Structural and dynamic changes associated with beneficial engineered single-amino-acid deletion mutations in enhanced green fluorescent protein
- Cardiff University, Park Place, Cardiff CF10 3AT Wales (United Kingdom)
The beneficial engineered single-amino-acid deletion variants EGFP{sup D190Δ} and EGFP{sup A227Δ} have been studied. 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{sup D190Δ} containing a deletion within a loop revealed propagated changes only after the deleted residue. The structure of EGFP{sup 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.
- OSTI ID:
- 22347778
- Journal Information:
- Acta Crystallographica. Section D: Biological Crystallography, Vol. 70, Issue Pt 8; Other Information: PMCID: PMC4118826; PMID: 25084334; PUBLISHER-ID: qh5010; OAI: oai:pubmedcentral.nih.gov:4118826; Copyright (c) Arpino et al. 2014; This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.; Country of input: International Atomic Energy Agency (IAEA); ISSN 0907-4449
- Country of Publication:
- Denmark
- Language:
- English
Similar Records
A donor splice mutation and a single-base deletion produce two carboxyl-terminal variants of human serum albumin
Characterization of the nuclear localization signal of high risk HPV16 E2 protein