Structural and dynamic changes associated with beneficial engineered single-amino-acid deletion mutations in enhanced green fluorescent protein.
Author(s)
Arpino, JA
Rizkallah, PJ
Jones, DD
Type
Journal Article
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.
Date Issued
2014-07-25
Date Acceptance
2014-05-31
Citation
Acta Crystallographica Section D - Biological Crystallography, 2014, 70 (Pt 8), pp.2152-2162
ISSN
0907-4449
Publisher
International Union of Crystallography
Start Page
2152
End Page
2162
Journal / Book Title
Acta Crystallographica Section D - Biological Crystallography
Volume
70
Issue
Pt 8
Copyright Statement
© 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.
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.
License URL
Identifier
PII: S139900471401267X
Subjects
enhanced green fluorescent protein
protein engineering
single-amino-acid deletions
Amino Acids
Crystallization
Green Fluorescent Proteins
Protein Conformation
Protein Engineering
Publication Status
Published