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Quantitative comparisons of intrinsically disordered proteins (IDPs) with similar sequences, such as mutant forms of the same protein, may provide insights into IDP aggregation—a process that plays a role in several neurodegenerative disorders. Here we describe an approach for modeling IDPs with similar sequences that simplifies the comparison of the ensembles by utilizing a single library of structures. The relative population weights of the structures are estimated using a Bayesian formalism, which provides measures of uncertainty in the resulting ensembles. We applied this approach to the comparison of ensembles for Aβ40 and Aβ42. Bayesian hypothesis testing finds that although both Aβ species sample β-rich conformations in solution that may represent prefibrillar intermediates, the probability that Aβ42 samples these prefibrillar states is roughly an order of magnitude larger than the frequency in which Aβ40 samples such structures. Moreover, the structure of the soluble prefibrillar state in our ensembles is similar to the experimentally determined structure of Aβ that has been implicated as an intermediate in the aggregation pathway. Overall, our approach for comparative studies of IDPs with similar sequences provides a platform for future studies on the effect of mutations on the structure and function of disordered proteins.

Studies on collagen and collagen-like peptides suggest that triple-helical stability can vary along the amino acid chain. In this regard, it has been shown that lysine residues in the Y position and acidic residues in the X′ position of (GPO)[subscript 3]GXYGX′Y′(GPO)[subscript 3] peptides lead to triple-helical structures with melting temperatures similar to (GPO)[subscript 8] (where O is hydroxyproline), which is generally regarded as the most stable collagen-like sequence of this length. This enhanced stability has been attributed to the formation of salt bridges between adjacent collagen chains. In this study, we explore the relationship between interchain salt bridge formation and triple-helical stability using detailed molecular simulations. Although our results confirm that salt bridges promote triple-helical stability, we find that not all salt bridges are created equal. In particular, lysine-glutamate salt bridges are most stabilizing when formed between residues in the middle strand (B) and the trailing strand (C), whereas lysine-aspartate salt bridges are most stabilizing when formed between residues in the leading (A) and middle (B) strand—the latter observation being consistent with recent NMR data on a heterotrimeric model peptide. Overall, we believe these data clarify the role of salt bridges in modulating triple-helical stability and can be used to guide the design of collagen-like peptides that have specific interchain interactions., National Science Foundation (U.S.) (Grant 0745638), National Science Foundation (U.S.) (Grant 0821391)

Intrinsic disorder plays a key role in the regulation of cell death by bacterial toxin-antitoxin (TA) modules. In TA modules, an unstable antitoxin normally inhibits a protein toxin. Cellular stressors trigger increased degradation of the labile antitoxin, thereby releasing the toxin. When not inhibited, the toxin disrupts essential cellular processes, causing cell death or quiescence. The CcdA-CcdB TA module in E. coli consists of the antitoxin CcdA and the toxin CcdB. CcdA is comprised of a folded DNA-binding domain and two intrinsically disordered regions (IDRs), which regulate binding to CcdB. NMR studies suggest that CcdA, in the absence of CcdB, predominantly samples conformations belonging to either a closed or an open state, distinguished by the distance from the IDR termini to the folded domain. The potential for CcdA to sample multiple partially stable states provokes the question of the role these states play in facilitating the IDRs’ functions. We use all-atom explicit-water molecular dynamics (MD) simulations and native ion mobility-mass spectrometry (MS) to determine the conformational ensemble of unbound CcdA, with the goal of inferring functional roles from structural details. Both MD and MS indicate that CcdA samples metastable states of varying compactness. In one state CcdA can adopt compact, relatively closed conformations, as predicted by NMR. In a separate state, that has similar energy, one IDR extends away from the central folded domain. Further analysis of IDR helicity and solvent exposure within each substate provides insight into the functional role of these states. As intrinsically disordered antitoxin proteins like CcdA are plentiful in bacteria, understanding how disorder facilitates their functions could lead to novel antibiotics that harness TA modules to kill bacterial cells.

Prior investigations into the effects hyperglycemia on collagen degradation have yielded conflicting results. We present a new formalism for understanding the biochemistry of collagenolysis and the effects of hyperglycemia on collagen degradation. The analysis is based on an understanding of environments that affect the conformational stability of collagen. We suggest that collagen can exist in two distinct conformational states—a native state and a vulnerable state. Vulnerable collagen corresponds to a non-native conformation where partially unfolded regions near collagenase cleavage sites enable collagenases to efficiently degrade collagen. Theoretical calculations on collagen-like model peptides suggest that relatively short periods of hyperglycemia can alter the equilibrium distribution of states to favor vulnerable states of collagen. These data provide new insights into the mechanism of collagenolysis and resolve apparently discrepant experimental data on the effects of hyperglycemia on collagen degradation.

The classical structure-function paradigm states that the amino acid sequence of a protein encodes a unique 3-dimensional structure that determines the protein's biological function. The view that most proteins adopt an ordered native conformation has been bolstered by the success of X-ray crystallography in determining high-resolution protein structures. Nevertheless, evidence from nuclear magnetic resonance spectroscopy, single molecule experiments and other techniques suggests that proteins exhibit a high degree of flexibility, which is critical for processes like molecular recognition. An information theoretic order parameter that describes the degree of heterogeneity in a protein conformational ensemble was used to characterize the flexibility of representative protein folds using computer simulations and a large sample of crystallographic B-factors from high-resolution protein structures in the Protein Data Bank. These data demonstrate that many proteins which are typically described by an ordered native structure, in fact, populate multiple conformational states.

Alpha-Synuclein is an intrinsically disordered protein (IDP) shown to be implicated in the pathogenesis of Parkinson's disease. IDPs do not adopt a well defined secondary structure; instead they are thought to adopt a heterogeneous collection of conformations in solution. A combination of a collection of conformations and their relative stabilities - an ensemble - allows insight into the complex energy landscape of IDPs. In this work we use a Bayesian weighting algorithm developed in our lab to model the ensemble of alpha-Synuclein. We incorporate experimental data from previously published work together with molecular dynamics simulations to form an ensemble that agrees with experiment. By using the Bayesian approach we do not rely on a single set of weights but rather on a distribution over possible sets of weights. In addition the Baysian approach allows for a novel uncertainty measure, by which we can quantify our confidence in predictions made using the ensemble. Alpha-Synuclein was shown to aggregate in vitro and was found to be the major component in neuronal inclusions known as Lewy bodies. It is believed that the formation of alpha-Synuclein aggregates has a toxic effect on dopaminergic neurons. Residues 68-78 in alpha-Synuclein were shown to be a critical component for alpha-Synuclein aggregation and toxicity. We present preliminary results analyzing long-range contacts within alpha-Synuclein. We find alpha-Synuclein forms multiple long-range contacts throughout the sequence; however, residues 68-78 form comparatively few long-range contacts with distant residues within alpha-Synuclein. In addition we find that this region has a rather large solvent accessible surface area of 60% its maximum value.

A number of disorders such as tumor metastasis, arthritis, and atherosclerotic heart disease are related to excessive collagen degradation. Therefore methods that further our understanding of collagenolysis are of particular interest. However, as the collagenase active site is too small to accommodate the triple-helical structure of collagen and the scissile bond within collagen is not accessible to the collagenase catalytic site, the precise molecular mechanism of collagenolysis is unclear. Prior experiments have been interpreted to mean that collagenases actively unfold collagen, in a process that requires intact full length collagenase - which is typically a multi-domain protein, containing both a catalytic and hemopexin-like domain - leading to exposure of the scissile bond. Here we demonstrate that collagen types I and III can be degraded by the catalytic domain alone of either MMP1 or MMP8 at temperatures far below the melting temperature of collagen. These data argue that active unwinding of collagen is not required for collagenolysis to occur in solution. Molecular simulations further suggest that normal thermal fluctuations in the structure of the triple-helical structure of collagen lead to the protein sampling states where the scissile bond is relatively exposed and hence accessible to collagenase active site. Taken together, these data suggest that collagen degradation is the result of the interaction of preformed locally unfolded states of collagen and collagenases, rather than a mechanism that involves active collagenase-mediated unfolding.

Implicit solvent models approximate the effects of solvent through a potential of mean force and therefore make solvated simulations computationally efficient. Yet despite their computational efficiency, the inherent approximations made by implicit solvent models can sometimes lead to inaccurate results. To test the accuracy of a number of popular implicit solvent models, we determined whether implicit solvent simulations can reproduce the set of potential energy minima obtained from explicit solvent simulations. For these studies, we focus on a six-residue amino-acid sequence, referred to as the paired helical filament 6 (PHF6), which may play an important role in the formation of intracellular aggregates in patients with Alzheimer’s disease. Several implicit solvent models form the basis of this work—two based on the generalized Born formalism, and one based on a Gaussian solvent-exclusion model. All three implicit solvent models generate minima that are in good agreement with minima obtained from simulations with explicit solvent. Moreover, free-energy profiles generated with each implicit solvent model agree with free-energy profiles obtained with explicit solvent. For the Gaussian solvent-exclusion model, we demonstrate that a straightforward ranking of the relative stability of each minimum suggests that the most stable structure is extended, a result in excellent agreement with the free-energy profiles. Overall, our data demonstrate that for some peptides like PHF6, implicit solvent can accurately reproduce the set of local energy minimum arising from quenched dynamics simulations with explicit solvent. More importantly, all solvent models predict that PHF6 forms extended β-structures in solution, a finding consistent with the notion that PHF6 initiates neurofibrillary tangle formation in patients with Alzheimer’s disease.

Nickel homeostasis in gram-negative bacteria is often regulated at the transcriptional level by the nickel-dependent protein NikR, which belongs to the ribbon-helix-helix DNA-binding protein family. Generally, NikR is found as a homotetramer, with four nickel ions bound of the tetrameric interface at a metal binding domain (MBD). In Helicobacter pylori, NikR (HpNikR), unlike homologous NikR proteins, sits at a regulatory hub and has been found to have a variety of regulatory roles. HpNikR has been implicated in the regulation of proteins that function in acid acclimation, nickel uptake, virulence, and stress response. There are a number of questions about the nickel coordination environment in HpNikR due to a series of puzzling and sometimes contradictory structural and experimental results. Here we present results from complimentary computational and experimental techniques to help further resolve these questions. Specifically, we use molecular dynamics simulations and spectroscopic experiments to investigate the nickel coordination environment. We use circular dichroism to observe differences in the thermal stability of HpNikR depending on the nickel:protein stoichiometry, with stabilization increasing as nickel concentration increases. Stability of HpNikR is found to be pH dependent. Elucidation of details about nickel coordination in HpNikR will improve our understanding of the functional role of genetic regulatory pathways in H. pylori pathogenicity.

Escherichia coli NikR (EcNikR) regulates cellular nickel uptake by binding free nickel ions at high affinity metal binding sites in the protein, which induces EcNikR binding to the nik operon - a process that leads to suppression of the gene encoding the nickel uptake transporter, NikABCDE. A structure of the EcNikR-DNA complex suggests that a second metal binding site is present in addition to the high affinity sites, and raises the question of which metal occupies this second site under physiologic conditions: K+, which is present in the crystal structure, or Ni+2. To determine which ion is preferred at the second metal binding site, and the reason for any preference of one ion over another, we calculated the electrostatic free energy of EcNikR binding to DNA with either K+ or Ni+2 in the second site. While the interaction between EcNikR and DNA is more favorable when the second site contains Ni+2, the large desolvation penalty associated with moving Ni+2 from solution to the relatively buried second site offsets this favorable interaction. Consequently, our data suggest that EcNikR binding to DNA is more favorable when the second site contains a K+ ion. Moreover, additional calculations indicate that the second site is best suited for an ion having the size of K+ and not Ni+2, suggesting that the second site is optimized for K+. Taken together, our data suggest that the second metal binding site contains K+ and explain why K+ is preferred over Ni+2 at this site.

Ribonucleotide Reductase (RNR) is an enzyme that catalyzes the production of deoxyribonucletides from ribonucleotides. The active form of E. coli RNR is comprised of two homodimeric subunits, α2 and β2, and catalysis involves radical transfer between Y122 on the β2 subunit and C439 on the α2 subunit. By contrast, recent structural data suggest that the dATP-inhibited (inactive) form of the enzyme corresponds to an α4β4 ring. In this structure Y122 on the β2 subunit and C439 on the α2 subunit are separated by more than 60 angstroms - a finding that explains, in part, why this structure is inactive. Interestingly, in crystal structures of the inactive complex, ∼27 residues near the C-terminus the β2 subunit do not have electron density, suggesting that these residues are disordered in the inactive form of the enzyme. This missing “linker region” contains a tyrosine residue (Y356) that plays a crucial role in radical transfer in the active complex. In this work we use molecular simulations to explore the conformational space of these disordered residues in both active and inactive models of E. coli RNR. Our data suggest that Y356 adopts states that are inconsistent with radical transfer in the inactive complex. These observations argue that inactivity is explained both by the increased distance required for radical transfer, and by the relative position of crucial residues in the flexible linker region in the inactive state.

Collagen is an important structural biomolecule that is abundant in all animals. Proper degradation and remodeling of collagen is crucial to an organism's survival; moreover, excessive degradation of collagen is involved in many disease processes such as atherosclerosis, cancer metastasis, and arthritis. While a number of studies have shed light on the mechanism of collagenolysis in solution, the precise mechanism of collagen degradation in the native fiber remains mysterious. In this work, we use molecular dynamics simulations on a model of native collagen fibrils, obtained from recent fiber diffraction data, to understand the mechanism of fibrillar degradation. Our data suggest that the unique collagenase cleavage site in native fibrillar state behaves much like it does in solutions composed of tropocollagen molecules. In particular, in the fibrillar state the region about the collagenase cleavage site can adopt two distinct conformational states - one where the atoms comprising the scissile bond are relatively hidden and another state where the scissile bond is relatively exposed, or vulnerable, to collagenolysis. These data extends our previous work and suggests that conformational flexibility plays an important role in determining the rate of collagenolysis in collagen fibrils.

Amyloid-β, an intrinsically disordered protein, forms amyloid fibrils in the brains of patients with Alzheimer's disease. A recent study suggests that fibrils can catalyze the formation of neurotoxic soluble oligomeric species. We therefore explored the folding free energy landscape of Amyloid-β42 using extensive molecular dynamics (>50 μs) coupled with umbrella sampling. We find that fibril elongation occurs through a funnel-shaped, downhill free energy pathway, and we identify a key on-pathway intermediate that involves the interaction of a beta hairpin with the fibril core. Furthermore, an analysis of the minimum-energy pathway suggests two potential mechanisms by which fibrils can act as catalysts for soluble oligomer formation: 1) pre-existing fibrils provide a nucleation site at which the local concentration of monomers is increased and; 2) fibril elongation involves monomeric intermediates that can be incorporated into soluble oligomers. These data provide the first detailed mechanistic description of the elongation of amyloid β fibrils and provide a link between the disordered free monomeric protein on the one hand and the folded fibrillar state on the other. In addition, they suggest a rational drug design strategy that aims to stabilize the fibril-associated hairpin conformation in order to prevent elongation of pre-existing fibrils.