A Global Experimental Analysis of Protein Function: a Case Study in the PDZ Domain

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Title: A Global Experimental Analysis of Protein Function: a Case Study in the PDZ Domain
Author: McLaughlin, Richard Noel Jr.
Abstract: A complete understanding of the energetic architecture of a protein can be achieved only with a comprehensive description of the interaction of every amino acid with every other amino acid . Many efforts to understand the apparent complexity of protein function have attempted to address this problem with limited mutagenesis studies . A global computational description of amino acid interactions , Statistical Coupling Analysis has shown the existence of a contiguous subset of positions within a protein that displays significant co -evolution , termed protein sectors . Limited mutagenesis studies have shown sectors to be networks of higher -order interaction crucial for protein function ; however , a theory of such global scope requires validation with a global experiment . Here , we design an assay system that measures the cellular function of a PDZ domain in a high -throughput and quantitative manner . We perform a comprehensive single amino acid mutagenesis experiment to show that most positions in the protein are robust to most mutations , and the set of positions that shows sensitivity to mutation is enriched for sector positions . Further , we perform a global pairwise epistasis experiment in which we measure the way in which every amino acid mutation in the PDZ domain feels the effect of a second mutation at a key specificity and affinity determining position in the peptide ligand of the PDZ domain . We find that those positions that show strong non -additivity in the context of the peptide mutation are all contained within the PDZ sector . Further , these sector positions that display strong non -additivity all display the property of rapidly changing specificity upon mutation . That is , any mutation at these sector positions has a negative functional effect in the context of the endogenous peptide . However , these positions appear to be spring -loaded for change since these same mutations enhance function in the context of an alternative peptide . We hypothesize that proteins are robust as shown by their insensitivity to general mutation . However , proteins are simultaneously fragile as shown by their sensitivity to specific mutagenesis at sector positions . This fragility , however , is strongly coupled to evolvability as shown by the enhancement of alternative function endowed by these endogenously detrimental mutations .
URI: http : / /hdl .handle .net /2152 .5 /860
Date: 2011-02-01

Citation

A Global Experimental Analysis of Protein Function: a Case Study in the PDZ Domain. Graduate School of Biomedical Sciences. Available electronically from http : / /hdl .handle .net /2152 .5 /860 .

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