Conformational sampling of metastable states: Tq-REM as a novel replica exchange method.

Although the replica exchange methods (REMs) were developed as efficient conformational sampling methods for bio-molecular simulations, their application to very large bio-systems is somewhat limited. We propose a new replica exchange scheme (Tq-REM) created by combining the conventional temperature-REM (T-REM) and one of the Hamiltonian-REMs, q-REM, using the effective potential with reduced barriers. In the proposed Tq-REM scheme, high temperature replicas in T-REM are substituted with q-replicas. This combined scheme is expected to exploit advantages of the T-REM and q-REM resulting in improved sampling efficiency while minimizing the drawbacks of both approaches. We investigated the performance of Tq-REM compared with T-REM by performing all-atom MD simulations on Met-enkephalin, (AAQAA)3, and Trpzip2. It was found that convergence of the free energy surfaces was improved by Tq-REM over the conventional T-REM. In particular, the trajectories of Tq-REM were able to sample the relevant conformations for all of the metastable folding intermediates, while some of the local minimum structures are poorly represented by T-REM. The results of the present study suggest that Tq-REM can provide useful tools to investigate systems where metastable states play important roles.

Understanding Antimicrobial Peptide Synergy: Differential Binding Interactions and Their Impact on Membrane Integrity.

Research on antimicrobial peptides (AMPs) has been conducted as a solution to overcome antibiotic resistance. In particular, the synergistic effect that appears when two or more AMPs are used in combination has been observed. To find an effective synergistic combination, it is necessary to understand the underlying mechanism. However, a consistent explanation for this phenomenon has not yet been provided due to limitations in experimentally determining or predicting the structure of the heteroaggregates formed by the interactions between different AMPs and the interaction of the aggregate surface with the lipid membrane surface. In this study, we conducted molecular dynamics simulations for two heterogeneous aggregates of melittin-indolicidin and pexiganan-indolicidin to observe their structures in the solution phase and their interactions with the lipid membrane. We aimed to determine how the surfaces of these aggregates interact with the lipid membrane. Due to the different amino acid residue sequence characteristics of melittin and pexiganan, we found that when the two AMPs bind to indolicidin, they form aggregates with completely different structural characteristics. Accordingly, the sequence characteristics of pexiganan, which exhibits a relatively unstable structure compared to melittin in aqueous solution or on lipid membranes, allow for a more stable interaction with the lipid membrane when forming aggregates with indolicidin, effectively inhibiting the integrity of the lipid membranes. We also found that the amino acid residues forming the surface of the AMP aggregate show differential binding strengths to different lipid species forming the lipid membrane, thereby disrupting the membrane in a way that weakens its integrity. Through this, we provided insight into the basic principle of how the synergistic effect of AMPs occurs.

Urea-induced denaturation of preQ1-riboswitch.

Urea, a polar molecule with a large dipole moment, not only destabilizes folded RNA structures but can also enhance the folding rates of large ribozymes. Unlike the mechanism of urea-induced unfolding of proteins, which is well understood, the action of urea on RNA has barely been explored. We performed extensive all-atom molecular dynamics simulations to determine the molecular underpinnings of urea-induced RNA denaturation. Urea displays its denaturing power in both secondary and tertiary motifs of the riboswitch structure. Our simulations reveal that the denaturation of RNA structures is mainly driven by the hydrogen-bonding and stacking interactions of urea with the bases. Through detailed studies of the simulation trajectories, we found that geminate pairs between urea and bases due to hydrogen bonds and stacks persist only ~0.1-1 ns, which suggests that the urea-base interaction is highly dynamic. Most importantly, the early stage of base-pair disruption is triggered by penetration of water molecules into the hydrophobic domain between the RNA bases. The infiltration of water into the narrow space between base pairs is critical in increasing the accessibility of urea to transiently disrupted bases, thus allowing urea to displace inter-base hydrogen bonds. This mechanism--water-induced disruption of base pairs resulting in the formation of a "wet" destabilized RNA followed by solvation by urea--is the exact opposite of the two-stage denaturation of proteins by urea. In the latter case, initial urea penetration creates a dry globule, which is subsequently solvated by water, leading to global protein unfolding. Our work shows that the ability to interact with both water and polar or nonpolar components of nucleotides makes urea a powerful chemical denaturant for nucleic acids.

In silico investigation of the structural stability as the origin of the pathogenicity of α-synuclein protofibrils.

α-Synuclein is a presynaptic neuronal protein. The fibril form of α-synuclein is a major constituent of the intraneuronal inclusion called Lewy body, a characteristic hallmark of Parkinson's disease. Recent ssNMR and cryo-EM experiments of wild-type α-synuclein fibrils have shown polymorphism and observed two major polymorphs, rod and twister. To associate the cytotoxicity of α-synuclein fibrils with their structural features, it is essential to understand the origins of their structural stability. In this study, we performed molecular dynamics simulations of the two major polymorphs of wild-type α-synuclein fibrils. The predominance of specific fibril polymorphs was rationalized in terms of relative structural stability in aqueous environments, which was attributed to the cooperative contributions of various stabilizing features. The results of the simulations indicated that highly stable structures in aqueous environments could be maintained by the cooperation of compact sidechain packing in the hydrophobic core, backbone geometry of the maximal ß-sheet content wrapping the hydrophobic core, and solvent-exposed sidechains with large fluctuations maximizing the solvation entropy. The paired structure of the two protofilaments provides additional stability, especially at the interface region, by forming steric zipper interactions and hiding the hydrophobic residues from exposure to water. The sidechain interaction analyses and pulling simulations showed that the rod polymorph has stronger sidechain interactions and exhibits higher dissociation energy than the twister polymorph. It is expected that our study will provide a basis for understanding the pathogenic behaviors of diverse amyloid strains in terms of their structural properties.Communicated by Ramaswamy H. Sarma.

Computational Insights into the Aggregation Pathway of Self-Assembled Nanotubules.

We performed molecular dynamics simulations of self-assembled supramolecular nanotubules constructed from amphiphiles with bent-shaped rods. By systematically examining the structure from dimeric aggregates to the fully developed nanotubule, we identified the basic building block of the nanotubule and the optimal dimensions of its stable structure which are consistent with experimental findings. Moreover, we demonstrate that the cooperative interplay of different interactions drives aggregation by selecting and stabilizing the optimal self-assembled structures for various intermediates through a complex pathway. Additionally, contraction of the nanotubule, which accompanies the dehydration process, was observed upon heating. It is suggested that the optimal stability of the self-assembled aggregates is achieved by balancing entropic and enthalpic contributions, of which the ratio is a critical factor that drives the aggregation pathway.

Computational Study on Structure and Aggregation Pathway of Aß42 Amyloid Protofibril.

Amyloid deposits of Aß protein in neuronal cells are known to be a major symptom of Alzheimer's disease. In particular, Aß42 shows relatively high toxicity among the different Aß isoforms, and its toxicity is thought to be because of its structural features. Recent ssNMR and cryo-EM experiments identified that Aß42 shows an S-shaped triple-ß structure, in contrast to the previously suggested U-shaped ß-arch structure. In order to associate the high toxicity of Aß42 with its structural features, it is essential to explain the conformational stability and aggregation mechanisms of this triple-ß motif. We utilized several different simulation methods, including extensive straight molecular dynamics simulation, steered molecular dynamics simulation, and replica-exchange molecular dynamics simulation. The S-shaped triple-ß motif showed remarkable structural stability because of its complex residual interactions that form stable hydrophobic cores. The triple-ß structure of Aß42 is primarily made up of three ß-sheet regions and two hydrophobic cores formed between ß-sheet regions. Our analysis of ß-sheet rupture patterns between adjacent chains showed that its two hydrophobic cores have different degrees of stability, indicating a lock phase mechanism. Our analysis of the docking pathway of monomeric Aß42 to the fibril motif using REMD simulations showed that each of the three ß-sheet sequences plays a distinct role in the docking process by changing their conformational features. Our results provide an understanding for the stability and consequent high toxicity of the triple-ß structure Aß42.

Conformational characteristics of unstructured peptides: alpha-synuclein.

We have performed replica-exchange molecular dynamics simulations on 41 residue peptides containing NAC region of alpha-synuclein in various force fields and solvent conditions. Alpha-synuclein is known to be the major cause of Parkinson's disease by amyloid-like aggregation, and one of the natively unfolded proteins. To investigate conformational characteristics of intrinsically unstructured peptides, we carried out structural analysis by introducing 'representative structure' for ensemble of structures occurring during the overall trajectory. Representative structures may be defined by using either coordinate averaging or distance averaging. When applied to the natively folded proteins such as villin headpiece, structural analysis based on representative structure was found to yield consistent results with those obtained from conventional analysis. Individual conformations obtained from the simulations of NAC peptide for various conditions show flexible structures close to random coil. Secondary structure contents and free energy surfaces showed dependency on solvent conditions, which may be interpreted as another manifestation of structural diversity. It is found that representative structures can provide useful information about structural characteristics of intrinsically unstructured proteins.

The role of the acidic domain of α-synuclein in amyloid fibril formation: a molecular dynamics study.

The detailed mechanism of the pathology of α-synuclein in the Parkinson's disease has not been clearly elucidated. Recent studies suggested a possible chaperone-like role of the acidic C-terminal region of α-synuclein in the formation of amyloid fibrils. It was also previously demonstrated that the α-synuclein amyloid fibril formation is accelerated by mutations of proline residues to alanine in the acidic region. We performed replica exchange molecular dynamics simulations of the acidic and nonamyloid component (NAC) domains of the wild type and proline-to-alanine mutants of α-synuclein under various conditions. Our results showed that structural changes induced by a change in pH or an introduction of mutations lead to a reduction in mutual contacts between the NAC and acidic regions. Our data suggest that the highly charged acidic region of α-synuclein may act as an intramolecular chaperone by protecting the hydrophobic domain from aggregation. Understanding the function of such chaperone-like parts of fibril-forming proteins may provide novel insights into the mechanism of amyloid formation.

Using simulations and kinetic network models to reveal the dynamics and functions of riboswitches.

Riboswitches, RNA elements found in the untranslated region, regulate gene expression by binding to target metaboloites with exquisite specificity. Binding of metabolites to the conserved aptamer domain allosterically alters the conformation in the downstream expression platform. The fate of gene expression is determined by the changes in the downstream RNA sequence. As the metabolite-dependent cotranscriptional folding and unfolding dynamics of riboswitches are the key determinant of gene expression, it is important to investigate both the thermodynamics and kinetics of riboswitches both in the presence and absence of metabolite. Single molecule force experiments that decipher the free energy landscape of riboswitches from their mechanical responses, theoretical and computational studies have recently shed light on the distinct mechanism of folding dynamics in different classes of riboswitches. Here, we first discuss the dynamics of water around riboswitch, highlighting that water dynamics can enhance the fluctuation of nucleic acid structure. To go beyond native state fluctuations, we used the Self-Organized Polymer model to predict the dynamics of add adenine riboswitch under mechanical forces. In addition to quantitatively predicting the folding landscape of add-riboswitch, our simulations also explain the difference in the dynamics between pbuE adenine- and add adenine-riboswitches. In order to probe the function in vivo, we use the folding landscape to propose a system level kinetic network model to quantitatively predict how gene expression is regulated for riboswitches that are under kinetic control.

Dynamical transition and heterogeneous hydration dynamics in RNA.

Enhanced dynamical fluctuations of RNAs, facilitated by a network of water molecules with strong interactions with RNA, are suspected to be critical in their ability to respond to a variety of cellular signals. Using atomically detailed molecular dynamics simulations at various temperatures of purine (adenine) and preQ1 sensing riboswitch aptamers, which control gene expression by sensing and binding to metabolites, we show that water molecules in the vicinity of RNAs undergo complex dynamics depending on the local structures of the RNAs. The overall lifetimes of hydrogen bonds (HBs) of surface-bound waters are more than at least 1-2 orders of magnitude longer than those of bulk water. Slow hydration dynamics, revealed in the non-Arrhenius behavior of the relaxation time, arises from high activation barriers to break water HBs with a nucleotide and by reduced diffusion of water. The relaxation kinetics at specific locations in the two RNAs show a broad spectrum of time scales reminiscent of glass-like behavior, suggesting that the hydration dynamics is highly heterogeneous. Both RNAs undergo dynamic transition at T = TD ≳ 200 K, as assessed by the mean-square fluctuation of hydrogen atoms ⟨x(2)⟩, which undergoes an abrupt harmonic-to-anharmonic transition at TD. The near-universal value of TD found for these RNAs and previously for tRNA is strongly correlated with changes in hydration dynamics as T is altered. Hierarchical dynamics of waters associated with the RNA surface, revealed in the motions of distinct classes of water with well-separated time scales, reflects the heterogeneous local environment on the molecular surface of RNA. At low temperatures, slow water dynamics predominates over structural transitions. Our study demonstrates that the complex interplay of dynamics between water and the local environment in the RNA structures could be a key determinant of the functional activities of RNA.

Hidden complexity in the isomerization dynamics of Holliday junctions.

A plausible consequence of the rugged folding energy landscapes inherent to biomolecules is that there may be more than one functionally competent folded state. Indeed, molecule-to-molecule variations in the folding dynamics of enzymes and ribozymes have recently been identified in single-molecule experiments, but without systematic quantification or an understanding of their structural origin. Here, using concepts from glass physics and complementary clustering analysis, we provide a quantitative method to analyse single-molecule fluorescence resonance energy transfer (smFRET) data, thereby probing the isomerization dynamics of Holliday junctions, which display such heterogeneous dynamics over a long observation time (T(obs) ≈ 40 s). We show that the ergodicity of Holliday junction dynamics is effectively broken and that their conformational space is partitioned into a folding network of kinetically disconnected clusters. Theory suggests that the persistent heterogeneity of Holliday junction dynamics is a consequence of internal multiloops with varying sizes and flexibilities frozen by Mg(2+) ions. An annealing experiment using Mg(2+) pulses lends support to this idea by explicitly showing that interconversions between trajectories with different patterns can be induced.

Simulation studies on the stabilities of aggregates formed by fibril-forming segments of alpha-Synuclein.

We performed molecular dynamics simulations for various oligomers with different beta-sheet conformations consisting of alpha-Synuclein 71-82 residues using an all atom force field and explicit water model. Tetramers of antiparallel beta-sheet are shown to be stable, whereas parallel sheets are highly unstable due to the repulsive interactions between bulky and polar side chains as well as the weaker backbone hydrogen bonds. We also investigated the stabilities of double antiparallel beta-sheets stacked with asymmetric and symmetric geometries. Our results show that this 12 amino acid residue peptide can form stable beta-sheet conformers at 320K and higher temperatures. The backbone hydrogen bonds in beta-sheet and the steric packing between hydrophobic side chains between beta-sheets are shown to give conformational stabilities.