6 research outputs found

    Validating Molecular Dynamics Simulations against Experimental Observables in Light of Underlying Conformational Ensembles

    No full text
    Far from the static, idealized conformations deposited into structural databases, proteins are highly dynamic molecules that undergo conformational changes on temporal and spatial scales that may span several orders of magnitude. These conformational changes, often intimately connected to the functional roles that proteins play, may be obscured by traditional biophysical techniques. Over the past 40 years, molecular dynamics (MD) simulations have complemented these techniques by providing the “hidden” atomistic details that underlie protein dynamics. However, there are limitations of the degree to which molecular simulations accurately and quantitatively describe protein motions. Here we show that although four molecular dynamics simulation packages (AMBER, GROMACS, NAMD, and <i>il</i>mm) reproduced a variety of experimental observables for two different proteins (engrailed homeodomain and RNase H) equally well overall at room temperature, there were subtle differences in the underlying conformational distributions and the extent of conformational sampling obtained. This leads to ambiguity about which results are correct, as experiment cannot always provide the necessary detailed information to distinguish between the underlying conformational ensembles. However, the results with different packages diverged more when considering larger amplitude motion, for example, the thermal unfolding process and conformational states sampled, with some packages failing to allow the protein to unfold at high temperature or providing results at odds with experiment. While most differences between MD simulations performed with different packages are attributed to the force fields themselves, there are many other factors that influence the outcome, including the water model, algorithms that constrain motion, how atomic interactions are handled, and the simulation ensemble employed. Here four different MD packages were tested each using best practices as established by the developers, utilizing three different protein force fields and three different water models. Differences between the simulated protein behavior using two different packages but the same force field, as well as two different packages with different force fields but the same water models and approaches to restraining motion, show how other factors can influence the behavior, and it is incorrect to place all the blame for deviations and errors on force fields or to expect improvements in force fields alone to solve such problems

    Validating Molecular Dynamics Simulations against Experimental Observables in Light of Underlying Conformational Ensembles

    No full text
    Far from the static, idealized conformations deposited into structural databases, proteins are highly dynamic molecules that undergo conformational changes on temporal and spatial scales that may span several orders of magnitude. These conformational changes, often intimately connected to the functional roles that proteins play, may be obscured by traditional biophysical techniques. Over the past 40 years, molecular dynamics (MD) simulations have complemented these techniques by providing the “hidden” atomistic details that underlie protein dynamics. However, there are limitations of the degree to which molecular simulations accurately and quantitatively describe protein motions. Here we show that although four molecular dynamics simulation packages (AMBER, GROMACS, NAMD, and <i>il</i>mm) reproduced a variety of experimental observables for two different proteins (engrailed homeodomain and RNase H) equally well overall at room temperature, there were subtle differences in the underlying conformational distributions and the extent of conformational sampling obtained. This leads to ambiguity about which results are correct, as experiment cannot always provide the necessary detailed information to distinguish between the underlying conformational ensembles. However, the results with different packages diverged more when considering larger amplitude motion, for example, the thermal unfolding process and conformational states sampled, with some packages failing to allow the protein to unfold at high temperature or providing results at odds with experiment. While most differences between MD simulations performed with different packages are attributed to the force fields themselves, there are many other factors that influence the outcome, including the water model, algorithms that constrain motion, how atomic interactions are handled, and the simulation ensemble employed. Here four different MD packages were tested each using best practices as established by the developers, utilizing three different protein force fields and three different water models. Differences between the simulated protein behavior using two different packages but the same force field, as well as two different packages with different force fields but the same water models and approaches to restraining motion, show how other factors can influence the behavior, and it is incorrect to place all the blame for deviations and errors on force fields or to expect improvements in force fields alone to solve such problems

    Designed Trpzip‑3 β‑Hairpin Inhibits Amyloid Formation in Two Different Amyloid Systems

    No full text
    The trpzip peptides are small, monomeric, and extremely stable β-hairpins that have become valuable tools for studying protein folding. Here, we show that trpzip-3 inhibits aggregation in two very different amyloid systems: transthyretin and Aβ(1–42). Interestingly, Trp → Leu mutations renders the peptide ineffective against transthyretin, but Aβ inhibition remains. Computational docking was used to predict the interactions between trpzip-3 and transthyretin, suggesting that inhibition occurs via binding to the outer region of the thyroxine-binding site, which is supported by dye displacement experiments

    Chemical and Physical Variability in Structural Isomers of an l/d α‑Sheet Peptide Designed To Inhibit Amyloidogenesis

    No full text
    There has been much interest in synthetic peptides as inhibitors of aggregation associated with amyloid diseases. Of particular interest are compounds that target the cytotoxic soluble oligomers preceding the formation of mature, nontoxic fibrils. This study explores physical and chemical differences between two <i>de novo</i>-designed peptides that share an identical primary structure but differ in backbone chirality at six key positions. We show that the presence of alternating l/d-amino acid motifs dramatically increases aqueous solubility, enforces α-sheet secondary structure, and inhibits aggregation of the β-amyloid peptide implicated in Alzheimer’s disease, in addition to neutralizing its cytotoxicity. In contrast, the all-l-amino acid isomer does not form α-sheet structure and is insoluble and inactive

    Structural and Functional Consequences of the Cardiac Troponin C L48Q Ca<sup>2+</sup>-Sensitizing Mutation

    No full text
    Calcium binding to the regulatory domain of cardiac troponin C (cNTnC) causes a conformational change that exposes a hydrophobic surface to which troponin I (cTnI) binds, prompting a series of protein–protein interactions that culminate in muscle contraction. A number of cTnC variants that alter the Ca<sup>2+</sup> sensitivity of the thin filament have been linked to disease. Tikunova and Davis engineered a series of cNTnC mutations that altered Ca<sup>2+</sup> binding properties and studied the effects on the Ca<sup>2+</sup> sensitivity of the thin filament and contraction [Tikunova, S. B., and Davis, J. P. (2004) <i>J. Biol. Chem. 279</i>, 35341–35352]. One of the mutations they engineered, the L48Q variant, resulted in a pronounced increase in the cNTnC Ca<sup>2+</sup> binding affinity and Ca<sup>2+</sup> sensitivity of cardiac muscle force development. In this work, we sought structural and mechanistic explanations for the increased Ca<sup>2+</sup> sensitivity of contraction for the L48Q cNTnC variant, using an array of biophysical techniques. We found that the L48Q mutation enhanced binding of both Ca<sup>2+</sup> and cTnI to cTnC. Nuclear magnetic resonance chemical shift and relaxation data provided evidence that the cNTnC hydrophobic core is more exposed with the L48Q variant. Molecular dynamics simulations suggest that the mutation disrupts a network of crucial hydrophobic interactions so that the closed form of cNTnC is destabilized. The findings emphasize the importance of cNTnC’s conformation in the regulation of contraction and suggest that mutations in cNTnC that alter myofilament Ca<sup>2+</sup> sensitivity can do so by modulating Ca<sup>2+</sup> and cTnI binding

    Structural and Functional Consequences of the Cardiac Troponin C L48Q Ca<sup>2+</sup>-Sensitizing Mutation

    No full text
    Calcium binding to the regulatory domain of cardiac troponin C (cNTnC) causes a conformational change that exposes a hydrophobic surface to which troponin I (cTnI) binds, prompting a series of protein–protein interactions that culminate in muscle contraction. A number of cTnC variants that alter the Ca<sup>2+</sup> sensitivity of the thin filament have been linked to disease. Tikunova and Davis engineered a series of cNTnC mutations that altered Ca<sup>2+</sup> binding properties and studied the effects on the Ca<sup>2+</sup> sensitivity of the thin filament and contraction [Tikunova, S. B., and Davis, J. P. (2004) <i>J. Biol. Chem. 279</i>, 35341–35352]. One of the mutations they engineered, the L48Q variant, resulted in a pronounced increase in the cNTnC Ca<sup>2+</sup> binding affinity and Ca<sup>2+</sup> sensitivity of cardiac muscle force development. In this work, we sought structural and mechanistic explanations for the increased Ca<sup>2+</sup> sensitivity of contraction for the L48Q cNTnC variant, using an array of biophysical techniques. We found that the L48Q mutation enhanced binding of both Ca<sup>2+</sup> and cTnI to cTnC. Nuclear magnetic resonance chemical shift and relaxation data provided evidence that the cNTnC hydrophobic core is more exposed with the L48Q variant. Molecular dynamics simulations suggest that the mutation disrupts a network of crucial hydrophobic interactions so that the closed form of cNTnC is destabilized. The findings emphasize the importance of cNTnC’s conformation in the regulation of contraction and suggest that mutations in cNTnC that alter myofilament Ca<sup>2+</sup> sensitivity can do so by modulating Ca<sup>2+</sup> and cTnI binding
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