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

Abstract

SummaryAltering 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.

Computational prediction of the tolerance to amino-acid deletion in green-fluorescent protein

“…Similar to 24 observations from computational studies, most functional deletion mutants were located 25 in unstructured loop regions, as opposed to in highly structured regions comprised of 26 β-sheets and α-helices. In addition, Arpino et al observed that non-functional mutants 27 were more likely to occur in buried regions than were functional mutants [16]. 28 Using the data from Arpino et al, we investigate here how well quantities known to 29 co-vary with amino-acid substitution rates can predict the functional consequences of 30 deletions in eGFP.…”

“…Furthermore, indel events may be 14 more likely to occur on the protein surface, where residues are more exposed to solvent, 15 rather than in a protein's core [13,14]. Thus, constraints on indel events appear to 16 mirror those on substitution events, in that amino-acid substitutions are most frequent 17 in unstructured regions and on the protein surface [1]. In spite of these observations, 18 however, the fundamental structural properties which govern whether indel events are 19 tolerated remain largely uncharacterized, ultimately hindering a complete 20 understanding of how protein structure influences protein evolution.…”

“…In spite of these observations, 18 however, the fundamental structural properties which govern whether indel events are 19 tolerated remain largely uncharacterized, ultimately hindering a complete 20 understanding of how protein structure influences protein evolution. 21 Recently, Arpino et al [16] experimentally determined the functional consequences 22 of single deletions in enhanced green fluorescent protein (eGFP) by systematically 23 deleting individual residues from eGFP and then assaying for function. Similar to 24 observations from computational studies, most functional deletion mutants were located 25 in unstructured loop regions, as opposed to in highly structured regions comprised of 26 β-sheets and α-helices.…”

“…All functional data corresponding to each mutant was taken from Arpino et al [16]. In 52 brief, Arpino et al [16] made tri-nucleotide deletions in the eGFP coding sequence using 53 a transposon-mediated directed evolution tri-nucleotide deletion experimental 54 approach [20,21].…”

“…In 52 brief, Arpino et al [16] made tri-nucleotide deletions in the eGFP coding sequence using 53 a transposon-mediated directed evolution tri-nucleotide deletion experimental 54 approach [20,21]. Their approach produced mutants with either a single amino-acid 55 deletion, multiple adjacent amino-acid deletions, or an amino-acid deletion with an 56 adjacent non-synonymous (i.e.…”