Systematic Mapping of Protein Mutational Space by Prolonged Drift Reveals the Deleterious Effects of Seemingly Neutral Mutations

Abstract

Systematic mappings of the effects of protein mutations are becoming increasingly popular. Unexpectedly, these experiments often find that proteins are tolerant to most amino acid substitutions, including substitutions in positions that are highly conserved in nature. To obtain a more realistic distribution of the effects of protein mutations, we applied a laboratory drift comprising 17 rounds of random mutagenesis and selection of M.HaeIII, a DNA methyltransferase. During this drift, multiple mutations gradually accumulated. Deep sequencing of the drifted gene ensembles allowed determination of the relative effects of all possible single nucleotide mutations. Despite being averaged across many different genetic backgrounds, about 67% of all nonsynonymous, missense mutations were evidently deleterious, and an additional 16% were likely to be deleterious. In the early generations, the frequency of most deleterious mutations remained high. However, by the 17th generation, their frequency was consistently reduced, and those remaining were accepted alongside compensatory mutations. The tolerance to mutations measured in this laboratory drift correlated with sequence exchanges seen in M.HaeIII’s natural orthologs. The biophysical constraints dictating purging in nature and in this laboratory drift also seemed to overlap. Our experiment therefore provides an improved method for measuring the effects of protein mutations that more closely replicates the natural evolutionary forces, and thereby a more realistic view of the mutational space of proteins.

Author Summary

Understanding and predicting the effects of single nucleotide polymorphisms (SNPs) is of fundamental importance in many fields. Systematic experimental mappings of the effects of such mutations within a given gene/protein comprise an essential experimental tool for determining protein function and for refining models of protein evolution, as well as an important resource for improving prediction algorithms. Here, we present the results of a laboratory system that mimics the manner by which protein sequences diverge in nature: a prolonged process of gradually accumulating random mutations that retain the protein’s structure and function. The change in frequencies of mutations over generations, as obtained by deep sequencing, enabled us to assess the relative effects of all possible SNPs at the background of an accumulating number of mutations. Compared to previous reports, we found that > 80% of all possible amino acid exchanges have potential deleterious effects, with 67% being clearly deleterious. Tolerance vs. purging of mutations in our prolonged drift also showed better correlation with natural diversity. Overall, our experimental setup provides a better understanding of how protein sequences diverge in nature, plus a new basis for improving the prediction accuracy of the effects of protein mutations, and specifically of SNPs.