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Random Single Amino Acid Deletion Sampling Unveils Structural Tolerance and the Benefits of Helical Registry Shift on GFP Folding and Structure

DOI: 10.1016/j.str.2014.03.014 DOI Help
PMID: 24856363 PMID Help

Authors: James A. J. Arpino (Cardiff University, U.K.) , Samuel C. Reddington (Cardiff University, U.K.) , Lisa m. Halliwell (Cardiff University, U.K.) , Pierre J. Rizkallah (Cardiff University) , D. Dafydd Jones (Cardiff University, U.K.)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Structure , VOL 22 (6) , PAGES 889 - 898

State: Published (Approved)
Published: June 2014

Open Access Open Access

Abstract: Altering a protein’s backbone through amino acid deletion is a common evolutionary mutational mechanism, but is generally ignored during protein engineering primarily because its effect on the folding-structure-function relationship is difficult to predict. Using directed evolution, enhanced green fluorescent protein (EGFP) was observed to tolerate residue deletion across the breadth of the protein, particularly within short and long loops, helical elements, and at the termini of strands. A variant with G4 removed from a helix (EGFPG4Δ) conferred significantly higher cellular fluorescence. Folding analysis revealed that EGFPG4Δ retained more structure upon unfolding and refolded with almost 100% efficiency but at the expense of thermodynamic stability. The EGFPG4Δ structure revealed that G4 deletion caused a beneficial helical registry shift resulting in a new polar interaction network, which potentially stabilizes a cis proline peptide bond and links secondary structure elements. Thus, deletion mutations and registry shifts can enhance proteins through structural rearrangements not possible by substitution mutations alone.

Journal Keywords: Crystallography; X-Ray; Directed; Fluorescence; Green; Kinetics; Models; Molecular; Protein; Secondary; Protein; Tertiary; Recombinant; Sequence Deletion

Subject Areas: Biology and Bio-materials


Instruments: I03-Macromolecular Crystallography

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