Conformational Manifold Sampled by Two Short Linear Motif Segments Probed by Circular Dichroism, Vibrational, and Nuclear Magnetic Resonance Spectroscopy.

Disordered protein segments called short linear motifs (SLiM) serve as recognition sites for a variety of biological processes and act as targeting signals, modification, and ligand binding sites. While SLiMs do not adopt one of the known regular secondary structures, the conformational distribution might still reflect the structural propensities of their amino acid residues and possible interactions between them. In the past, conformational analyses of short peptides provided compelling evidence for the notion that individual residues are less conformationally flexible than locally expected for a random coil. Here, we combined various spectroscopies (NMR, IR, vibrational, and UV circular dichroism) to determine the Ramachandran plots of two SLiM motifs, i.e., GRRDSG and GRRTSG. They are two representatives of RxxS motifs that are capable of being phosphorylated by protein kinase A, an enzyme that plays a fundamental role in a variety of biological processes. Our results reveal that the nearest and non-nearest interactions between residues cause redistributions between polyproline II and ß-strand basins while concomitantly stabilizing extended relative to turn-forming and helical structures. They also cause shifts in basin positions. With increasing temperature, ß-strand populations become more populated at the expense of polyproline II. While molecular dynamics simulations with Amber ff14SB and CHARMM 36m force fields indicate residue-residue interactions, they do not account for the observed structural changes.

How do salt and lipids affect conformational dynamics of Aß42 monomers in water?

It is well established that amyloid ß-protein (Aß) self-assembly is involved in triggering of Alzheimer's disease. On the other hand, evidence of physiological function of Aß interacting with lipids has only begun to emerge. Details of Aß-lipid interactions, which may underlie physiological and pathological activities of Aß, are not well understood. Here, the effects of salt and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipids on conformational dynamics of Aß42 monomer in water are examined by all-atom molecular dynamics (MD). We acquired six sets of 250 ns long MD trajectories for each of the three lipid concentrations (0, 27, and 109 mM) in the absence and presence of 150 mM salt. Ten replica trajectories per set are used to enhance sampling of Aß42 conformational space. We show that salt facilitates long-range tertiary contacts in Aß42, resulting in more compact Aß42 conformations. By contrast, addition of lipids results in lipid-concentration dependent Aß42 unfolding concomitant with enhanced stability of the turn in the A21-A30 region. At the high lipid concentration, salt enables the N-terminal region of Aß42 to form long-range tertiary contacts and interact with lipids, which results in formation of a parallel ß-strand. Aß42 forms stable lipid-protein complexes whereby the protein is adhered to the lipid cluster rather than embedded into it. We propose that the inability of Aß42 monomer to get embedded into the lipid cluster may be important for facilitating repair of leaks in the blood-brain barrier without penetrating and damaging cellular membranes.

Do molecular dynamics force fields accurately model Ramachandran distributions of amino acid residues in water?

Molecular dynamics (MD) is a powerful tool for studying intrinsically disordered proteins, however, its reliability depends on the accuracy of the force field. We assess Amber ff19SB, Amber ff14SB, OPLS-AA/M, and CHARMM36m with respect to their capacity to capture intrinsic conformational dynamics of 14 guest residues x (=G, A, L, V, I, F, Y, DP, EP, R, C, N, S, T) in GxG peptides in water. The MD-derived Ramachandran distribution of each guest residue is used to calculate 5 J-coupling constants and amide I' band profiles to facilitate a comparison to spectroscopic data through reduced χ2 functions. We show that the Gaussian model, optimized to best fit the experimental data, outperforms all MD force fields by an order of magnitude. The weaknesses of the MD force fields are: (i) insufficient variability of the polyproline II (pPII) population among the guest residues; (ii) oversampling of antiparallel at the expense of transitional ß-strand region; (iii) inadequate sampling of turn-forming conformations for ionizable and polar residues; and (iv) insufficient guest residue-specificity of the Ramachandran distributions. Whereas Amber ff19SB performs worse than the other three force fields with respect to χ2 values, it accounts for residue-specific pPII content better than the other three force fields. Additional testing of residue-specific RSFF1 and Amber ff14SB combined with TIP4P/2005 on six guest residues x (=A, I, F, DP, R, S) reveals that residue specificity derived from protein coil libraries or an improved water model alone do not result in significantly lower χ2 values.

Short peptides as predictors for the structure of polyarginine sequences in disordered proteins.

Intrinsically disordered proteins and intrinsically disordered regions are frequently enriched in charged amino acids. Intrinsically disordered regions are regularly involved in important biological processes in which one or more charged residues is the driving force behind a protein-biomolecule interaction. Several lines of experimental and computational evidence suggest that polypeptides and proteins that carry high net charges have a high preference for extended conformations with average end-to-end distances exceeding expectations for self-avoiding random coils. Here, we show that charged arginine residues even in short glycine-capped model peptides (GRRG and GRRRG) significantly affect the conformational propensities of each other when compared with the intrinsic propensities of a mostly unperturbed arginine in the tripeptide GRG. A conformational analysis based on experimentally determined J-coupling constants from heteronuclear NMR spectroscopy and amide I' band profiles from vibrational spectroscopy reveals that nearest-neighbor interactions stabilize extended ß-strand conformations at the expense of polyproline II and turn conformations. The results from molecular dynamics simulations with a CHARMM36m force field and TIP3P water reproduce our results only to a limited extent. The use of the Ramachandran distribution of the central residue of GRRRG in a calculation of end-to-end distances of polyarginines of different length yielded the expected power law behavior. The scaling coefficient of 0.66 suggests that such peptides would be more extended than predicted by a self-avoiding random walk. Our findings thus support in principle theoretical predictions.

Insulin Inhibits Aß42 Aggregation and Prevents Aß42-Induced Membrane Disruption.

Alzheimer's disease (AD) is associated with self-assembly of amyloid ß-protein (Aß) into soluble oligomers. Of the two predominant Aß alloforms, Aß40 and Aß42, the latter is particularly strongly linked to AD. Longitudinal studies revealed a correlation between AD and type 2 diabetes (T2D), characterized by abnormal insulin levels and insulin resistance. Although administration of intranasal insulin is explored as a therapy against AD, the extent to which insulin affects Aß dynamics and activity is unclear. We here investigate the effect of insulin on Aß42 self-assembly and characterize the capacity of insulin, Aß42, and Aß42 co-incubated with insulin to disrupt the integrity of biomimetic lipid vesicles. We demonstrate that quiescently incubated insulin, which does not form amyloid fibrils, over time develops membrane-disrupting capacity, which we propose to originate in misfolded insulin monomers. These hypothetically toxic misfolded monomers might contribute to the development of insulin resistance in early stages of T2D that are associated with abnormally high insulin levels. We show that in contrast to quiescent incubation, insulin incubated under agitated conditions readily forms amyloid fibrils, which protect against membrane permeation. Insulin quiescently incubated with Aß42 attenuates both Aß42 fibril formation and the ability of Aß42 to disrupt membranes in a concentration-dependent manner. Our findings offer insights into interactions between insulin and Aß42 that are relevant to understanding the molecular basis of intranasal insulin as a therapy against Aß-induced AD pathology, thereby elucidating a plausible mechanism underlying the observed correlations between AD and T2D.

Stabilization of native amyloid ß-protein oligomers by Copper and Hydrogen peroxide Induced Cross-linking of Unmodified Proteins (CHICUP).

Oligomeric assemblies are postulated to be proximate neurotoxic species in human diseases associated with aberrant protein aggregation. Their heterogeneous and transient nature makes their structural characterization difficult. Size distributions of oligomers of several amyloidogenic proteins, including amyloid ß-protein (Aß) relevant to Alzheimer's disease (AD), have been previously characterized in vitro by photo-induced cross-linking of unmodified proteins (PICUP) followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Due to non-physiological conditions associated with the PICUP chemistry, Aß oligomers cross-linked by PICUP may not be representative of in vivo conditions. Here, we examine an alternative Copper and Hydrogen peroxide Induced Cross-linking of Unmodified Proteins (CHICUP), which utilizes naturally occurring divalent copper ions and hydrogen peroxide and does not require photo activation. Our results demonstrate that CHICUP and PICUP applied to the two predominant Aß alloforms, Aß40 and Aß42, result in similar oligomer size distributions. Thioflavin T fluorescence data and atomic force microscopy images demonstrate that both CHICUP and PICUP stabilize Aß oligomers and attenuate fibril formation. Relative to noncross-linked peptides, CHICUP-treated Aß40 and Aß42 cause prolonged disruption to biomimetic lipid vesicles. CHICUP-stabilized Aß oligomers link the amyloid cascade, metal, and oxidative stress hypotheses of AD into a more comprehensive understanding of the molecular basis of AD pathology. Because copper and hydrogen peroxide are elevated in the AD brain, CHICUP-stabilized Aß oligomers are biologically relevant and should be further explored as a new therapeutic target.

Elucidation of insulin assembly at acidic and neutral pH: Characterization of low molecular weight oligomers.

Deficiency in insulin secretion and function that characterize type 2 diabetes often requires administration of extraneous insulin, leading to injection-site amyloidosis. Insulin aggregation at neutral pH is not well understood. Although oligomer formation is believed to play an important role, insulin oligomers have not been fully characterized yet. Here, we elucidate similarities and differences between in vitro insulin aggregation at acidic and neutral pH for a range of insulin concentrations (2.5-100 µM) by using kinetic thioflavin T fluorescence, circular dichroism, atomic force and electron microscopy imaging. Importantly, we characterize the size distribution of insulin oligomers at different assembly stages by the application of covalent cross-linking and gel electrophoresis. Our results show that at the earliest assembly stage, oligomers comprise up to 40% and 70% of soluble insulin at acidic and neutral pH, respectively. While the highest oligomer order increases with insulin concentration at acidic pH, the opposite tendency is observed at neutral pH, where oligomers up to heptamers are formed in 10 µM insulin. These findings suggest that oligomers may be on- and off-pathway assemblies for insulin at acidic and neutral pH, respectively. Agitation, which is required to induce insulin aggregation at neutral pH, is shown to increase fibril formation rate and fibrillar mass both by an order of magnitude. Insulin incubated under agitated conditions at neutral pH rapidly aggregates into large micrometer-sized aggregates, which may be of physiological relevance and provides insight into injection-site amyloidosis and toxic pulmonary aggregates induced by administration of extraneous insulin.

Amino acid substitutions [K16A] and [K28A] distinctly affect amyloid ß-protein oligomerization.

Amyloid ß-protein (A ß) assembles into oligomers that play a seminal role in Alzheimer's disease (AD), a leading cause of dementia among the elderly. Despite undisputed importance of A ß oligomers, their structure and the basis of their toxicity remain elusive. Previous experimental studies revealed that the [K16A] substitution strongly inhibits toxicity of the two predominant A ß alloforms in the brain, A ß 40 and A ß 42, whereas the [K28A] substitution exerts only a moderate effect. Here, folding and oligomerization of [A16]A ß 40, [A28]A ß 40, [A16]A ß 42, and [A28]A ß 42 are examined by discrete molecular dynamics (DMD) combined with a four-bead implicit solvent force field, DMD4B-HYDRA, and compared to A ß 40 and A ß 42 oligomer formation. Our results show that both substitutions promote A ß 40 and A ß 42 oligomerization and that structural changes to oligomers are substitution- and alloform-specific. The [K28A] substitution increases solvent-accessible surface area of hydrophobic residues and the intrapeptide N-to-C terminal distance within oligomers more than the [K16A] substitution. The [K16A] substitution decreases the overall ß-strand content, whereas the [K28A] substitution exerts only a modest change. Substitution-specific tertiary and quaternary structure changes indicate that the [K16A] substitution induces formation of more compact oligomers than the [K28A] substitution. If the structure-function paradigm applies to A ß oligomers, then the observed substitution-specific structural changes in A ß 40 and A ß 42 oligomers are critical for understanding the structural basis of A ß oligomer toxicity and correct identification of therapeutic targets against AD.

A computational model for the loss of neuronal organization in microcolumns.

A population of neurons in the cerebral cortex of humans and other mammals organize themselves into vertical microcolumns perpendicular to the pial surface. Anatomical changes to these microcolumns have been correlated with neurological diseases and normal aging; in particular, in area 46 of the rhesus monkey brain, the strength of microcolumns was shown to decrease with age. These changes can be caused by alterations in the spatial distribution of the neurons in microcolumns and/or neuronal loss. Using a three-dimensional computational model of neuronal arrangements derived from thin tissue sections and validated in brain tissue from rhesus monkeys, we show that neuronal loss is inconsistent with the findings in aged individuals. In contrast, a model of simple random neuronal displacements, constrained in magnitude by restorative harmonic forces, is consistent with observed changes and provides mechanistic insights into the age-induced loss of microcolumnar structure. Connection of the model to normal aging and disease are discussed.

Oligomer Formation by Physiologically Relevant C-Terminal Isoforms of Amyloid ß-Protein.

Alzheimer's disease (AD) is a neurological disorder associated with amyloid ß-protein (Aß) assembly into toxic oligomers. In addition to the two predominant alloforms, Aß1-40 and Aß1-42, other C-terminally truncated Aß peptides, including Aß1-38 and Aß1-43, are produced in the brain. Here, we use discrete molecular dynamics (DMD) and a four-bead protein model with amino acid-specific hydropathic interactions, DMD4B-HYDRA, to examine oligomer formation of Aß1-38, Aß1-40, Aß1-42, and Aß1-43. Self-assembly of 32 unstructured monomer peptides into oligomers is examined using 32 replica DMD trajectories for each of the four peptides. In a quasi-steady state, Aß1-38 and Aß1-40 adopt similar unimodal oligomer size distributions with a maximum at trimers, whereas Aß1-42 and Aß1-43 oligomer size distributions are multimodal with the dominant maximum at trimers or tetramers, and additional maxima at hexamers and unidecamers (for Aß1-42) or octamers and pentadecamers (for Aß1-43). The free energy landscapes reveal isoform- and oligomer-order specific structural and morphological features of oligomer ensembles. Our results show that oligomers of each of the four isoforms have unique features, with Aß1-42 alone resulting in oligomers with disordered and solvent-exposed N-termini. Our findings help unravel the structure-function paradigm governing oligomers formed by various Aß isoforms.

Intrinsic Conformational Dynamics of Glycine and Alanine in Polarizable Molecular Dynamics Force Fields: Comparison to Spectroscopic Data.

Molecular dynamics (MD) is a great tool for elucidating conformational dynamics of proteins and peptides in water at the atomistic level that often surpasses the level of detail available experimentally. Structure predictions, however, are limited by the accuracy of the underlying MD force field. This limitation is particularly stark in the case of intrinsically disordered peptides and proteins, which are characterized by solvent-accessible and disordered peptide regions and domains. Recent studies show that most additive MD force fields, including CHARMM36m, do not reproduce the intrinsic conformational distributions of guest amino acid residues x in cationic GxG peptides in water in line with experimental data. Positing that a lack of polarizability in additive MD force fields may be the culprit for the reported discrepancies, we here examine the conformational dynamics of guest glycine and alanine residues in cationic GxG peptides in water using two polarizable MD force fields, CHARMM Drude and AMOEBA. Our results indicate that while AMOEBA captures the experimental data better than CHARMM Drude, neither of the two polarizable force fields offers an improvement of the Ramachandran distributions of glycine and alanine residues in cationic GGG and GAG peptides, respectively, over CHARMM36m.

Aß42 oligomers, but not fibrils, simultaneously bind to and cause damage to ganglioside-containing lipid membranes.

Aß (amyloid-ß peptide) assembles to form amyloid fibres that accumulate in senile plaques associated with AD (Alzheimer's disease). The major constituent, a 42-residue Aß, has the propensity to assemble and form soluble and potentially cytotoxic oligomers, as well as ordered stable amyloid fibres. It is widely believed that the cytotoxicity is a result of the formation of transient soluble oligomers. This observed toxicity may be associated with the ability of oligomers to associate with and cause permeation of lipid membranes. In the present study, we have investigated the ability of oligomeric and fibrillar Aß42 to simultaneously associate with and affect the integrity of biomimetic membranes in vitro. Surface plasmon field-enhanced fluorescence spectroscopy reveals that the binding of the freshly dissolved oligomeric 42-residue peptide binds with a two-step association with the lipid bilayer, and causes disruption of the membrane resulting in leakage from vesicles. In contrast, fibrils bind with a 2-fold reduced avidity, and their addition results in approximately 2-fold less fluorophore leakage compared with oligomeric Aß. Binding of the oligomers may be, in part, mediated by the GM1 ganglioside receptors as there is a 1.8-fold increase in oligomeric Aß binding and a 2-fold increase in permeation compared with when GM1 is not present. Atomic force microscopy reveals the formation of defects and holes in response to oligomeric Aß, but not preformed fibrillar Aß. The results of the present study indicate that significant membrane disruption arises from association of low-molecular-mass Aß and this may be mediated by mechanical damage to the membranes by Aß aggregation. This membrane disruption may play a key role in the mechanism of Aß-related cell toxicity in AD.

Folding of pig gastric mucin non-glycosylated domains: a discrete molecular dynamics study.

Mucin glycoproteins consist of tandem-repeating glycosylated regions flanked by non-repetitive protein domains with little glycosylation. These non-repetitive domains are involved in polymerization of mucin and play an important role in the pH-dependent gelation of gastric mucin, which is essential for protecting the stomach from autodigestion. We examine folding of the non-repetitive sequence of PGM-2X (242 amino acids) and the von Willebrand factor vWF-C1 domain (67 amino acids) at neutral and low pH using discrete molecular dynamics (DMD) in an implicit solvent combined with a four-bead peptide model. Using the same implicit solvent parameters, folding of both domains is simulated at neutral and low pH. In contrast to vWF-C1, PGM-2X folding is strongly affected by pH as indicated by changes in the contact order, radius of gyration, free-energy landscape, and the secondary structure. Whereas the free-energy landscape of vWF-C1 shows a single minimum at both neutral and low pH, the free-energy landscape of PGM-2X is characterized by multiple minima that are more numerous and shallower at low pH. Detailed structural analysis shows that PGM-2X partially unfolds at low pH. This partial unfolding is facilitated by the C-terminal region GLU236-PRO242, which loses contact with the rest of the domain due to effective "mean-field" repulsion among highly positively charged N- and C-terminal regions. Consequently, at low pH, hydrophobic amino acids are more exposed to the solvent. In vWF-C1, low pH induces some structural changes, including an increased exposure of CYS at position 67, but these changes are small compared to those found in PGM-2X. For PGM-2X, the DMD-derived average ß-strand propensity increases from 0.26 ± 0.01 at neutral pH to 0.38 ± 0.01 at low pH. For vWF-C1, the DMD-derived average ß-strand propensity is 0.32 ± 0.02 at neutral pH and 0.35 ± 0.02 at low pH. The DMD-derived structural information provides insight into pH-induced changes in the folding of two distinct mucin domains and suggests plausible mechanisms of the aggregation/gelation of mucin.

C-terminal peptides coassemble into Abeta42 oligomers and protect neurons against Abeta42-induced neurotoxicity.

Alzheimer's disease (AD) is an age-related disorder that threatens to become an epidemic as the world population ages. Neurotoxic oligomers of Abeta42 are believed to be the main cause of AD; therefore, disruption of Abeta oligomerization is a promising approach for developing therapeutics for AD. Formation of Abeta42 oligomers is mediated by intermolecular interactions in which the C terminus plays a central role. We hypothesized that peptides derived from the C terminus of Abeta42 may get incorporated into oligomers of Abeta42, disrupt their structure, and thereby inhibit their toxicity. We tested this hypothesis using Abeta fragments with the general formula Abeta(x-42) (x = 28-39). A cell viability screen identified Abeta(31-42) as the most potent inhibitor. In addition, the shortest peptide, Abeta(39-42), also had high activity. Both Abeta(31-42) and Abeta(39-42) inhibited Abeta-induced cell death and rescued disruption of synaptic activity by Abeta42 oligomers at micromolar concentrations. Biophysical characterization indicated that the action of these peptides likely involved stabilization of Abeta42 in nontoxic oligomers. Computer simulations suggested a mechanism by which the fragments coassembled with Abeta42 to form heterooligomers. Thus, Abeta(31-42) and Abeta(39-42) are leads for obtaining mechanism-based drugs for treatment of AD using a systematic structure-activity approach.

Cross-Linked Amyloid ß-Protein Oligomers: A Missing Link in Alzheimer's Disease Pathology?

Amyloid ß-protein (Aß) oligomers are broadly viewed as the proximate mediators of toxicity in Alzheimer's disease (AD). Recent studies, however, provide substantial evidence that Aß is involved in protection and repair of the central nervous system whereby Aß oligomer and subsequent fibril formation are integral to its normal antimicrobial and antiviral function. These developments raise a question of what exactly makes Aß oligomers toxic in the context of AD. This Perspective describes a paradigm shift in the search for toxic Aß oligomer species that involves oxidative-stress-induced stabilization of Aß oligomers via cross-linking and reviews most recent research elucidating structural aspects of cross-linked Aß oligomers and potential inhibition of their toxicity.

Soluble State of Villin Headpiece Protein as a Tool in the Assessment of MD Force Fields.

Protein self-assembly plays an important role in cellular processes. Whereas molecular dynamics (MD) represents a powerful tool in studying assembly mechanisms, its predictions depend on the accuracy of underlying force fields, which are known to overly promote protein assembly. We here examine villin headpiece domain, HP36, which remains soluble at concentrations amenable to MD studies. The experimental characterization of soluble HP36 at concentrations of 0.05 to 1 mM reveals concentration-independent 90% monomeric and 10% dimeric populations. Extensive all-atom MD simulations at two protein concentrations, 0.9 and 8.5 mM, probe the HP36 dimer population, stability, and kinetics of dimer formation within two MD force fields, Amber ff14SB and CHARMM36m. MD results demonstrate that whereas CHARMM36m captures experimental HP36 monomer populations at the lower concentration, both force fields overly promote HP36 association at the higher concentration. Moreover, contacts stabilizing HP36 dimers are force-field-dependent. CHARMM36m produces consistently higher HP36 monomer populations, lower association rates, and weaker dependence of these quantities on the protein concentration than Amber ff14SB. Nonetheless, the highest monomer populations and dissociation constants are observed when the TIP3P water model in Amber ff14SB is replaced by TIP4P/2005, showcasing the critical role of the water model in addressing the protein solubility problem in MD.

Biophysical characterization of Abeta42 C-terminal fragments: inhibitors of Abeta42 neurotoxicity.

A key event in Alzheimer's disease (AD) is age-dependent, brain accumulation of amyloid beta-protein (Abeta) leading to Abeta self-association into neurotoxic oligomers. Previously, we showed that Abeta oligomerization and neurotoxicity could be inhibited by C-terminal fragments (CTFs) of Abeta42. Because these CTFs are highly hydrophobic, we asked if they themselves aggregated and, if so, what parameters regulated their aggregation. To answer these questions, we investigated the dependence of CTF aqueous solubility, aggregation kinetics, and morphology on peptide length and sequence and the correlation between these characteristics and inhibition of Abeta42-induced toxicity. We found that CTFs up to 8 residues long were soluble at concentrations >100 microM and had a low propensity to aggregate. Longer CTFs were soluble at approximately 1-80 microM, and most, but not all, readily formed beta-sheet-rich fibrils. Comparison to Abeta40-derived CTFs showed that the C-terminal dipeptide I41-A42 strongly promoted aggregation. Aggregation propensity correlated with the previously reported tendency to form beta-hairpin conformation but not with inhibition of Abeta42-induced neurotoxicity. The data enhance our understanding of the physical characteristics that affect CTF activity and advance our ability to design, synthesize, and test future generations of inhibitors.

Mechanistic investigation of the inhibition of Abeta42 assembly and neurotoxicity by Abeta42 C-terminal fragments.

Oligomeric forms of amyloid beta-protein (Abeta) are key neurotoxins in Alzheimer's disease (AD). Previously, we found that C-terminal fragments (CTFs) of Abeta42 interfered with assembly of full-length Abeta42 and inhibited Abeta42-induced toxicity. To decipher the mechanism(s) by which CTFs affect Abeta42 assembly and neurotoxicity, here, we investigated the interaction between Abeta42 and CTFs using photoinduced cross-linking and dynamic light scattering. The results demonstrate that distinct parameters control CTF inhibition of Abeta42 assembly and Abeta42-induced toxicity. Inhibition of Abeta42-induced toxicity was found to correlate with stabilization of oligomers with a hydrodynamic radius (R(H)) of 8-12 nm and attenuation of formation of oligomers with an R(H) of 20-60 nm. In contrast, inhibition of Abeta42 paranucleus formation correlated with CTF solubility and the degree to which CTFs formed amyloid fibrils themselves but did not correlate with inhibition of Abeta42-induced toxicity. Our findings provide important insight into the mechanisms by which different CTFs inhibit the toxic effect of Abeta42 and suggest that stabilization of nontoxic Abeta42 oligomers is a promising strategy for designing inhibitors of Abeta42 neurotoxicity.

Do Molecular Dynamics Force Fields Capture Conformational Dynamics of Alanine in Water?

We examine the ability of six molecular dynamics (MD) force fields (Amber ff14SB, Amber ff99SBnmr1, Amber ff03ws, OPLS-AA/L, OPLS-AA/M, and CHARMM36) to reproduce conformational ensembles of the central alanine in GAG and AAA in a way that is consistent with five (GAG) or six (AAA) J coupling constants and amide I' profiles. MD-derived Ramachandran plots for all six force fields under study differ from those obtained by the Gaussian fit to experimental data in three major ways: (i) the polyproline II (pPII) basin in the Ramachandran plot is too concentrated, (ii) the antiparallel ß (aß) basin is overpopulated, and (iii) the transitional ß (ßt) basin is underpopulated. Amber ff14SB outperforms the other five MD force fields and yields the highest pPII populations of the central alanine residue in GAG (55%) and AAA (63%), in good agreement with the predictions of the Gaussian model (59 and 76%). The analysis of the hydration layer around the central alanine residue reveals considerable reorientation of water molecules and reduction in both the average number of water molecules and the average number of water-water hydrogen bonds when glycines (in GAG) are replaced by alanines (in AAA), elucidating water-mediated nearest neighbor effects on alanine's conformational dynamics.

Intrinsic Conformational Dynamics of Alanine in Water/Ethanol Mixtures: An Experiment-Driven Molecular Dynamics Study.

In vitro, cationic glycylalanylglycine (GAG) forms a hydrogel in binary mixtures of water and ethanol. In water, alanine residue is known for its high polyproline II (pPII) content. Spectroscopic data, including three J-coupling constants and amide I' profiles, indicate that addition of 42% ethanol to water significantly reduces the pPII content of alanine residue in GAG. Here, experiment-based Gaussian Ramachandran distributions of alanine in GAG at different ethanol fractions are examined and three MD force fields are evaluated with respect to their ability to capture these ethanol-induced conformational changes. MD simulations on monomeric GAG in eight different water/ethanol mixtures within Amber ff14SB, OPLS-AA/M, and CHARMM36m reveal that only Amber ff14SB partially captures the ethanol-induced conformational changes of alanine residue in monomeric GAG when 42% ethanol is added to water. MD simulations of 200 mM GAG ensembles in pure water and in the aqueous solution with 42% ethanol showcase the ability of CHARMM36m to capture the effect of ethanol on the average pPII content of alanine in GAG and provide a plausible explanation for this effect, which may stem from an increased propensity of GAG to form oligomers in the presence of ethanol.