8 research outputs found

    Global computational mutagenesis provides a critical stability framework in protein structures

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    <div><p>A protein’s amino acid sequence dictates the folds and final structure the macromolecule will form. We propose that by identifying critical residues in a protein’s atomic structure, we can select a critical stability framework within the protein structure essential to proper protein folding. We use global computational mutagenesis based on the unfolding mutation screen to test the effect of every possible missense mutation on the protein structure to identify the residues that cannot tolerate a substitution without causing protein misfolding. This method was tested using molecular dynamics to simulate the stability effects of mutating critical residues in proteins involved in inherited disease, such as myoglobin, p53, and the 15<sup>th</sup> sushi domain of complement factor H. In addition we prove that when the critical residues are in place, other residues may be changed within the structure without a stability loss. We validate that critical residues are conserved using myoglobin to show that critical residues are the same for crystal structures of 6 different species and comparing conservation indices to critical residues in 9 eye disease-related proteins. Our studies demonstrate that by using a selection of critical elements in a protein structure we can identify a critical protein stability framework. The frame of critical residues can be used in genetic engineering to improve small molecule binding for drug studies, identify loss-of-function disease-causing missense mutations in genetics studies, and aide in identifying templates for homology modeling.</p></div

    Comparison of the foldability and conservation index parameters.

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    <p>Comparison of the foldability and conservation index parameters.</p

    Critical residue and foldability comparison across myoglobin for 6 species.

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    <p><b>A)</b> The output colored structure from UMS analysis of the 6 proteins. The red residues represent the critical residues, while the blue shows the residues that may be substituted with other residues. <b>B)</b> Pairwise comparison of human myoglobin with the 5 other species. The black outlines represent a 95% confidence interval for the data. The statistics of the graph are summarized in <b>C)</b>. <b>D)</b> The density plot shows the distribution of foldabilties in each of the structures.</p

    Molecular dynamics (MD) were used to simulate the affect of mutating protiens to CS and ΔCS.

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    <p>Critical residues for each of the structure are red and were calculated independently. <b>A)</b> 52% of noncritical human myoglobin residues were changed. The CS structure is superimposed on top of the WT human myoglobin structure. <b>B)</b> The critical residues of human myoglobin were changed to alanine residues, accounting for 12% of the residues in the structure. The ΔCS protein is superimposed on top of the WT human myoglobin structure. <b>C)</b> The RMSD for CS and ΔCS myoglobin is plotted for the MD simulation. <b>D)</b> The CS p53 with 53% of WT residues changed superimposed on the WT protein. <b>E)</b> The ΔCS p53 with 15% of residues changed superimposed on the WT protein. <b>F)</b> The RMSD for CS and ΔCS p53 is plotted for the MD simulation. <b>G)</b> The CS sushi domain 15 of complement factor H with 47% of WT residues changed superimposed on the WT protein. <b>H)</b> The ΔCS sushi domain 15 of complement factor H with 23% of residues changed superimposed on the WT protein. <b>I)</b> The RMSD for CS and ΔCS sushi domain 15 of complement factor H is plotted for the MD simulation.</p

    Comparison of stability between the CS and ΔCS proteins using Ramachandran plots and residue-residue distances.

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    <p><b>A)</b> The plots for both the CS and ΔCS myoglobin structures. <b>B)</b> The plots for both the CS and ΔCS p53 structures. <b>C)</b> The plots for both the CS and ΔCS sushi domain 15 of complement factor H structures.</p

    Construction and testing of the critical structure (CS) and the changed critical structure (ΔCS) proteins.

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    <p><b>A)</b> The wild type protein structure is obtained from RCSB Protein Data Bank. <b>B)</b> The WT protein is run through UMS to identify the critical residues (shown in red). <b>C)</b> For the CS protein, the critical residues are kept in place and the remaining residues are mutated according to the rules of the allowed substitutions list (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189064#pone.0189064.s004" target="_blank">S4 Fig</a>). <b>D)</b> For the ΔCS protein, each of the critical residues is mutated to alanine. <b>E)</b> Both the CS and the ΔCS structures were equilibrated in water for 100 ns as described in Methods section. <b>F)</b> The CS structure is run through UMS to identify consistencies in critical residues. <b>G)</b> The ΔCS structure is run through UMS to identify changes in critical residues.</p

    The critical residues frame for atomic protein structures.

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    <p>The <b>A)</b> p53, <b>B)</b> domain S15 of complement factor H, <b>C)</b> alpha-tocopherol transfer proteins are shown. The red residues represent the critical residues within the structures.</p
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