On the Validation of Protein Force Fields Based on Structural Criteria.

Molecular dynamics simulations depend critically on the quality of the force field used to describe the interatomic interactions and the extent to which it has been validated for use in a specific application. Using a curated test set of 52 high-resolution structures, 39 derived from X-ray diffraction and 13 solved using NMR, we consider the extent to which different parameter sets of the GROMOS protein force field can be distinguished based on comparing a range of structural criteria, including the number of backbone hydrogen bonds, the number of native hydrogen bonds, polar and nonpolar solvent-accessible surface area, radius of gyration, the prevalence of secondary structure elements, J-coupling constants, nuclear Overhauser effect (NOE) intensities, positional root-mean-square deviations (RMSD), and the distribution of backbone ϕ and ψ dihedral angles. It is shown that while statistically significant differences between the average values of individual metrics could be detected, these were in general small. Furthermore, improvements in agreement in one metric were often offset by loss of agreement in another. The work establishes a framework and test set against which protein force fields can be validated. It also highlights the danger of inferring the relative quality of a given force field based on a small range of structural properties or small number of proteins.

Facilitating the structural characterisation of non-canonical amino acids in biomolecular NMR.

Peptides and proteins containing non-canonical amino acids (ncAAs) are a large and important class of biopolymers. They include non-ribosomally synthesised peptides, post-translationally modified proteins, expressed or synthesised proteins containing unnatural amino acids, and peptides and proteins that are chemically modified. Here, we describe a general procedure for generating atomic descriptions required to incorporate ncAAs within popular NMR structure determination software such as CYANA, CNS, Xplor-NIH and ARIA. This procedure is made publicly available via the existing Automated Topology Builder (ATB) server (https://atb.uq.edu.au, last access: 17 February 2023) with all submitted ncAAs stored in a dedicated database. The described procedure also includes a general method for linking of side chains of amino acids from CYANA templates. To ensure compatibility with other systems, atom names comply with IUPAC guidelines. In addition to describing the workflow, 3D models of complex natural products generated by CYANA are presented, including vancomycin. In order to demonstrate the manner in which the templates for ncAAs generated by the ATB can be used in practice, we use a combination of CYANA and CNS to solve the structure of a synthetic peptide designed to disrupt Alzheimer-related protein-protein interactions. Automating the generation of structural templates for ncAAs will extend the utility of NMR spectroscopy to studies of more complex biomolecules, with applications in the rapidly growing fields of synthetic biology and chemical biology. The procedures we outline can also be used to standardise the creation of structural templates for any amino acid and thus have the potential to impact structural biology more generally.

Engineering Transferable Atomic Force Fields: Empirical Optimization of Hydrocarbon Lennard-Jones Interactions by Direct Mapping of Parameter Space.

The utility of atomistic simulations depends on the accuracy of the force field used to represent the potential energy landscape, the consistency with which interaction parameters can be assigned, and the extent to which parameters can be transferred between chemical entities. Here, parameter space mapping, a simple and robust procedure for atom typing (parameter assignment) and parameter optimization, is used to identify a minimal set of parameters capable of simultaneously reproducing the density, heat of vaporization, and solvation free energies for a targeted set of simple hydrocarbons. Using an atom-centered fixed charge model and a 6-12 Lennard-Jones potential, the experimental densities and the heats of vaporization for 22 hydrocarbons (linear, cyclic, and aromatic) could be predicted with high precision: average unsigned error (AUE) of 6.1 kg/m3 and 0.5 kJ/mol, respectively, and R2 values of 0.991 and 0.999, respectively. For the 17 compounds with experimental solvation free energy values in water, the AUE was 1.3 kJ/mol, and the slope and R2 for the line of best fit were 0.968 and 0.991, respectively. A key element in ensuring transferability in this work was minimizing confounding variables by ensuring that the calculation of observables was independent of the precise choice of simulation settings (cutoff, bond constraints, etc.) and the explicit consideration of correlations between parameters.

OFraMP: a fragment-based tool to facilitate the parametrization of large molecules.

An Online tool for Fragment-based Molecule Parametrization (OFraMP) is described. OFraMP is a web application for assigning atomic interaction parameters to large molecules by matching sub-fragments within the target molecule to equivalent sub-fragments within the Automated Topology Builder (ATB, atb.uq.edu.au) database. OFraMP identifies and compares alternative molecular fragments from the ATB database, which contains over 890,000 pre-parameterized molecules, using a novel hierarchical matching procedure. Atoms are considered within the context of an extended local environment (buffer region) with the degree of similarity between an atom in the target molecule and that in the proposed match controlled by varying the size of the buffer region. Adjacent matching atoms are combined into progressively larger matched sub-structures. The user then selects the most appropriate match. OFraMP also allows users to manually alter interaction parameters and automates the submission of missing substructures to the ATB in order to generate parameters for atoms in environments not represented in the existing database. The utility of OFraMP is illustrated using the anti-cancer agent paclitaxel and a dendrimer used in organic semiconductor devices. OFraMP applied to paclitaxel (ATB ID 35922).

Self-cyclisation as a general and efficient platform for peptide and protein macrocyclisation.

Macrocyclisation of proteins and peptides results in a remarkable increase in structural stability, making cyclic peptides and proteins of great interest in drug discovery-either directly as drug leads or as in the case of cyclised nanodiscs (cNDs), as tools for studies of trans-membrane receptors and membrane-active peptides. Various biological methods have been developed that are capable of yielding head-to-tail macrocyclised products. Recent advances in enzyme-catalysed macrocyclisation include discovery of new enzymes or design of new engineered enzymes. Here, we describe the engineering of a self-cyclising "autocyclase" protein, capable of performing a controllable unimolecular reaction for generation of cyclic biomolecules in high yield. We characterise the self-cyclisation reaction mechanism, and demonstrate how the unimolecular reaction path provides alternative avenues for addressing existing challenges in enzymatic cyclisation. We use the method to produce several notable cyclic peptides and proteins, demonstrating how autocyclases offer a simple, alternative way to access a vast diversity of macrocyclic biomolecules.

PyThinFilm: Automated Molecular Dynamics Simulation Protocols for the Generation of Thin Film Morphologies.

The performance of organic optoelectronic devices, such as organic light-emitting diodes (OLEDs) and organic solar cells (OSCs), is intrinsically related to the molecular-scale morphology of the thin films from which they are composed. However, the experimental characterization of morphology at the molecular level is challenging due to the often amorphous or at best semicrystalline nature of these films. Classical molecular modeling techniques, such as molecular dynamics (MD) simulation, are increasingly used to understand the relationship between morphology and the properties of thin-film devices. PyThinFilm (github.com/ATB-UQ/PyThinFilm) is an open-source Python package which allows fully automated MD simulations of thin film growth to be performed using vacuum and/or solution deposition processes. PyThinFilm utilizes the GROMACS simulation package in combination with interaction parameters from the Automated Topology Builder (atb.uq.edu.au). Here, PyThinFilm is described along with an overview of applications in which PyThinFilm has been used to study the thin films of organic semiconductor materials typically used in OLEDs and OSCs.

Understanding the Effect of pH on the Solubility and Aggregation Extent of Humic Acid in Solution by Combining Simulation and the Experiment.

Molecular dynamics (MD) simulations were performed to investigate the dynamics of humic acid (HA) in an aqueous solution and the influence of pH, temperature, and HA concentration. The HA model employed in MD simulations was chosen and validated using experimental chemical composition data and Fourier transform infrared (FTIR) spectra. The simulations showed that the HA molecule has a strong propensity to adopt a compact conformation in water independent of pH, while the aggregation of HA was found to be pH-dependent. At high pH, the ionized HAs assembled into a thread-like structure, maximizing contact with water. At low pH, the neutral HAs formed a droplet-like aggregate, minimizing contact with the solvent. The simulation results are consistent with experimental data from dynamic light scattering (DLS) measurements and transmission electron microscopy (TEM) imaging. This work provides new insight into the folding and aggregation of HA as a function of pH and a molecular-level understanding of the relationship between the acidity and the structure, solubility, and aggregation of HA, with direct implications for HA-based remediation strategies of contaminated sites.

Curved or linear? Predicting the 3-dimensional structure of α-helical antimicrobial peptides in an amphipathic environment.

α-Helical membrane-active antimicrobial peptides (AMPs) are known to act via a range of mechanisms, including the formation of barrel-stave and toroidal pores and the micellisation of the membrane (carpet mechanism). Different mechanisms imply that the peptides adopt different 3D structures when bound at the water-membrane interface, a highly amphipathic environment. Here, an evolutionary algorithm is used to predict the 3D structure of a range of α-helical membrane-active AMPs at the water-membrane interface by optimising amphipathicity. This amphipathic structure prediction (ASP) is capable of distinguishing between curved and linear peptides solved experimentally, potentially allowing the activity and mechanism of action of different membrane-active AMPs to be predicted. The ASP algorithm is accessible via a web interface at http://atb.uq.edu.au/asp/.

Automated Topology Builder Version 3.0: Prediction of Solvation Free Enthalpies in Water and Hexane.

The ability of atomic interaction parameters generated using the Automated Topology Builder and Repository version 3.0 (ATB3.0) to predict experimental hydration free enthalpies (Δ Gwater) and solvation free enthalpies in the apolar solvent hexane (Δ Ghexane) is presented. For a validation set of 685 molecules the average unsigned error (AUE) between Δ Gwater values calculated using the ATB3.0 and experiment is 3.8 kJ·mol-1. The slope of the line of best fit is 1.00, the intercept -1.0 kJ·mol-1, and the R2 0.90. For the more restricted set of 239 molecules used to validate OPLS3 ( J. Chem. Theory Comput. 2016 , 12 , 281 - 296 , DOI: 10.1021/acs.jctc.5b00864 ) the AUE using the ATB3.0 is just 2.7 kJ·mol-1 and the R2 0.93. A roadmap for further improvement of the ATB parameters is presented together with a discussion of the challenges of validating force fields against the available experimental data.

Predicting the Prevalence of Alternative Warfarin Tautomers in Solution.

Warfarin, a widely used oral anticoagulant, is prescribed as a racemic mixture. Each enantiomer of neutral Warfarin can exist in 20 possible tautomeric states leading to complex pharmacokinetics and uncertainty as to the relevant species under different conditions. Here, the ability of alternative computational approaches to predict the preferred tautomeric form(s) of neutral Warfarin in different solvents is examined. It is shown that varying the method used to estimate the heat of formation in vacuum (direct or via homodesmic reactions), whether entropic corrections were included, and the method used to estimate the free enthalpy of solvation (i.e., PCM, COSMO, or SMD implicit models or explicit solvent) lead to large differences in the predicted rank and relative populations of the tautomers. In this case, only a combination of the enthalpy of formation using homodesmic reactions and explicit solvent to estimate the free enthalpy of solvation yielded results compatible with the available experimental data. The work also suggests that a small but significant subset of the possible Warfarin tautomers are likely to be physiologically relevant.

Optimization of Empirical Force Fields by Parameter Space Mapping: A Single-Step Perturbation Approach.

A general method for parametrizing atomic interaction functions is presented. The method is based on an analysis of surfaces corresponding to the difference between calculated and target data as a function of alternative combinations of parameters (parameter space mapping). The consideration of surfaces in parameter space as opposed to local values or gradients leads to a better understanding of the relationships between the parameters being optimized and a given set of target data. This in turn enables for a range of target data from multiple molecules to be combined in a robust manner and for the optimal region of parameter space to be trivially identified. The effectiveness of the approach is illustrated by using the method to refine the chlorine 6-12 Lennard-Jones parameters against experimental solvation free enthalpies in water and hexane as well as the density and heat of vaporization of the liquid at atmospheric pressure for a set of 10 aromatic-chloro compounds simultaneously. Single-step perturbation is used to efficiently calculate solvation free enthalpies for a wide range of parameter combinations. The capacity of this approach to parametrize accurate and transferrable force fields is discussed.

The Molecular Origin of Anisotropic Emission in an Organic Light-Emitting Diode.

Atomistic nonequilibrium molecular dynamics simulations have been used to model the induction of molecular orientation anisotropy within the emission layer of an organic light-emitting diode (OLED) formed by vapor deposition. Two emitter species were compared: racemic fac-tris(2-phenylpyridine)iridium(III) (Ir(ppy)3) and trans-bis(2-phenylpyridine)(acetylacetonate)iridium(III) (Ir(ppy)2(acac)). The simulations show that the molecular symmetry axes of both emitters preferentially align perpendicular to the surface during deposition. The molecular arrangement formed on deposition combined with consideration of the transition dipole moments provides insight into experimental reports that Ir(ppy)3 shows isotropic emission, while Ir(ppy)2(acac) displays improved efficiency due to an apparent preferential alignment of the transition dipole vectors parallel to the substrate. The simulations indicate that this difference is not due to differences in the extent of emitter alignment, but rather differences in the direction of the transition dipoles within the two complexes.

Real Cost of Speed: The Effect of a Time-Saving Multiple-Time-Stepping Algorithm on the Accuracy of Molecular Dynamics Simulations.

To enhance efficiency in molecular dynamics simulations, forces that vary slowly are often evaluated less often than those that vary rapidly. We show that the multiple-time-step algorithm implemented in recent versions of GROMACS results in significant differences in the collective properties of a system under conditions where the system was previously stable. The implications of changing the simulation algorithm without assessment of potential artifacts on the parametrization and transferability of effective force fields are discussed.

Elucidating the Spatial Arrangement of Emitter Molecules in Organic Light-Emitting Diode Films.

The effect of varying the emitter concentration on the structural properties of an archetypal phosphorescent blend consisting of 4,4'-bis(N-carbazolyl)biphenyl and tris(2-phenylpyridyl)iridium(III) has been investigated using non-equilibrium molecular dynamics (MD) simulations that mimic the process of vacuum deposition. By comparison with reflectometry measurements, we show that the simulations provide an accurate model of the average density of such films. The emitter molecules were found not to be evenly distributed throughout film, but rather they can form networks that provide charge and/or energy migration pathways, even at emitter concentrations as low as ≈5 weight percent. At slightly higher concentrations, percolated networks form that span the entire system. While such networks would give improved charge transport, they could also lead to more non-radiative pathways for the emissive state and a resultant loss of efficiency.

Testing and validation of the Automated Topology Builder (ATB) version 2.0: prediction of hydration free enthalpies.

To test and validate the Automated force field Topology Builder and Repository (ATB; http://compbio.biosci.uq.edu.au/atb/ ) the hydration free enthalpies for a set of 214 drug-like molecules, including 47 molecules that form part of the SAMPL4 challenge have been estimated using thermodynamic integration and compared to experiment. The calculations were performed using a fully automated protocol that incorporated a dynamic analysis of the convergence and integration error in the selection of intermediate points. The system has been designed and implemented such that hydration free enthalpies can be obtained without manual intervention following the submission of a molecule to the ATB. The overall average unsigned error (AUE) using ATB 2.0 topologies for the complete set of 214 molecules was 6.7 kJ/mol and for molecules within the SAMPL4 7.5 kJ/mol. The root mean square error (RMSE) was 9.5 and 10.0 kJ/mol respectively. However, for molecules containing functional groups that form part of the main GROMOS force field the AUE was 3.4 kJ/mol and the RMSE was 4.0 kJ/mol. This suggests it will be possible to further refine the parameters provided by the ATB based on hydration free enthalpies.

An Automated Force Field Topology Builder (ATB) and Repository: Version 1.0.

The Automated force field Topology Builder (ATB, http://compbio.biosci.uq.edu.au/atb ) is a Web-accessible server that can provide topologies and parameters for a wide range of molecules appropriate for use in molecular simulations, computational drug design, and X-ray refinement. The ATB has three primary functions: (1) to act as a repository for molecules that have been parametrized as part of the GROMOS family of force fields, (2) to act as a repository for pre-equilibrated systems for use as starting configurations in molecular dynamics simulations (solvent mixtures, lipid systems pre-equilibrated to adopt a specific phase, etc.), and (3) to generate force field descriptions of novel molecules compatible with the GROMOS family of force fields in a variety of formats (GROMOS, GROMACS, and CNS). Force field descriptions of novel molecules are derived using a multistep process in which results from quantum mechanical (QM) calculations are combined with a knowledge-based approach to ensure compatibility (as far as possible) with a specific parameter set of the GROMOS force field. The ATB has several unique features: (1) It requires that the user stipulate the protonation and tautomeric states of the molecule. (2) The symmetry of the molecule is analyzed to ensure that equivalent atoms are assigned identical parameters. (3) Charge groups are assigned automatically. (4) Where the assignment of a given parameter is ambiguous, a range of possible alternatives is provided. The ATB also provides several validation tools to assist the user to assess the degree to which the topology generated may be appropriate for a given task. In addition to detailing the steps involved in generating a force field topology compatible with a specific GROMOS parameter set (GROMOS 53A6), the challenges involved in the automatic generation of force field parameters for atomic simulations in general are discussed.