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

Summary

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 (EGFP ^G4Δ) conferred significantly higher cellular fluorescence. Folding analysis revealed that EGFP ^G4Δ retained more structure upon unfolding and refolded with almost 100% efficiency but at the expense of thermodynamic stability. The EGFP ^G4Δ 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.

Graphical Abstract

Highlights

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  Using directed evolution, the impact of amino acid deletion on EGFP is explored

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  Loops, helices, and strand termini are especially tolerant to amino acid deletion

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  A deletion mutant that enhances cellular production and fluorescence is identified

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  Structure reveals that a helical registry shift creates a new polar network

Abstract

Using directed evolution, Arpino et al. examine the impact of amino acid deletion on EGFP and find that loops, helices, and strand termini are especially tolerant to amino acid deletion. Structural work provides a molecular explanation for this observation.