Characterizing amyloid‐beta protein misfolding from molecular dynamics simulations with explicit water

Extracellular deposition of amyloid‐beta (Aβ) protein, a fragment of membrane glycoprotein called β‐amyloid precursor transmembrane protein (βAPP), is the major characteristic for the Alzheimer's disease (AD). However, the structural and mechanistic information of forming Aβ protein aggregates in a lag phase in cell exterior has been still limited. Here, we have performed multiple all‐atom molecular dynamics simulations for physiological 42‐residue amyloid‐beta protein (Aβ42) in explicit water to characterize most plausible aggregation‐prone structure (APS) for the monomer and the very early conformational transitions for Aβ42 protein misfolding process in a lag phase. Monitoring the early sequential conformational transitions of Aβ42 misfolding in water, the APS for Aβ42 monomer is characterized by the observed correlation between the nonlocal backbone H‐bond formation and the hydrophobic side‐chain exposure. Characteristics on the nature of the APS of Aβ42 allow us to provide new insight into the higher aggregation propensity of Aβ42 over Aβ40, which is in agreement with the experiments. On the basis of the structural features of APS, we propose a plausible aggregation mechanism from APS of Aβ42 to form fibril. The structural and mechanistic observations based on these simulations agree with the recent NMR experiments and provide the driving force and structural origin for the Aβ42 aggregation process to cause AD. © 2010 Wiley Periodicals, Inc. J Comput Chem, 2011     

DIRECT‐ID: An automated method to identify and quantify conformational variations—application to β2‐adrenergic GPCR

The conformational dynamics of a macromolecule can be modulated by a number of factors, including changes in environment, ligand binding, and interactions with other macromolecules, among others. We present a method that quantifies the differences in macromolecular conformational dynamics and automatically extracts the structural features responsible for these changes. Given a set of molecular dynamics (MD) simulations of a macromolecule, the norms of the differences in covariance matrices are calculated for each pair of trajectories. A matrix of these norms thus quantifies the differences in conformational dynamics across the set of simulations. For each pair of trajectories, covariance difference matrices are parsed to extract structural elements that undergo changes in conformational properties. As a demonstration of its applicability to biomacromolecular systems, the method, referred to as DIRECT‐ID, was used to identify relevant ligand‐modulated structural variations in the β₂‐adrenergic (β₂AR) G‐protein coupled receptor. Micro‐second MD simulations of the β₂AR in an explicit lipid bilayer were run in the apo state and complexed with the ligands: BI‐167107 (agonist), epinephrine (agonist), salbutamol (long‐acting partial agonist), or carazolol (inverse agonist). Each ligand modulated the conformational dynamics of β₂AR differently and DIRECT‐ID analysis of the inverse‐agonist vs. agonist‐modulated β₂AR identified residues known through previous studies to selectively propagate deactivation/activation information, along with some previously unidentified ligand‐specific microswitches across the GPCR. This study demonstrates the utility of DIRECT‐ID to rapidly extract functionally relevant conformational dynamics information from extended MD simulations of large and complex macromolecular systems. © 2015 Wiley Periodicals, Inc.     

Protein structure predictions by parallel simulated annealing molecular dynamics using genetic crossover

We propose a conformational search method to find a global minimum energy structure for protein systems. The simulated annealing is a powerful method for local conformational search. On the other hand, the genetic crossover can search the global conformational space. Our method incorporates these attractive features of the simulated annealing and genetic crossover. In the previous works, we have been using the Monte Carlo algorithm for simulated annealing. In the present work, we use the molecular dynamics algorithm instead. To examine the effectiveness of our method, we compared our results with those of the normal simulated annealing molecular dynamics simulations by using an α-helical miniprotein. We used genetic two-point crossover here. The conformations, which have lower energy than those obtained from the conventional simulated annealing, were obtained.     

Helix formation in a pentapeptide: experiment and force-field dependent dynamics

We used a combined approach of experiment and simulation to determine the helical population and folding pathway of a small helix forming blocked pentapeptide, Ac-(Ala)(5)-NH(2). Experimental structural characterization of this blocked peptide was carried out with far UV circular dichroism spectroscopy, FTIR, and NMR measurements. These measurements confirm the presence of the α-helical state in a buffer solution. Direct molecular dynamics and replica-exchange simulations of the pentapeptide were performed using several popular force fields with explicit solvent. The simulations yielded statistically reliable estimates of helix populations, melting curves, folding, and nucleation times. The distributions of conformer populations are used to measure folding cooperativity. Finally, a statistical analysis of the sample of helix-coil transition paths was performed. The details of the calculated helix populations, folding kinetics and pathways vary with the employed force field. Interestingly, the helix populations, folding, and unfolding times obtained from most of the studied force fields are in qualitative agreement with each other and with available experimental data, with the deviations corresponding to several kcal/mol in energy at 300 K. Most of the force fields also predict qualitatively similar transition paths, with unfolding initiated at the C-terminus. Accuracy of potential energy parameters, rather than conformational sampling may be the limiting factor in current molecular simulations.     

Direct observations of conformational distributions of intrinsically disordered p53 peptides using UV Raman and explicit solvent simulations

We report the first experimental measurements of Ramachandran Ψ-angle distributions for intrinsically disordered peptides: the N-terminal peptide fragment of tumor suppressor p53 and its P27S mutant form. To provide atomically detailed views of the conformational distributions, we performed classical, explicit-solvent molecular dynamics simulations on the microsecond time scale. Upon binding its partner protein, MDM2, wild-type p53 peptide adopts an α-helical conformation. Mutation of Pro27 to serine results in the highest affinity yet observed for MDM2-binding of the p53 peptide. Both UV resonance Raman spectroscopy (UVRR) and simulations reveal that the P27S mutation decreases the extent of PPII helical content and increases the probability for conformations that are similar to the α-helical MDM2-bound conformation. In addition, UVRR measurements were performed on peptides that were isotopically labeled at the Leu26 residue preceding the Pro27 in order to determine the conformational distributions of Leu26 in the wild-type and mutant peptides. The UVRR and simulation results are in quantitative agreement in terms of the change in the population of non-PPII conformations involving Leu26 upon mutation of Pro27 to serine. Finally, our simulations reveal that the MDM2-bound conformation of the peptide is significantly populated in both the wild-type and mutant isolated peptide ensembles in their unbound states, suggesting that MDM2 binding of the p53 peptides may involve conformational selection.     

Ultrafast dynamics of nonequilibrium resonance energy transfer and probing globular protein flexibility of myoglobin

Protein structural plasticity is critical to many biological activities and accurate determination of its temporal and spatial fluctuations is challenging and difficult. Here, we report our extensive characterization of global flexibility of a globular heme protein of myoglobin using resonance energy transfer as a molecular ruler. With site-directed mutagenesis, we use a tryptophan scan to examine local structural fluctuations from B to H helices utilizing 10 tryptophan-heme energy transfer pairs with femtosecond resolution. We observed ultrafast resonance energy transfer dynamics by following a nearly single exponential behavior in 10-100 ps, strongly indicating that the globular structure of myoglobin is relatively rigid, with no observable static or slow dynamic conformational heterogeneity. The observation is against our molecular dynamics simulations, which show large local fluctuations and give multiple exponential energy transfer behaviors, suggesting too flexible of the global structure and thus raising a serious issue of the force fields used in simulations. Finally, these ultrafast energy transfer dynamics all occur on the similar time scales of local environmental relaxations (solvation), leading to nonexponential processes caused by energy relaxations, not structural fluctuations. Our analyses of such processes reveal an intrinsic compressed- and/or stretched-exponential behaviors and elucidate the nature of inherent nonequilibrium of ultrafast resonance energy transfer in proteins. This new concept of compressed nonequilibrium transfer dynamics should be applied to all protein studies by time-resolved F?rster resonance energy transfer (FRET).     

Structural dissimilarity sampling with dynamically self‐guiding selection

Structural dissimilarity sampling (SDS) has been proposed as an enhanced conformational sampling method for reproducing the structural transitions of a given protein. SDS consists of cycles of two steps: (1) Selections of initial structures with structural dissimilarities by referring to a measure. (2) Conformational resampling by restarting short‐time molecular dynamics (MD) simulations from the initial structures. In the present study, an efficient measure is proposed as a dynamically self‐guiding selection to accelerate the structural transitions from a reactant state to a product state as an extension to the original SDS. In the extended SDS, the inner product (IP ) between the reactant and the snapshots generated by short‐time MD simulations are evaluated and ranked according to the IP s at every cycle. Then, the snapshots with low IP s are selected as initial structures for the short‐time MD simulations. This scheme enables one to choose dissimilar and distant initial structures from the reactant, and thus the initial structures dynamically head towards the product, promoting structural transitions from the reactant. To confirm the conformational sampling efficiency, the extended SDS was applied to maltodextrin binding protein (MBP), and we successfully reproduced the structural transition from the open to closed states with submicrosecond‐order simulation times. However, a conventional long‐time MD simulation failed to reproduce the same structural transition. We also compared the performance with that obtained by the ordinary SDS and other sampling techniques that have been developed by us to characterize the possible utility of the extended SDS for actual applications. © 2017 Wiley Periodicals, Inc.     

Modeling of the transition temperature for the helical denaturation of α-keratin intermediate filaments

Simulations of the stability of the secondary and tertiary structure of the α-keratin intermediate filament (IF) monomeric unit of wool are reported. Based on the assumed secondary structure three segments of the primary structure were selected: 1A, L12, and a part of 2B. Starting with an ideal α-helical conformation for each IF-segment, molecular dynamics simulations were carried out on the atomistic level at various temperatures in vaccum using the CFF91 force field. In either simulation the expected destabilization of the helical structure with increasing simulation temperature was observed. By use of different procedures of analysis, transition temperatures for the α-helical denaturation were determined that are significantly higher for the supposedly α-helical segments 1A and 2B than for the linker segment L12. The different stabilities of segments 1A and L12 were further verified through simulations in water environment that show the linker segment to be non-helical at room temperature. The lower transition temperature of segment L12 confirms the expectation that its amino acid sequence leads to increased conformational flexibility. The mobility of the water molecules surrounding the IF-segment is found to be significantly decreased by protein/water interactions.     

Energy landscape analyses of disordered histone tails reveal special organization of their conformational dynamics

Histone tails are highly flexible N- or C-terminal protrusions of histone proteins which facilitate the compaction of DNA into dense superstructures known as chromatin. On a molecular scale histone tails are polyelectrolytes with high degree of conformational disorder which allows them to function as biomolecular "switches", regulating various genetic processes. Unfortunately, their intrinsically disordered nature creates obstacles for comprehensive experimental investigation of both the structural and dynamical aspects of histone tails, because of which their conformational behaviors are still not well understood. In this work we have carried out ～3 microsecond long all atom replica exchange molecular dynamics (REMD) simulations for each of four histone tails, H4, H3, H2B, and H2A, and probed their intrinsic conformational preferences. Our subsequent free energy landscape analysis demonstrated that most tails are not fully disordered, but show distinct conformational organization, containing specific flickering secondary structural elements. In particular, H4 forms β-hairpins, H3 and H2B adopt α-helical elements, while H2A is fully disordered. We rationalized observed patterns of conformational dynamics of various histone tails using ideas from physics of polyelectrolytes and disordered systems. We also discovered an intriguing re-entrant contraction-expansion of the tails upon heating, which is caused by subtle interplay between ionic screening and chain entropy.     

Vibrational circular dichroism study of solvent- and temperature-induced conformational changes in poly-γ-benzyl-l-glutamate and poly-β-benzyl-l-aspartate

Vibrational circular dichroism (VCD) spectroscopy was used to study the effect of the different composition of mixed solvents and temperature on the conformation and aggregation states of two synthetically prepared polypeptides, poly-γ-benzyl-l-glutamate (PBLG) and poly-β-benzyl-l-aspartate (PBLA).Additions of trifluoroacetic acid (TFA) into a solution of heligenic solvents trichloromethane and benzene-d6 caused the conformational change from the α-helical to polyproline II-like for both of the polypeptides, which represented interesting transition previously mostly observed in aqueous solutions rather than in organic solvents. The VCD method proved a lower stability of the α-helical conformation of PBLA than PBLG and the structural differences between these polypeptides.The variation of temperature in the region 13–50 °C induced atypical conformational transformations in the PBLG/trichloromethane/TFA and PBLG/benzene-d6/TFA systems. The usually more stable α-helical conformation was observed at higher temperatures than the polyproline II-like conformation.     

The use of a generalized born model for the analysis of protein conformational transitions: a comparative study with explicit solvent simulations for chemotaxis Y protein (CheY)

To investigate whether implicit solvent models are appropriate for mechanistic studies of conformational transition in proteins, a recently developed generalized Born model (GBSW) was applied to a small signaling protein, chemotaxis protein Y (CheY), with different combinations of the phosphorylation state and conformation of the system; the results were compared to explicit solvent simulations using a stochastic boundary condition. The subtle but distinct conformational transitions involved in CheY activation makes the system ideally suited for comparing implicit and explicit solvent models because these conformational transitions are potentially accessible in both types of simulations. The structural and dynamical properties analyzed include not only those localized to the active site region but also throughout the protein, such as sidechain methyl group order parameters, backbone hydrogen bonding lifetime and occupancy as well as principal components of the trajectories. Overall, many properties were well reproduced by the GBSW simulations when compared with the explicit solvent calculations, although a number of observations consistently point to the suggestion that the current parameterization of the GBSW model tends to overestimate hydrogen-bonding interactions involving both charged groups and (charge-neutral) backbone atoms. This deficiency led to overstabilization of certain secondary structural motifs and more importantly, qualitatively different behaviors for the active site groups (Thr 87, Ala 88, the beta4-alpha4 loop) in response to phosphorylation, when compared with explicit solvent simulations. The current study highlights the value of carrying out both explicit and implicit solvent simulations for complementary mechanistic insights in the analysis of conformational transition in biomolecules.     

Co-operative intra-protein structural response due to protein–protein complexation revealed through thermodynamic quantification: study of MDM2-p53 binding

The p53 protein activation protects the organism from propagation of cells with damaged DNA having oncogenic mutations. In normal cells, activity of p53 is controlled by interaction with MDM2. The well understood p53-MDM2 interaction facilitates design of ligands that could potentially disrupt or prevent the complexation owing to its emergence as an important objective for cancer therapy. However, thermodynamic quantification of the p53-peptide induced structural changes of the MDM2-protein remains an area to be explored. This study attempts to understand the conformational free energy and entropy costs due to this complex formation from the histograms of dihedral angles generated from molecular dynamics simulations. Residue-specific quantification illustrates that, hydrophobic residues of the protein contribute maximum to the conformational thermodynamic changes. Thermodynamic quantification of structural changes of the protein unfold the fact that, p53 binding provides a source of inter-element cooperativity among the protein secondary structural elements, where the highest affected structural elements (α2 and α4) found at the binding site of the protein affects faraway structural elements (β1 and Loop1) of the protein. The communication perhaps involves water mediated hydrogen bonded network formation. Further, we infer that in inhibitory F19A mutation of P53, though Phe19 is important in the recognition process, it has less prominent contribution in the stability of the complex. Collectively, this study provides vivid microscopic understanding of the interaction within the protein complex along with exploring mutation sites, which will contribute further to engineer the protein function and binding affinity.     

Complex ion effects on polypeptide conformational stability: chloride and sulfate salts of guanidinium and tetrapropylammonium

The effects of chloride and sulfate salts of tetrapropylammonium (TPA(+)) and guanidinium (Gdm(+)) on the conformational stabilities of tryptophan zipper (trpzip) and α-helical (alahel) peptides were measured by circular dichroism spectroscopy. Like Gdm(+), TPA(+) interacts with the planar tryptophan indole group, perturbing the conformational stability of trpzip peptides. TPA(+) effects are largely unaffected by sulfate, indicating an absence of the heteroion pairing that is observed in concentrated Gdm(2)SO(4) solutions. TPA(+) stabilizes helical conformations in alahel peptides, indicating exclusion from the peptide bond. The observations are broadly consistent with predictions of molecular dynamics simulations [Mason, P. E.; et al. J. Phys. Chem. B2009, 113, 3227-3234], indicating that the effects of complex ions on proteins are increasingly predictable in terms of ion hydration, complementary interactions with specific protein groups, and ion-pairing contributions.     

Replica Exchange and Multicanonical Algorithms with the coarse-grained UNRES force field

Three algorithms, namely a Replica Exchange method (REM), a Replica Exchange Multicanonical method (REMUCA), and Replica Exchange Multicanonical with Replica Exchange (REMUCAREM), were implemented with the coarse-grained united-residue force field (UNRES) in both Monte Carlo and Molecular Dynamics versions. The MD algorithms use the constant-temperature Berendsen thermostat, with the velocity Verlet algorithm and variable time step. The algorithms were applied to one peptide (20 residues of Alanine with free ends; ala(20)) and two small proteins, namely an α-helical protein of 46 residues (the B-domain of the staphylococal protein A; 1BDD), and an α+β-protein of 48 residues (the E. Coli Mltd Lysm Domain; 1E0G). Calculated thermodynamic averages, such as canonical average energy and heat capacity, are in good agreement among all simulations for poly-L-alanine, showing that the algorithms were implemented correctly, and that all three algorithms are equally effective for small systems. For protein A, all algorithms performed reasonably well, although some variability in the calculated results was observed whereas, for a more complicated α+β-protein (1E0G), only Replica Exchange was capable of producing reliable statistics for calculating thermodynamic quantities. Finally, from the Replica Exchange molecular dynamics results, we calculated free energy maps as functions of RMSD and radius of gyration for different temperatures. The free energy calculations show correct folding behavior for poly-L-alanine and protein A while, for 1E0G, the native structure had the lowest free energy only at very low temperatures. Hence, the entropy contribution for 1E0G is larger than that for protein A at the same temperature. A larger contribution from entropy means that there are more accessible conformations at a given temperature, making it more difficult to obtain an efficient coverage of conformational space to obtain reliable thermodynamic properties. At the same temperature, ala(20) has the smallest entropy contribution, followed by protein A, and then by 1E0G.     

Molecular structures and dynamics of the stepwise activation mechanism of a matrix metalloproteinase zymogen: challenging the cysteine switch dogma

Activation of matrix metalloproteinase zymogen (pro-MMP) is a vital homeostatic process, yet its molecular basis remains unresolved. Using stopped-flow X-ray spectroscopy of the active site zinc ion, we determined the temporal sequence of pro-MMP-9 activation catalyzed by tissue kallikrein protease in milliseconds to several minutes. The identity of three intermediates seen by X-ray spectroscopy was corroborated by molecular dynamics simulations and quantum mechanics/molecular mechanics calculations. The cysteine-zinc interaction that maintains enzyme latency is disrupted via active-site proton transfers that mediate transient metal-protein coordination events and eventual binding of water. Unexpectedly, these events ensue as a direct result of complexation of pro-MMP-9 and kallikrein and occur before proteolysis and eventual dissociation of the pro-peptide from the catalytic site. Here we demonstrate the synergism among long-range protein conformational transitions, local structural rearrangements, and fine atomic events in the process of zymogen activation.     

Ultrafast spectroscopy of a photoswitchable 30-amino acid de novo synthesized peptide

The ultrafast photoresponse of small, often cyclic peptides with azobenzene units has widely been investigated during the last years. Both the photoisomerization of the optical switch as well as the different conformational states of the peptide moiety can be characterized by optical spectroscopy. Here, we investigate the fast photoisomerization dynamics of an α-helical 30mer azobenzene peptide. The peptide is based on a construct used for the assembly of di-heme-binding maquettes. The femtosecond to picosecond photodynamics for the trans to cis isomerization of the optical switch was found to occur slower upon its insertion in the peptide construct. Both isomers are sufficiently photostable to allow spectroscopic analysis of conformational states, since the thermal cis–trans relaxation occurs over a period of several hours. This approach thus offers the possibility for the de novo design of photoresponsive chromopeptides which could be instrumental in unravelling fundamental dynamic features of assembly/disassembly triggered by fast photoswitches.     

Polarization Dependent Time-Resolved Infrared Spectroscopy and Its Applications

Polarization dependent time-resolved infrared (TRIR) spectroscopy has proven to be a useful technique to study the structural dynamics in a photochemical process. The angular information of transient species is obtainable in this measurement, which makes it a valuable technique for the investigation of electron distribution, molecular structure, and conformational dynamics. In this review, we briefly introduce the principles and applications of polarization dependent TRIR spectroscopy. We mainly focused on the following topics: (i) an overview of TRIR spectroscopy, (ii) principles of TRIR spectroscopy and its advantages compared to the other ultrafast techniques, (iii) examples that use polarization dependent TRIR spectroscopy to probe a variety of chemical and dynamical phenomena including protein conformational dynamics, excited state electron localization, and photoisomerization, (iv) the limitations and prospects of TRIR spectroscopy.     

The native ensemble and folding of a protein molten-globule: functional consequence of downhill folding

The continually emerging functional significance of intrinsic disorder and conformational flexibility in proteins has challenged the long-standing dogma of a well-defined structure contributing to a specific function. Molten-globular states, a class of proteins with significant secondary-structure but a fluid hydrophobic core, is one such example. They have however been difficult to characterize due to the complexity of experimental data and lack of computational avenues. Here, we dissect the folding mechanism of the α-helical molten-globular protein NCBD from three fundamentally different approaches: statistical-mechanical variable barrier model, C(α)-based Gō-model and explicit water all-atom molecular dynamics (MD) simulations. We find that NCBD displays the characteristics of a one-state globally downhill folder but is significantly destabilized. Using simulation techniques, we generate a highly constrained but a heterogeneous native ensemble of the molten-globule for the first time that is consistent with experimental data including small angle X-ray scattering (SAXS), circular dichroism (CD), and nuclear magnetic resonance (NMR). The resulting native ensemble populates conformations reported in other bound-forms providing direct evidence to the mechanism of conformational selection for binding multiple partners in this domain. Importantly, our simulations reveal a connection between downhill folding and large conformational flexibility in this domain that has been evolutionarily selected and functionally exploited resulting in large binding promiscuity. Finally, the multimodel approach we employ here serves as a powerful methodology to study mechanisms and suggests that the thermodynamic features of molten-globules fall within the array of folding mechanisms available to small single-domain proteins.     

Structural and thermodynamic investigations on the aggregation and folding of acylphosphatase by molecular dynamics simulations and solvation free energy analysis

Protein engineering method to study the mutation effects on muscle acylphosphatase (AcP) has been actively applied to describe kinetics and thermodynamics associated with AcP aggregation as well as folding processes. Despite the extensive mutation experiments, the molecular origin and the structural motifs for aggregation and folding kinetics as well as thermodynamics of AcP have not been rationalized at the atomic resolution. To this end, we have investigated the mutation effects on the structures and thermodynamics for the aggregation and folding of AcP by using the combination of fully atomistic, explicit-water molecular dynamics simulations, and three-dimensional reference interaction site model theory. The results indicate that the A30G mutant with the fastest experimental aggregation rate displays considerably decreased α1-helical contents as well as disrupted hydrophobic core compared to the wild-type AcP. Increased solvation free energy as well as hydrophobicity upon A30G mutation is achieved due to the dehydration of hydrophilic side chains in the disrupted α1-helix region of A30G. In contrast, the Y91Q mutant with the slowest aggregation rate shows a non-native H-bonding network spanning the mutation site to hydrophobic core and α1-helix region, which rigidifies the native state protein conformation with the enhanced α1-helicity. Furthermore, Y91Q exhibits decreased solvation free energy and hydrophobicity compared to wild type due to more exposed and solvated hydrophilic side chains in the α1-region. On the other hand, the experimentally observed slower folding rates in both mutants are accompanied by decreased helicity in α2-helix upon mutation. We here provide the atomic-level structures and thermodynamic quantities of AcP mutants and rationalize the structural origin for the changes that occur upon introduction of those mutations along the AcP aggregation and folding processes.