36 research outputs found

    Unfolding Simulations of Holomyoglobin from Four Mammals: Identification of Intermediates and β-Sheet Formation from Partially Unfolded States

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    Myoglobin (Mb) is a centrally important, widely studied mammalian protein. While much work has investigated multi-step unfolding of apoMb using acid or denaturant, holomyoglobin unfolding is poorly understood despite its biological relevance. We present here the first systematic unfolding simulations of holoMb and the first comparative study of unfolding of protein orthologs from different species (sperm whale, pig, horse, and harbor seal). We also provide new interpretations of experimental mean molecular ellipticities of myoglobin intermediates, notably correcting for random coil and number of helices in intermediates. The simulated holoproteins at 310 K displayed structures and dynamics in agreement with crystal structures (R g ~1.48-1.51 nm, helicity ~75%). At 400 K, heme was not lost, but some helix loss was observed in pig and horse, suggesting that these helices are less stable in terrestrial species. At 500 K, heme was lost within 1.0-3.7 ns. All four proteins displayed exponentially decaying helix structure within 20 ns. The C- and F-helices were lost quickly in all cases. Heme delayed helix loss, and sperm whale myoglobin exhibited highest retention of heme and D/E helices. Persistence of conformation (RMSD), secondary structure, and ellipticity between 2-11 ns was interpreted as intermediates of holoMb unfolding in all four species. The intermediates resemble those of apoMb notably in A and H helices, but differ substantially in the D-, E- and F-helices, which interact with heme. The identified mechanisms cast light on the role of metal/cofactor in poorly understood holoMb unfolding. We also observed β-sheet formation of several myoglobins at 500 K as seen experimentally, occurring after disruption of helices to a partially unfolded, globally disordered state; heme reduced this tendency and sperm-whale did not display any sheet propensity during the simulations

    Implications of the model relating to experimental observations.

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    <p>Implications of the model relating to experimental observations.</p

    Experimental Data for Myoglobin States from the Literature.

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    <p><sup>a</sup> For HoloMb, average based on crystal structures 1A6M <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080308#pone.0080308-Ozdemir1" target="_blank">[84]</a>, 1MBO <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080308#pone.0080308-Hirst2" target="_blank">[85]</a>, 1U7S <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080308#pone.0080308-VanDerSpoel1" target="_blank">[65]</a>, and 1U7R <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080308#pone.0080308-VanDerSpoel1" target="_blank">[65]</a>.</p><p><sup>b</sup> Estimated from NMR data, see text.</p

    Transient Violation of Le Chatelier's Principle for a Network of Water Molecules

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    Selection spaces <i>s</i><sub>i</sub> (fitness-differences normalized to wild-type fitness) for mutations causing increased mutant protein expression (<i>A</i><sub>i,mutant</sub>).

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    <p>(<b>A</b>) Selection acts against increased protein abundance of mutant vs. wild-type (<i>A</i><sub>i,WT</sub>) (Default values of parameters from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090504#pone-0090504-t001" target="_blank">Table 1</a>). (<b>B</b>) High-turnover proteins (with large values of <i>k</i><sub>d,i</sub>) are under stronger selection pressure to perform optimally. (<b>C</b>) For a high-turnover protein (life time ∼1 minute, <i>k</i><sub>d</sub> = 0.01 s<sup>−1</sup>, larger proteins are under stronger selection to perform optimally, <i>ceteris paribus</i>. (<b>D</b>) Selection pressure is stronger for proteins that are synthetically expensive, as measured by <i>C</i><sub>s,i</sub> (<i>k</i><sub>d</sub> = 0.01 s<sup>−1</sup>).</p
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