changes to the fluorescence parameters was the mechanism that was likely to be responsible. The structure EGFPD190 containing a deletion within a loop revealed propagated changes only after the deleted residue. The structure of EGFPA227 revealed that a `flipping' mechanism was used to adjust for residue deletion at the end of a -strand, with amino acids C-terminal to the deletion site repositioning to take the place of the deleted amino acid. In both variants new networks of short-range and long-range interactions are generated while maintaining the integrity of the hydrophobic core. Both deletion variants also displayed significant local and long-range changes in dynamics, as evident by changes in B factors compared with EGFP. Rather than being detrimental, deletion mutations can introduce beneficial structural effects through altering core protein properties, folding and dynamics, as well as function.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Biosciences
Medicine
Subjects: Q Science > Q Science (General)
Uncontrolled Keywords: Enhanced green fluorescent protein; single-amino-acid deletions; protein engineering.
Publisher: International Union of Crystallography
ISSN: 1399-0047
Funders: BBSRC
Date of First Compliant Deposit: 30 March 2016
Date of Acceptance: 31 May 2014
Last Modified: 17 May 2019 22:09
URI: http://orca.cf.ac.uk/id/eprint/63695

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