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Acta Crystallogr D Biol Crystallogr. 2014 Aug;70(Pt 8):2152-62. doi: 10.1107/S139900471401267X. Epub 2014 Jul 25.

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

Author information

1
School of Biosciences, Cardiff University, Park Place, Cardiff CF10 3AT, Wales.
2
School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, Wales.

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 EGFP(D190Δ) containing a deletion within a loop revealed propagated changes only after the deleted residue. The structure of EGFP(A227Δ) revealed that a `flipping' mechanism was used to adjust for residue deletion at the end of a β-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.

KEYWORDS:

enhanced green fluorescent protein; protein engineering; single-amino-acid deletions

PMID:
25084334
PMCID:
PMC4118826
DOI:
10.1107/S139900471401267X
[Indexed for MEDLINE]
Free PMC Article

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