Synthetic NAC 71-82 Peptides Designed to Produce Fibrils with Different Protofilament Interface Contacts.

Alpha-synucleinopathies are featured by fibrillar inclusions in brain cells. Although α-synuclein fibrils display structural diversity, the origin of this diversity is not fully understood. We used molecular dynamics simulations to design synthetic peptides, based on the NAC 71-82 amino acid fragment of α-synuclein, that govern protofilament contacts and generation of twisted fibrillar polymorphs. Four peptides with structures based on either single or double fragments and capped or non-capped ends were selected for further analysis. We determined the fibrillar yield and the structures from these peptides found in the solution after fibrillisation using protein concentration determination assay and circular dichroism spectroscopy. In addition, we characterised secondary structures formed by individual fibrillar complexes using laser-tweezers Raman spectroscopy. Results suggest less mature fibrils, based on the lower relative ß-sheet content for double- than single-fragment peptide fibrils. We confirmed this structural difference by TEM analysis which revealed, in addition to short protofibrils, more elongated, twisted and rod-like fibril structures in non-capped and capped double-fragment peptide systems, respectively. Finally, time-correlated single-photon counting demonstrated a difference in the Thioflavin T fluorescence lifetime profiles upon fibril binding. It could be proposed that this difference originated from morphological differences in the fibril samples. Altogether, these results highlight the potential of using peptide models for the generation of fibrils that share morphological features relevant for disease, e.g., twisted and rod-like polymorphs.

A Capped Peptide of the Aggregation Prone NAC 71-82 Amino Acid Stretch of α-Synuclein Folds into Soluble ß-Sheet Oligomers at Low and Elevated Peptide Concentrations.

Although Lewy bodies and Lewy neurites are hallmarks of Parkinson's disease (PD) and dementia with Lewy bodies (DLB), misfolded α-synuclein oligomers are nowadays believed to be key for the development of these diseases. Attempts to target soluble misfolded species of the full-length protein have been limited so far, probably due to the fast aggregation kinetics and burial of aggregation prone segments in final cross-ß-sheet fibrils. A previous characterisation study of fibrils prepared from a capped peptide of the non-amyloid ß-component (NAC) 71-82 amino acid stretch of α-synuclein demonstrated an increased aggregation propensity resulting in a cross-ß-structure that is also found in prion proteins. From this, it was suggested that capped NAC 71-82 peptide oligomers would provide interesting motifs with a capacity to regulate disease development. Here, we demonstrated, from a series of circular dichroism spectroscopic measurements and molecular dynamics simulations, the molecular-environment-sensitive behaviour of the capped NAC 71-82 peptide in a solution phase and the formation of ß-sheet oligomeric structures in the supernatant of a fibrillisation mixture. These results highlighted the use of the capped NAC 71-82 peptide as a motif in the preparation of oligomeric ß-sheet structures that potentially could be used in therapeutic strategies in the fight against progressive neurodegenerative disorders, such as PD and DLB.

Key Residues and Phosphate Release Routes in the Saccharomyces cerevisiae Pho84 Transceptor: THE ROLE OF TYR179 IN FUNCTIONAL REGULATION.

Pho84, a major facilitator superfamily (MFS) protein, is the main high-affinity Pi transceptor in Saccharomyces cerevisiae Although transport mechanisms have been suggested for other MFS members, the key residues and molecular events driving transport by Pi:H+ symporters are unclear. The current Pho84 transport model is based on the inward-facing occluded crystal structure of the Pho84 homologue PiPT in the fungus Piriformospora indica However, this model is limited by the lack of experimental data on the regulatory residues for each stage of the transport cycle. In this study, an open, inward-facing conformation of Pho84 was used to study the release of Pi A comparison of this conformation with the model for Pi release in PiPT revealed that Tyr179 in Pho84 (Tyr150 in PiPT) is not part of the Pi binding site. This difference may be due to a lack of detailed information on the Pi release step in PiPT. Molecular dynamics simulations of Pho84 in which a residue adjacent to Tyr179, Asp178, is protonated revealed a conformational change in Pho84 from an open, inward-facing state to an occluded state. Tyr179 then became part of the binding site as was observed in the PiPT crystal structure. The importance of Tyr179 in regulating Pi release was supported by site-directed mutagenesis and transport assays. Using trehalase activity measurements, we demonstrated that the release of Pi is a critical step for transceptor signaling. Our results add to previous studies on PiPT, creating a more complete picture of the proton-coupled Pi transport cycle of a transceptor.

In silico screening of molecular imprinting prepolymerization systems: oseltamivir selective polymers through full-system molecular dynamics-based studies.

All-component molecular dynamics studies were used to probe a library of oseltamivir molecularly imprinted polymer prepolymerization mixtures. Polymers included one of five functional monomers (acrylamide, hydroxyethylmethacrylate, methacrylic acid, 2-(triflouromethyl)acrylic acid, 4-vinylpyridine) and one of three porogens (acetonitrile, chloroform, methanol) combined with the crosslinking agent ethylene glycol dimethacrylate and initiator 2,2'-azobis(2-methylpropionitrile). Polymers were characterized by nitrogen gas sorption measurements and SEM, and affinity studies performed using radioligand binding in various media. In agreement with the predictions made from the simulations, polymers prepared in acetonitrile using either methacrylic or trifluoromethacrylic acid demonstrated the highest affinities for oseltamivir. Further, the ensemble of interactions observed in the methanol system provided an explanation for the morphology of polymers prepared in this solvent. The materials developed here offer potential for use in solid-phase extraction or for catalysis. The results illustrate the strength of this in silico strategy as a potential prognostic tool in molecularly imprinted polymer design.

Molecular dynamics approaches to the design and synthesis of PCB targeting molecularly imprinted polymers: interference to monomer-template interactions in imprinting of 1,2,3-trichlorobenzene.

The interactions between each component of the pre-polymerisation mixtures used in the synthesis of molecularly imprinted polymers (MIP) specific for 1,2,3,4,5-pentachlorobenzene (1) and 1,2,3-trichlorobenzene (2) were examined in four molecular dynamics simulations. These simulations revealed that the relative frequency of functional monomer-template (FM-T) interactions was consistent with results obtained by the synthesis and evaluation of the actual MIPs. The higher frequency of 1 interaction with trimethylstyrene (TMS; 54.7%) than 1 interaction with pentafluorostyrene (PFS; 44.7%) correlated with a higher imprinting factor (IF) of 2.1 vs. 1.7 for each functional monomer respectively. The higher frequency of PFS interactions with 2 (29.6%) than TMS interactions with 2 (1.9%) also correlated well with the observed differences in IF (3.7) of 2 MIPs imprinted using PFS as the FM than the IF (2.8) of 2 MIPs imprinted using TMS as the FM. The TMS-1 interaction dominated the molecular simulation due to high interaction energies, but the weaker TMS-2 resulted in low interaction maintenance, and thus lower IF values. Examination of the other pre-polymerisation mixture components revealed that the low levels of TMS-2 interaction was, in part, due to interference caused by the cross linker (CL) ethyleneglycol dimethylacrylate (EGDMA) interactions with TMS. The main reason was, however, attributed to MeOH interactions with TMS in both a hydrogen bond and perpendicular configuration. This positioned a MeOH directly above the π-orbital of all TMS for an average of 63.8% of MD2 creating significant interference to π-π stacking interactions between 2 and TMS. These findings are consistent with the deviation from the 'normal' molecularly imprinted polymer synthesis ratio of 1 : 4 : 20 (T : FM : CL) of 20 : 1 : 29 and 15 : 6 : 29 observed with 2 and TMS and PFS respectively. Our molecular dynamics simulations correctly predicted the high level of interference from other MIP synthesis components. The effect on PFS-1 interaction by MeOH was significantly lower and thus this system was not adversely affected.

A functional monomer is not enough: principal component analysis of the influence of template complexation in pre-polymerization mixtures on imprinted polymer recognition and morphology.

In this report, principal component analysis (PCA) has been used to explore the influence of template complexation in the pre-polymerization phase on template molecularly imprinted polymer (MIP) recognition and polymer morphology. A series of 16 bupivacaine MIPs were studied. The ethylene glycol dimethacrylate (EGDMA)-crosslinked polymers had either methacrylic acid (MAA) or methyl methacrylate (MMA) as the functional monomer, and the stoichiometry between template, functional monomer and crosslinker was varied. The polymers were characterized using radioligand equilibrium binding experiments, gas sorption measurements, swelling studies and data extracted from molecular dynamics (MD) simulations of all-component pre-polymerization mixtures. The molar fraction of the functional monomer in the MAA-polymers contributed to describing both the binding, surface area and pore volume. Interestingly, weak positive correlations between the swelling behavior and the rebinding characteristics of the MAA-MIPs were exposed. Polymers prepared with MMA as a functional monomer and a polymer prepared with only EGDMA were found to share the same characteristics, such as poor rebinding capacities, as well as similar surface area and pore volume, independent of the molar fraction MMA used in synthesis. The use of PCA for interpreting relationships between MD-derived descriptions of events in the pre-polymerization mixture, recognition properties and morphologies of the corresponding polymers illustrates the potential of PCA as a tool for better understanding these complex materials and for their rational design.

On the influence of crosslinker on template complexation in molecularly imprinted polymers: a computational study of prepolymerization mixture events with correlations to template-polymer recognition behavior and NMR spectroscopic studies.

Aspects of the molecular-level basis for the function of ethylene glycol dimethacrylate and trimethylolproprane trimethacrylate crosslinked methacrylic acid copolymers molecularly imprinted with (S)-propranolol have been studied using a series of all-component and all-atom molecular dynamics studies of the corresponding prepolymerization systems. The crosslinking agents were observed to contribute to template complexation, and the results were contrasted with previously reported template-recognition behavior of the corresponding polymers. Differences in the extent to which the two crosslinkers interacted with the functional monomer were identified, and correlations were made to polymer-ligand recognition behavior and the results of nuclear magnetic resonance spectroscopic studies studies. This study demonstrates the importance of considering the functional monomer-crosslinker interaction when designing molecularly imprinted polymers, and highlights the often neglected general contribution of crosslinker to determining the nature of molecularly imprinted polymer-template selectivity.

Towards global QSAR model building for acute toxicity: Munro database case study.

A series of 436 Munro database chemicals were studied with respect to their corresponding experimental LD50 values to investigate the possibility of establishing a global QSAR model for acute toxicity. Dragon molecular descriptors were used for the QSAR model development and genetic algorithms were used to select descriptors better correlated with toxicity data. Toxic values were discretized in a qualitative class on the basis of the Globally Harmonized Scheme: the 436 chemicals were divided into 3 classes based on their experimental LD50 values: highly toxic, intermediate toxic and low to non-toxic. The k-nearest neighbor (k-NN) classification method was calibrated on 25 molecular descriptors and gave a non-error rate (NER) equal to 0.66 and 0.57 for internal and external prediction sets, respectively. Even if the classification performances are not optimal, the subsequent analysis of the selected descriptors and their relationship with toxicity levels constitute a step towards the development of a global QSAR model for acute toxicity.

Consequences of morphology on molecularly imprinted polymer-ligand recognition.

The relationship between molecularly imprinted polymer (MIP) morphology and template-rebinding over a series of warfarin-imprinted methacrylic acid co(ethylene dimethacrylate) polymers has been explored. Detailed investigations of the nature of template recognition revealed that an optimal template binding was obtained with polymers possessing a narrow population of pores (~3-4 nm) in the mesopore size range. Importantly, the warfarin-polymer rebinding analyses suggest strategies for regulating ligand binding capacity and specificity through variation of the degree of cross-linking, where polymers prepared with a lower degree of cross-linking afford higher capacity though non-specific in character. In contrast, the co-existence of specific and non-specific binding was found in conjunction with higher degrees of cross-linking and resultant meso- and macropore size distributions.

Mechanisms underlying molecularly imprinted polymer molecular memory and the role of crosslinker: resolving debate on the nature of template recognition in phenylalanine anilide imprinted polymers.

A series of molecular dynamics simulations of prepolymerization mixtures for phenylalanine anilide imprinted co-(ethylene glycol dimethacrylate-methacrylic acid) molecularly imprinted polymers have been employed to investigate the mechanistic basis for template selective recognition in these systems. This has provided new insights on the mechanisms underlying template recognition, in particular the significant role played by the crosslinking agent. Importantly, the study supports the occurrence of template self-association events that allows us to resolve debate between the two previously proposed models used to explain this system's underlying recognition mechanisms. Moreover, the complexity of the molecular level events underlying template complexation is highlighted by this study, a factor that should be considered in rational molecularly imprinted polymer design, especially with respect to recognition site heterogeneity.

Spectroscopic evidence for the presence of the cyclic hemiketal form of warfarin in aqueous solution: consequences for bioavailability.

Warfarin is an important oral anticoagulant drug that demonstrates a molecular-environment dependent structural diversity. Previous investigations of warfarin's ensemble of isomers in organic solvent-based environments have pointed to the importance of the closed-ring cyclic hemiketal form of the drug in non-polar environments, e.g. the interior of proteins, enzymes and biomembranes. Detection of the presence of these isomers in polar environments has not yet been reported. Here, we demonstrate the presence of the cyclic hemiketal structural form of warfarin under aqueous conditions. This finding underscores the importance considering all structural isomers of this drug when making predictions on warfarin's bioavailability.

In situ detection of warfarin using time-correlated single-photon counting.

Here we report on a novel method for the direct in situ measurement of specific isomeric forms of the anticoagulant warfarin using time correlated single-photon counting (TCSPC) spectroscopy in conjunction with synthetic Sudlow I binding site receptors. The method is highly robust over the clinically significant concentration range, and demonstrates the potential of the binding site mimics in conjunction with the spectroscopic strategy employed here for the determination of this important pharmaceutical in clinical or even environmental samples.

Towards a synthetic avidin mimic.

A series of streptavidin-mimicking molecularly imprinted polymers has been developed and evaluated for their biotin binding characteristics. A combination of molecular dynamics and NMR spectroscopy was used to examine potential polymer systems, in particular with the functional monomers methacrylic acid and 2-acrylamidopyridine. The synthesis of copolymers of ethylene dimethacrylate and one or both of these functional monomers was performed. A combination of radioligand binding studies and surface area analyses demonstrated the presence of selectivity in polymers prepared using methacrylic acid as the functional monomer. This was predicted by the molecular dynamics studies showing the power of this methodology as a prognostic tool for predicting the behavior of molecularly imprinted polymers.

Rational design of biomimetic molecularly imprinted materials: theoretical and computational strategies for guiding nanoscale structured polymer development.

In principle, molecularly imprinted polymer science and technology provides a means for ready access to nano-structured polymeric materials of predetermined selectivity. The versatility of the technique has brought it to the attention of many working with the development of nanomaterials with biological or biomimetic properties for use as therapeutics or in medical devices. Nonetheless, the further evolution of the field necessitates the development of robust predictive tools capable of handling the complexity of molecular imprinting systems. The rapid growth in computer power and software over the past decade has opened new possibilities for simulating aspects of the complex molecular imprinting process. We present here a survey of the current status of the use of in silico-based approaches to aspects of molecular imprinting. Finally, we highlight areas where ongoing and future efforts should yield information critical to our understanding of the underlying mechanisms sufficient to permit the rational design of molecularly imprinted polymers.

Warfarin: an environment-dependent switchable molecular probe.

The complex nature of the structure of the anticoagulant warfarin is reflected in the diversity of binding modes observed in warfarin-protein recognition systems. A series of theoretical, (1)H-NMR and steady state and time resolved fluorescence spectroscopic studies, have been used to establish correlations between the molecular environment provided by various solvent systems and the relative concentrations of the various members of warfarin's ensemble of isomers. A consequence of these observations is that the judicious choice of solvent system or molecular environment of warfarin allows for manipulation of the position of the equilibrium between isomeric structures such as the hemiacetal and open phenol-keto forms, the latter even possible in a deprotonated form, where in each case unique spectroscopic properties are exhibited by the respective structures. Collectively, warfarin's capacity to adapt its structure as a function of environment in conjunction with the fluorescence behaviours of the various isomers together provide an environment-dependent molecular switch with reporter properties, which allows for the simultaneous detection of warfarin in different states with lifetimes spanning the range < 0.10-5.5 ns. These characteristics are here used to examine warfarin binding domains in a series of materials (solvents, protein, inorganic matrix and synthetic polymer). Moreover, these studies demonstrate the potential for using warfarin, or other switchable analogues thereof, as a tool for studying molecular level characteristics, for example local dielectricity.

Structure and dynamics of monomer-template complexation: an explanation for molecularly imprinted polymer recognition site heterogeneity.

We here present the first simulation of a complete molecularly imprinted polymer prepolymerization system. Molecular dynamics studies were performed for a system comprising a total of 1199 discrete molecules, replicating the components and concentrations employed in the corresponding polymer synthesis. The observed interactions correlate well with results obtained from (1)H NMR spectroscopic studies. Comparison with simulations performed in the absence of cross-linking agent (ethylene dimethacrylate) demonstrated its significance in the formation of ligand recognition sites. Moreover, the influence of events such as template-template (bupivacaine) and monomer-monomer (methacrylic acid) self-association, porogen-template interactions, and template conformational variability was revealed. The template recognition capacity of the modeled polymer system was verified by synthesis of imprinted and reference polymers and subsequent radioligand binding analysis. Collectively, through a series of statistical analyses of molecular trajectories in conjunction with spectroscopic data it was demonstrated that an ensemble of complex structures is present in the prepolymerization mixture and that this diversity is the basis for the binding site heterogeneity observed in molecularly imprinted polymers (MIPs) prepared using the noncovalent strategy.

Molecular insights on the two fluorescence lifetimes displayed by warfarin from fluorescence anisotropy and molecular dynamics studies.

A series of steady-state fluorescence anisotropy experiments has been performed to demonstrate the presence of a deprotonated open side chain form of warfarin in organic environments. We explain the observed emission-wavelength-dependent anisotropy of warfarin in ethanol, 2-propanol, and acetonitrile due to the coexistence of neutral isomers and deprotonated open side chain forms displaying different fluorescence decay kinetics. To investigate solvent-solute interactions in more detail, a series of molecular dynamics simulations was performed to study warfarin solvation and to predict the time scale of rotational diffusion displayed by this compound. Predictions obtained provide an explanation for the nonzero values in anisotropy observed for neutral isomers of warfarin associated with the short fluorescence lifetime (tau<0.1 ns) and for an approximately zero anisotropy observed for the deprotonated open side chain form, which is associated with the longer fluorescence lifetime (tau=0.5-1.6 ns). Finally, we address the potential use of fluorescence anisotropy for an increased understanding of the structural diversity of warfarin in protein binding pockets.

Amyloid fibrils prepared using an acetylated and methyl amidated peptide model of the α-Synuclein NAC 71-82 amino acid stretch contain an additional cross-ß structure also found in prion proteins.

The 71-82 fragment of the non-amyloid-ß component (NAC) region of the Parkinson's disease (PD) and dementia with Lewy bodies (DLB) related protein α-Synuclein, has been reported to be important during protein misfolding. Although reports have demonstrated the importance of this fragment for the aggregation properties of the full-length protein, its exact role in pre-fibrillar oligomerisation, fibrillar growth and morphology has not yet been fully elucidated. Here, we provide evidence that fibrils prepared from an acetylated and methyl amidated peptide of the NAC 71-82 amino acid stretch of α-Synuclein are amyloid and contain, in addition to the cross-ß structure detected in the full-length protein fibrils, a cross-ß structure previously observed in prion proteins. These results shed light on the aggregation propensity of the NAC 71-82 amino acid stretch of the full-length protein but also the roles of the N- and C-terminal domains of α-Synuclein in balancing this aggregation propensity. The results also suggest that early aggregated forms of the capped NAC 71-82 peptide generated structures were stabilised by an anti-parallel and twisted ß-sheet motif. Due to its expected toxicity, this ß-sheet motif may be a promising molecular target for the development of therapeutic strategies for PD and DLB.

Author Correction: Dilution of whisky - the molecular perspective.

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The spectrophysics of warfarin: implications for protein binding.

The photophysical behavior of the isomers of the anticoagulant drug warfarin in various solvents and solvent mixtures was investigated using absorption, 1H NMR, and steady-state and time-resolved fluorescence spectroscopies in conjunction with B3LYP-based theoretical treatments. Complex absorption patterns were observed, indicative of the presence of different isomers of warfarin in the various solvents studied. In alkaline aqueous solution, the deprotonated open side form of warfarin is highly dominant and only one S0-->S1 singlet transition could be observed in the absorption spectrum centered at 320 nm. These observations were supported by theoretical density functional calculations (B3LYP) in which the geometries of nine isomers of warfarin were optimized and their respective eight lowest singlet and three lowest triplet excitation energy levels were predicted. Examination of the fluorescence excitation and emission spectra of the isomers in nonpolar and polar organic solvents showed the presence of the deprotonated open side chain form of warfarin in 2-propanol, ethanol, and acetonitrile. Time-resolved fluorescence experiments revealed a short decay time constant, tau1, in all solvents studied while in more polar environments a second longer one, tau2, was evident varying between 0.5 and 1.6 ns depending on solvent polarity. The variation of number and length of fluorescence lifetimes as a function of solvent environment has provided a tool for examining warfarin protein binding. Studies on the binding of warfarin to human serum albumin (HSA) have been undertaken, and different modes of binding were observed which are indicative of binding to the anion-selective Sudlow I and, second, a lower affinity mode of interaction.