Molecular dynamics simulations of copper binding to amyloid-ß Glu22 mutants.

We report microsecond timescale ligand field molecular dynamics simulations of the copper complexes of three known mutants of the amyloid-ß peptide, E22G, E22Q and E22K, alongside the naturally occurring sequence. We find that all three mutants lead to formation of less compact structures than the wild-type: E22Q is the most similar to the native peptide, while E22G and especially E22K are markedly different in size, shape and stability. Turn and coil structures dominate all structures studied but subtle differences in helical and ß-sheet distribution are noted, especially in the C-terminal region. The origin of these changes is traced to disruption of key salt bridges: in particular, the Asp23-Lys28 bridge that is prevalent in the wild-type is absent in E22G and E22K, while Lys22 in the latter mutant forms a strong association with Asp23. We surmise that the drastically different pattern of salt bridges in the mutants lead to adoption of a different structural ensemble of the peptide backbone, and speculate that this might affect the ability of the mutant peptides to aggregate in the same manner as known for the wild-type.

Molecular dynamics simulation of aluminium binding to amyloid-ß and its effect on peptide structure.

Multiple microsecond-length molecular dynamics simulations of complexes of Al(III) with amyloid-ß (Aß) peptides of varying length are reported, employing a non-bonded model of Al-coordination to the peptide, which is modelled using the AMBER ff14SB forcefield. Individual simulations reach equilibrium within 100 to 400 ns, as determined by root mean square deviations, leading to between 2.1 and 2.7 µs of equilibrated data. These reveal a compact set of configurations, with radius of gyration similar to that of the metal free peptide but larger than complexes with Cu, Fe and Zn. Strong coordination through acidic residues Glu3, Asp7 and Glu11 is maintained throughout all trajectories, leading to average coordination numbers of approximately 4 to 5. Helical conformations predominate, particularly in the longer Al-Aß40 and Al-Aß42 peptides, while ß-strand forms are rare. Binding of the small, highly charged Al(III) ion to acidic residues in the N-terminus strongly disrupts their ability to engage in salt bridges, whereas residues outside the metal binding region engage in salt bridges to similar extent to the metal-free peptide, including the Asp23-Lys28 bridge known to be important for formation of fibrils. High helical content and disruption of salt bridges leads to characteristic tertiary structure, as shown by heat maps of contact between residues as well as representative clusters of trajectories.

Solution Structure of Mannobioses Unravelled by Means of Raman Optical Activity.

Structural analysis of carbohydrates is a complicated endeavour, due to the complexity and diversity of the samples at hand. Herein, we apply a combined computational and experimental approach, employing molecular dynamics (MD) and density functional theory (DFT) calculations together with NMR and Raman optical activity (ROA) measurements, in the structural study of three mannobiose disaccharides, consisting of two mannoses with varying glycosidic linkages. The disaccharide structures make up the scaffold of high mannose glycans and are therefore important targets for structural analysis. Based on the MD population analysis and NMR, the major conformers of each mannobiose were identified and used as input for DFT analysis. By systematically varying the solvent models used to describe water interacting with the molecules and applying overlap integral analysis to the resulting calculational ROA spectra, we found that a full quantum mechanical/molecular mechanical approach is required for an optimal calculation of the ROA parameters. Subsequent normal mode analysis of the predicted vibrational modes was attempted in order to identify possible marker bands for glycosidic linkages. However, the normal mode vibrations of the mannobioses are completely delocalised, presumably due to conformational flexibility in these compounds, rendering the identification of isolated marker bands unfeasible.

Replica exchange molecular dynamics simulation of the coordination of Pt(ii)-Phenanthroline to amyloid-ß.

We report replica exchange molecular dynamics (REMD) simulations of the complex formed between amyloid-ß peptides and platinum bound to a phenanthroline ligand, Pt(phen). After construction of an AMBER-style forcefield for the Pt complex, REMD simulation employing temperatures between 270 and 615 K was used to provide thorough sampling of the conformational freedom available to the peptide. We find that the full length peptide Aß42, in particular, frequently adopts a compact conformation with a large proportion of α- and 3,10-helix content, with smaller amounts of ß-strand in the C-terminal region of the peptide. Helical structures are more prevalent than in the metal-free peptide, while turn and strand conformations are markedly less common. Non-covalent interactions, including salt-bridges, hydrogen bonds, and π-stacking between aromatic residues and the phenanthroline ligand, are common, and markedly different from those seen in the amyloid-ß peptides alone.

Molecular dynamics simulation on the effect of transition metal binding to the N-terminal fragment of amyloid-ß.

We report molecular dynamics simulations of three possible adducts of Fe(II) to the N-terminal 1-16 fragments of the amyloid-ß peptide, along with analogous simulations of Cu(II) and Zn(II) adducts. We find that multiple simulations from different starting points reach pseudo-equilibration within 100-300 ns, leading to over 900 ns of equilibrated trajectory data for each system. The specifics of the coordination modes for Fe(II) have only a weak effect on peptide secondary and tertiary structures, and we therefore compare one of these with analogous models of Cu(II) and Zn(II) complexes. All share broadly similar structural features, with mixture of coil, turn and bend in the N-terminal region and helical structure for residues 11-16. Within this overall pattern, subtle effects due to changes in metal are evident: Fe(II) complexes are more compact and are more likely to occupy bridge and ribbon regions of Ramachandran maps, while Cu(II) coordination leads to greater occupancy of the poly-proline region. Analysis of representative clusters in terms of molecular mechanics energy and atoms-in-molecules properties indicates similarity of four-coordinate Cu and Zn complexes, compared to five-coordinate Fe complex that exhibits lower stability and weaker metal-ligand bonding. Communicated by Ramaswamy H. Sarma.

Metal Binding to Amyloid-ß1-42: A Ligand Field Molecular Dynamics Study.

Ligand field molecular mechanics simulation has been used to model the interactions of copper(II) and platinum(II) with the amyloid-ß1-42 peptide monomer. Molecular dynamics over several microseconds for both metalated systems are compared to analogous results for the free peptide. Significant differences in structural parameters are observed, both between Cu and Pt bound systems as well as between free and metal-bound peptide. Both metals stabilize the formation of helices in the peptide as well as reducing the content of ß secondary structural elements compared to the unbound monomer. This is in agreement with experimental reports of metals reducing ß-sheet structures, leading to formation of amorphous aggregates over amyloid fibrils. The shape and size of the peptide structures also undergo noteworthy change, with the free peptide exhibiting globular-like structure, platinum(II) system adopting extended structures, and copper(II) system resulting in a mixture of conformations similar to both of these. Salt bridge networks exhibit major differences: the Asp23-Lys28 salt bridge, known to be important in fibril formation, has a differing distance profile within all three systems studied. Salt bridges in the metal binding region of the peptide are strongly altered; in particular, the Arg5-Asp7 salt bridge, which has an occurrence of 71% in the free peptide, is reduced to zero in the presence of both metals.

Optically Active Vibrational Spectroscopy of α-Aminoisobutyric Acid Foldamers in Organic Solvents and Phospholipid Bilayers.

Helical α-aminoisobutyric acid (Aib) foldamers show great potential as devices for the communication of conformational information across phospholipid bilayers, but determining their conformation in bilayers remains a challenge. In the present study, Raman, Raman optical activity (ROA), infrared (IR) and vibrational circular dichroism (VCD) spectroscopies have been used to analyze the conformational preferences of Aib foldamers in solution and when interacting with bilayers. A 310 -helix marker band at 1665-1668 cm-1 in Raman spectra was used to show that net helical content increased strongly with oligomer length. ROA and VCD spectra of chiral Aib foldamers provided the chiroptical signature for both left- and right-handed 310 -helices in organic solvents, with VCD establishing that foldamer screw-sense was preserved when the foldamers became embedded within bilayers. However, the population distribution between different secondary structures was perturbed by the chiral phospholipid. These studies indicate that ROA and VCD spectroscopies are valuable tools for the study of biomimetic structures, such as artificial signal transduction molecules, in phospholipid bilayers.

Ligand field molecular dynamics simulation of Pt(II)-phenanthroline binding to N-terminal fragment of amyloid-ß peptide.

We report microsecond timescale molecular dynamics simulation of the complex formed between Pt(II)-phenanthroline and the 16 N-terminal residues of the Aß peptide that is implicated in the onset of Alzheimer's disease, along with equivalent simulations of the metal-free peptide. Simulations from a variety of starting points reach equilibrium within 100 ns, as judged by root mean square deviation and radius of gyration. Platinum-bound peptides deviate rather more from starting points, and adopt structures with larger radius of gyration, than their metal-free counterparts. Residues bound directly to Pt show smaller fluctuation, but others actually move more in the Pt-bound peptide. Hydrogen bonding within the peptide is disrupted by binding of Pt, whereas the presence of salt-bridges are enhanced.

Benchmarking of copper(II) LFMM parameters for studying amyloid-ß peptides.

Ligand field molecular mechanics (LFMM) parameters have been benchmarked for copper (II) bound to the amyloid-ß1-16 peptide fragment. Several density functional theory (DFT) optimised small test models, representative of different possible copper coordination modes, have been used to test the accuracy of the LFMM copper bond lengths and angles, resulting in errors typically less than 0.1 Å and 5°. Ligand field molecular dynamics (LFMD) simulations have been carried out on the copper bound amyloid-ß1-16 peptide and snapshots extracted from the subsequent trajectory. Snapshots have been optimised using DFT and the semi-empirical PM7 method resulting in good agreement against the LFMM calculated geometry. Analysis of substructures within snapshots shows that the larger contribution of geometrical difference, as measured by RMSD, lies within the peptide backbone, arising from differences in DFT and AMBER, and the copper coordination sphere is reproduced well by LFMM. PM7 performs excellently against LFMM with an average RMSD of 0.2 Å over 21 tested snapshots. Further analysis of the LFMD trajectory shows that copper bond lengths and angles have only small deviations from average values, with the exception of a carbonyl moiety from the N-terminus, which can act as a weakly bound fifth ligand.

Quantitative detection of codeine in human plasma using surface-enhanced Raman scattering via adaptation of the isotopic labelling principle.

In this study surface enhanced Raman scattering (SERS) combined with the isotopic labelling (IL) principle has been used for the quantification of codeine spiked into both water and human plasma. Multivariate statistical approaches were employed for the analysis of these SERS spectral data, particularly partial least squares regression (PLSR) which was used to generate models using the full SERS spectral data for quantification of codeine with, and without, an internal isotopic labelled standard. The PLSR models provided accurate codeine quantification in water and human plasma with high prediction accuracy (Q2). In addition, the employment of codeine-d6 as the internal standard further improved the accuracy of the model, by increasing the Q2 from 0.89 to 0.94 and decreasing the low root-mean-square error of predictions (RMSEP) from 11.36 to 8.44. Using the peak area at 1281 cm-1 assigned to C-N stretching, C-H wagging and ring breathing, the limit of detection was calculated in both water and human plasma to be 0.7 µM (209.55 ng mL-1) and 1.39 µM (416.12 ng mL-1), respectively. Due to a lack of definitive codeine vibrational assignments, density functional theory (DFT) calculations have also been used to assign the spectral bands with their corresponding vibrational modes, which were in excellent agreement with our experimental Raman and SERS findings. Thus, we have successfully demonstrated the application of SERS with isotope labelling for the absolute quantification of codeine in human plasma for the first time with a high degree of accuracy and reproducibility. The use of the IL principle which employs an isotopolog (that is to say, a molecule which is only different by the substitution of atoms by isotopes) improves quantification and reproducibility because the competition of the codeine and codeine-d6 for the metal surface used for SERS is equal and this will offset any difference in the number of particles under analysis or any fluctuations in laser fluence. It is our belief that this may open up new exciting opportunities for testing SERS in real-world samples and applications which would be an area of potential future studies.

Distinguishing Epimers Through Raman Optical Activity.

The Raman optical activity spectra of the epimers ß-D-glucose and ß-D-galactose, two monosaccharides of biological importance, have been calculated using molecular dynamics combined with a quantum mechanics/molecular mechanics approach. Good agreement between theoretical and experimental spectra is observed for both monosaccharides. Full band assignments have been carried out, which has not previously been possible for carbohydrate epimers. For the regions where the spectral features are opposite in sign, the differences in the vibrational modes have been noted and ascribed to the band sign changes.

The Raman optical activity of ß-D-xylose: where experiment and computation meet.

Besides its applications in bioenergy and biosynthesis, ß-d-xylose is a very simple monosaccharide that exhibits relatively high rigidity. As such, it provides the best basis to study the impact of different solvation shell radii on the computation of its Raman optical activity (ROA) spectrum. Indeed, this chiroptical spectroscopic technique provides exquisite sensitivity to stereochemistry, and benefits much from theoretical support for interpretation. Our simulation approach combines density functional theory (DFT) and molecular dynamics (MD) in order to efficiently account for the crucial hydration effects in the simulation of carbohydrates and their spectroscopic response predictions. Excellent agreement between the simulated spectrum and the experiment was obtained with a solvation radius of 10 Å. Vibrational bands have been resolved from the computed ROA data, and compared with previous results on different monosaccharides in order to identify specific structure-spectrum relationships and to investigate the effect of the solvation environment on the conformational dynamics of small sugars. From the comparison with ROA analytical results, a shortcoming of the classical force field used for the MD simulations has been identified and overcome, again highlighting the complementary role of experiment and theory in the structural characterisation of complex biomolecules. Indeed, due to unphysical puckering, a spurious ring conformation initially led to erroneous conformer ratios, which are used as weights for the averaging of the spectral average, and only by removing this contribution was near perfect comparison between theory and experiment achieved.

Calculation of Raman optical activity spectra for vibrational analysis.

By looking back on the history of Raman Optical Activity (ROA), the present article shows that the success of this analytical technique was for a long time hindered, paradoxically, by the deep level of detail and wealth of structural information it can provide. Basic principles of the underlying theory are discussed, to illustrate the technique's sensitivity due to its physical origins in the delicate response of molecular vibrations to electromagnetic properties. Following a short review of significant advances in the application of ROA by UK researchers, we dedicate two extensive sections to the technical and theoretical difficulties that were overcome to eventually provide predictive power to computational simulations in terms of ROA spectral calculation. In the last sections, we focus on a new modelling strategy that has been successful in coping with the dramatic impact of solvent effects on ROA analyses. This work emphasises the role of complementarity between experiment and theory for analysing the conformations and dynamics of biomolecules, so providing new perspectives for methodological improvements and molecular modelling development. For the latter, an example of a next-generation force-field for more accurate simulations and analysis of molecular behaviour is presented. By improving the accuracy of computational modelling, the analytical capabilities of ROA spectroscopy will be further developed so generating new insights into the complex behaviour of molecules.

Conformational dynamics of carbohydrates: Raman optical activity of D-glucuronic acid and N-acetyl-D-glucosamine using a combined molecular dynamics and quantum chemical approach.

As two biologically and medically relevant monosaccharides, the constituents of hyaluronic acid, d-glucuronic acid and N-acetyl-d-glucosamine, constitute perfect test cases for the development of carbohydrate-specific structural methods. These two molecules have been analysed by Raman optical activity (ROA), a spectroscopic technique exhibiting exquisite sensitivity to stereochemistry. We show that it is possible to support the experiment with a simulation approach combining density functional theory (DFT) and molecular dynamics (MD), both using explicit solvation. Thus, we have gained new insight into the crucial hydration effects that contribute to the conformational dynamics of carbohydrates and managed to characterize in detail the poorly understood vibrational nature of this class of biomolecules. Experimental and calculated ROA spectra of these two molecules are reported and excellent agreement has been found. More specifically, comparison has been made with the more commonly used gas phase and implicitly solvated calculation approaches, which offer poor or zero modelling of solvent interactions. The calculated spectra have been used to resolve the structural origins of the observed bands, a current challenge in the study of carbohydrates due to a lack of definitive vibrational assignments. We report and analyse major features in the fingerprint region of the ROA spectra, with recurrent structural and spectral features between the two monosaccharides observed.

DNA fragment conformations in adducts with Kiteplatin.

The anticancer activity of cisplatin is triggered by its formation of intrastrand adducts involving adjacent G residues of DNA. To obtain information on the different conformers that can be formed, carrier ligands such as 2,2'-bipiperidine, which provide large steric bulk near the platinum coordination plane and decrease the dynamic motion about the Pt-N7 bonds, were introduced ("retro-modelling" approach). In the present study we investigate the effect of cis-1,4-diaminocyclohexane (cis-1,4-DACH) on the formation, stability, and stereochemistry of (cis-1,4-DACH)Pt(ss-oligo) adducts (ss-oligo = d(GpG) with 3'- and/or 5'-substituents). Interesting features of this ligand, absent in previous retro-modelling studies, include the large bite angle (expected to impede the ease of interconversion between possible conformers), the presence of two protons on each nitrogen (a characteristic associated with antitumor activity), and the absence of chiral centres. The use of cis-1,4-DACH has made it possible to detect different conformers in a system containing a primary diamine carrier ligand associated with anticancer activity and to confirm the previous hypothesis that the coexistence of different conformers established in studies of retro models having relatively bulky ligands is not an artefact resulting from carrier-ligand bulk. Moreover, the data for the (cis-1,4-DACH)Pt(d(GpG)) and (cis-1,4-DACH)Pt(d(GGTTT)) adducts indicate that at a temperature close to the physiological one (40 °C) HH1 and ΔHT1 conformers are present in comparable amounts. In contrast, at low temperature (close to 0 °C) the equilibrium shifts dramatically toward the more stable HH1 conformer (for the (cis-1,4-DACH)Pt(d(TGGT)) adduct the HH1 conformer is always dominant, even at high temperature). Notably, (cis-1,4-DACH)PtCl2 (Kiteplatin) has been recently reinvestigated and found to be particularly active against colorectal cancer (including oxaliplatin-resistant phenotypes).

Computational evidence for structural consequences of kiteplatin damage on DNA.

The reaction of the potential anticancer drug kiteplatin, cis-[PtCl2(cis-1,4-DACH)], with oligomers of single- and double-stranded DNA ranging from 2 to 12 base pairs in length was performed as a model for DNA interaction. The potential for conformational flexibility of single-stranded adducts was examined with density functional theory (DFT) and compared with data from (1)H-NMR 1D and 2D spectroscopy. This indicates the presence of multiple conformations of an adduct with d(GpG), but only one form of the adduct with d(TGGT). The importance of a suitable theoretical model, and in particular basis set, in reproducing experimental data is demonstrated. The DFT theoretical model was extended to platinated base pair step (GG/CC), allowing a comparison to the related compounds cisplatin and oxaliplatin. Adducts of kiteplatin with larger fragments of double-stranded DNA, including tetramer, octamer, and dodecamer, were studied theoretically using hybrid quantum mechanics/molecular mechanics methods. Structural parameters of all the base-paired models were evaluated and binding energies calculated in gas phase and in solution; these are compared across the series and also with the related complexes cisplatin and oxaliplatin, thus revealing insights into how kiteplatin binds to DNA and similarities and differences between this and related compounds.

Density functional theory studies of interactions of ruthenium-arene complexes with base pair steps.

Density functional theory (DFT) calculations have been performed to determine the strength and geometry of intermolecular interactions of "piano-stool" ruthenium arene complexes, which show potential as anticancer treatments. Model complexes with methane and benzene indicate that the coordinated arene has C-H···π acceptor ability similar to that of free benzene, whereas this arene acts as a much stronger C-H donor or partner in π-stacking than free benzene. The source of these enhanced interactions is identified as a combination of electrostatic and dispersion effects. Complexes of Ru-arene complexes with base-pair step fragments of DNA, in which the arene has the potential to act as an intercalator, have also been investigated. Binding energies are found to be sensitive to the size and nature of the arene, with larger and more flexible arenes having stronger binding. π-stacking and C-H···π interactions between arene and DNA bases and hydrogen bonds from coordinated N-H to DNA oxygen atoms, as well as covalent Ru-N bonding, contribute to the overall binding. The effect of complexation on DNA structure is also examined, with larger rise and more negative slide values than canonical B-DNA observed in all cases.

Modulation of stacking interactions by transition-metal coordination: ab initio benchmark studies.

A series of ab initio calculations are used to determine the C--Hpi and pipi-stacking interactions of aromatic rings coordinated to transition-metal centres. Two model complexes have been employed, namely, ferrocene and chromium benzene tricarbonyl. Benchmark data obtained from extrapolation of MP2 energies to the basis set limit, coupled with CCSD(T) correction, indicate that coordinated aromatic rings are slightly weaker hydrogen-bond acceptors but are significantly stronger hydrogen-bond donors than uncomplexed rings. It is found that pipi stacking to a second benzene is stronger than in the free benzene dimer, especially in the chromium case. This is assigned, by use of energy partitioning in the local correlation method, to dispersion interactions between metal d and benzene pi orbitals. The benchmark data is also used to test the performance of more efficient theoretical methods, indicating that spin-component scaling of MP2 energies performs well in all cases, whereas various density functionals describe some complexes well, but others with errors of more than 1 kcal mol(-1).