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

DOI: 10.1107/S139900471401267X DOI Help
PMID: 25084334 PMID Help

Authors: James A. J. Arpino (Cardiff University, U.K.) , Pierre J. Rizkallah (Cardiff University, U.K.) , Dafydd Jones (Cardiff University, U.K.)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Acta Crystallographica Section D Biological Crystallography , VOL 70 , PAGES 2152 - 2162

State: Published (Approved)
Published: August 2014
Diamond Proposal Number(s): 6232

Open Access Open Access

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.

Journal Keywords: Enhanced Green Fluorescent Protein; Single-Amino-Acid Deletions; Protein Engineering.

Subject Areas: Biology and Bio-materials

Instruments: I03-Macromolecular Crystallography , I04-Macromolecular Crystallography

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