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

Abstract

Proteins evolve through two primary mechanisms: substitution, where mutations alter a protein's amino-acid sequence, and insertions and deletions (indels), where amino acids are either added to or removed from the sequence. Protein structure has been shown to influence the rate at which substitutions accumulate across sites in proteins, but whether structure similarly constrains the occurrence of indels has not been rigorously studied. Here, we investigate the extent to which structural properties known to covary with protein evolutionary rates might also predict protein tolerance to indels. Specifically, we analyze a publicly available dataset of single-amino-acid deletion mutations in enhanced green fluorescent protein (eGFP) to assess how well the functional effect of deletions can be predicted from protein structure. We find that weighted contact number (WCN), which measures how densely packed a residue is within the protein's three-dimensional structure, provides the best single predictor for whether eGFP will tolerate a given deletion. We additionally find that using protein design to explicitly model deletions results in improved predictions of functional status when combined with other structural predictors. Our work suggests that structure plays fundamental role in constraining deletions at sites in proteins, and further that similar biophysical constraints influence both substitutions and deletions. This study therefore provides a solid foundation for future work to examine how protein structure influences tolerance of more complex indel events, such as insertions or large deletions.

Fitness Effects of Single Amino Acid Insertions and Deletions in TEM-1 β-Lactamase

“…7e), though the R 2 is only 0.27. WCN was also the single best predictor of whether a deletion is tolerated in eGFP [17]. The lack of simple correlations between properties and insertion fitness were consistent with our expectation that the fitness effects of insertions have complex origins.…”

“…They found that the majority of tolerated deletions occurred in loops, while the rest were found equally distributed in helices and β-strands, with the termini of β-strands being more tolerant than the middle. Computational analysis of the EGFP found that structural properties such as relative solvent accessibility and weighted contact number (WCN) can be used to predict tolerance to deletions to some extent [17].…”

ΔFlucs: Brighter Photinus pyralis firefly luciferases identified by surveying consecutive single amino acid deletion mutations in a thermostable variant

“…Only two single ΔFlucs (ΔV384 and ΔR387) exhibited a near‐complete loss of activity, demonstrating that loop regions of ×11 Fluc are largely tolerant to single amino acid deletions in loops. Ordinarily deletions in proteins of residues with low residue solvent accessibility (RSA) such as Gly and Pro are better tolerated than larger residues, or those that make fewer contacts (Jackson, Spielman, & Wilke, ). Aside from in the N‐terminal, deletions of Pro (P353 and P359), Gly (G360 and G363), Ala (A361), Val (V362), Leu (L376), and Asp (D377) were best tolerated or enhanced brightness in vivo.…”

“…These were then inputted into a python script calc_RSA.py to generate a list of RSA values. Weighted contact numbers (WCN) calculated as described (Jackson et al, ) using script calc_WCN.py. Scripts are made free to use on GitHub page.…”

A probabilistic model for indel evolution: differentiating insertions from deletions

“…It is expected that different structural attributes do not share the same indel dynamics. For example, it was recently shown that for enhanced green fluorescent protein (eGFP), the packing density of a residue, as measured by the weighted contact number (Lin et al, 2008), considerably affects the probability that a single-residue deletion disrupts the protein’s function (Jackson et al, 2017). Relative surface accessibility and secondary structure attributes were also found to affect this probability.…”