6 research outputs found
Validating Molecular Dynamics Simulations against Experimental Observables in Light of Underlying Conformational Ensembles
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
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
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
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
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
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