Abstract
Relating a gene mutation to a phenotype is a common task in different disciplines such as protein biochemistry. In this endeavour, it is common to find false relationships arising from mutations introduced by cells that may be depurated using a phenotypic assay; yet, such phenotypic assays may introduce additional false relationships arising from experimental errors. Here we introduce the use of high-throughput DNA sequencers and statistical analysis aimed to identify incorrect DNA sequence-phenotype assignments and observed that 10–20% of these false assignments are expected in large screenings aimed to identify critical residues for protein function. We further show that this level of incorrect DNA sequence-phenotype assignments may significantly alter our understanding about the structure-function relationship of proteins. We have made available an implementation of our method at http://bis.ifc.unam.mx/en/software/chispas.
Highlights
The study of protein structure-function relationship involves the identification of residues indispensable for protein function; critical residues are commonly identified as those positions in proteins that result in loss of protein activity by affecting the proper protein folding, protein stability and/or the ability to perform a biochemical activity [1]
We performed mutagenesis experiments on hokC, an Escherichia coli gene that codes for a protein that induces the killing of its host upon expression [19]; in this case, cells expressing a wild-type copy of hokC should die upon its expression and those expressing mutated copies of hokC at a critical residue should survive
Of particular interest is the observation that mutations on the N-terminus of HokC did have an effect on its toxic activity. These results do not agree with previous results that suggested that only the C-terminus end of HokC is relevant for its activity [21]; many critical residues identified in this screening are located in non-conserved positions
Summary
The study of protein structure-function relationship involves the identification of residues indispensable for protein function (critical residues); critical residues are commonly identified as those positions in proteins that result in loss of protein activity by affecting the proper protein folding, protein stability and/or the ability to perform a biochemical activity [1]. Many protein coding genes have been subjected to site-directed mutagenesis experiments in the past with the aim of identifying the protein critical residues [2,3] and such information has been used to develop prediction methods useful to test our understanding about the function of these residues in proteins [4,5]. Directed evolution experiments circumvent our limitations to understand the structure-function relationship of proteins by discovering protein variants with valuable features [6]. It is important to validate the identity of the mutated residues to guarantee the reproducibility of the results and to reduce any bias on methods aimed to predict critical residues. The nature of mutations affecting protein function is established by sequencing the corresponding protein-coding DNA region. The relevance of the presence of DNA variants in a population for critical residue
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