Comparative Molecular Dynamics Analysis of RNase-S Complex Formation.

Limited proteolysis of RNase-A yields a short N-terminal S-peptide segment and the larger S-protein. Binding of S-peptide to S-protein results in the formation of an enzymatically active RNase-S protein. S-peptide undergoes a transition from intrinsic disorder to an ordered helical state upon association with S-protein to form RNase-S and is an excellent model system to study coupled folding and binding. To better understand the dynamics of the RNases-S complex and its isolated partners, comparative molecular dynamics simulations have been performed. In agreement with experiment, we find significant conformational fluctuations of the isolated S-peptide compatible with a disordered regime and only little residual helical structure. In the RNase-S complex, the N-terminal helix of S-peptide unfolds and refolds repeatedly on the microsecond timescale, indicating that the α-helical structure is only part of the equilibrium regime for these residues whereas the C-terminal residues are confined to the helical conformation that is found in the x-ray structure. This is also in line with systematic, in silico Alanine scanning free-energy simulations, which indicate that the major contribution to complex stability emerges from the C-terminal helical turn, consisting of residues 8-13 in S-peptide whereas the N-terminal S-peptide residues 1-7 make only minor contributions. Comparative simulations of S-protein in the presence and absence of S-peptide reveal that the isolated S-protein is significantly more flexible than in the complex, and undergoes a global pincerlike conformational change that narrows the S-peptide binding cleft. The narrowed binding cleft adds a barrier for complex formation likely influencing the binding kinetics. This conformational change is reversed by S-peptide association, which also stabilizes conformational fluctuations in S-protein. Such global motions associated with binding are also likely to play a role for other coupled peptide folding and binding processes at peptide binding regions on protein surfaces.

Protein-ligand docking using hamiltonian replica exchange simulations with soft core potentials.

Molecular dynamics (MD) simulations in explicit solvent allow studying receptor-ligand binding processes including full flexibility of the binding partners and an explicit inclusion of solvation effects. However, in MD simulations, the search for an optimal ligand-receptor complex geometry is frequently trapped in locally stable non-native binding geometries. A Hamiltonian replica-exchange (H-REMD)-based protocol has been designed to enhance the sampling of putative ligand-receptor complexes. It is based on softening nonbonded ligand-receptor interactions along the replicas and one reference replica under the control of the original force field. The efficiency of the method has been evaluated on two receptor-ligand systems and one protein-peptide complex. Starting from misplaced initial docking geometries, the H-REMD method reached in each case the known binding geometry significantly faster than a standard MD simulation. The approach could also be useful to identify and evaluate alternative binding geometries in a given binding region with small relative differences in binding free energy.

Role of tyrosine hot-spot residues at the interface of colicin E9 and immunity protein 9: a comparative free energy simulation study.

The endonuclease activity of the bacterial colicin 9 enzyme is controlled by the specific and high-affinity binding of immunity protein 9 (Im9). Molecular dynamics simulation studies in explicit solvent were used to investigate the free energy change associated with the mutation of two hot-spot interface residues [tyrosine (Tyr): Tyr54 and Tyr55] of Im9 to Ala. In addition, the effect of several other mutations (Leu33Ala, Leu52Ala, Val34Ala, Val37Ala, Ser48Ala, and Ile53Ala) with smaller influence on binding affinity was also studied. Good qualitative agreement of calculated free energy changes and experimental data on binding affinity of the mutations was observed. The simulation studies can help to elucidate the molecular details on how the mutations influence protein-protein binding affinity. The role of solvent and conformational flexibility of the partner proteins was studied by comparing the results in the presence or absence of solvent and with or without positional restraints. Restriction of the conformational mobility of protein partners resulted in significant changes of the calculated free energies but of similar magnitude for isolated Im9 and for the complex and therefore in only modest changes of binding free energy differences. Although the overall binding free energy change was similar for the two Tyr-Ala mutations, the physical origin appeared to be different with solvation changes contributing significantly to the Tyr55Ala mutation and to a loss of direct protein-protein interactions dominating the free energy change due to the Tyr54Ala mutation.

In Situ Actuators with Gallium Liquid Metal Alloys and Polypyrrole-Coated Electrodes.

Gallium liquid metal alloys (GLMAs) such as Galinstan and gallium-indium eutectic (EGaIn) are interesting materials due to their high surface tensions, low viscosities, and electrical conductivities comparable to classical solid metals. They have been used for applications in microelectromechanical systems (MEMS) and, more recently, liquid metal microfluidics (LMMF) for setting up devices like actuators. However, their high tendency to alloy with the most common metals used for electrodes such as gold (Au), platinum (Pt), titanium (Ti), nickel (Ni), and tungsten-titanium (WTi) is a major problem limiting the scaleup and applicability, e.g., liquid metal actuators. Stable electrodes are key elements for many applications and thus, the lack of an electrode material compatible with GLMAs is detrimental for many potential application scenarios. In this work, we study the effect of actuating Galinstan on various solid metal electrodes and present an electrode protection methodology that, first, prevents alloying and, second, prevents electrode corrosion. We demonstrate reproducible actuation of GLMA segments in LMMF, showcasing the stability of the proposed protective coating. We investigated a range of electrode materials including Au, Pt, Ti, Ni, and WTi, all in aqueous environments, and present the resulting corrosion/alloying effects by studying the interface morphology. Our proposed protective coating is based on a simple method to electrodeposit electrically conductive polypyrrole (PPy) on the electrodes to provide a conductive alloying-barrier layer for applications involving direct contact between GLMAs and electrodes. We demonstrate the versatility of this approach by direct three-dimensional (3D) printing of a 500 µm microfluidic chip on a set of electrodes onto which PPy is electrodeposited in situ for actuation of Galinstan plugs. The developed protection protocol will provide a generic, widely applicable strategy to protect a wide range of electrodes from alloying and corrosion and thus form a key element in future applications of GLMAs.

Volumetric additive manufacturing of silica glass with microscale computed axial lithography.

Glass is increasingly desired as a material for manufacturing complex microscopic geometries, from the micro-optics in compact consumer products to microfluidic systems for chemical synthesis and biological analyses. As the size, geometric, surface roughness, and mechanical strength requirements of glass evolve, conventional processing methods are challenged. We introduce microscale computed axial lithography (micro-CAL) of fused silica components, by tomographically illuminating a photopolymer-silica nanocomposite that is then sintered. We fabricated three-dimensional microfluidics with internal diameters of 150 micrometers, free-form micro-optical elements with a surface roughness of 6 nanometers, and complex high-strength trusses and lattice structures with minimum feature sizes of 50 micrometers. As a high-speed, layer-free digital light manufacturing process, micro-CAL can process nanocomposites with high solids content and high geometric freedom, enabling new device structures and applications.

Replicative manufacturing of metal moulds for low surface roughness polymer replication.

Tool based manufacturing processes like injection moulding allow fast and high-quality mass-market production, but for optical polymer components the production of the necessary tools is time-consuming and expensive. In this paper a process to fabricate metal-inserts for tool based manufacturing with smooth surfaces via a casting and replication process from fused silica templates is presented. Bronze, brass and cobalt-chromium could be successfully replicated from shaped fused silica replications achieving a surface roughnesses of Rq 8 nm and microstructures in the range of 5 µm. Injection moulding was successfully performed, using a commercially available injection moulding system, with thousands of replicas generated from the same tool. In addition, three-dimensional bodies in metal could be realised with 3D-Printing of fused silica casting moulds. This work thus represents an approach to high-quality moulding tools via a scalable facile and cost-effective route surpassing the currently employed cost-, labour- and equipment-intensive machining techniques.

Injection Molding of Magnesium Aluminate Spinel Nanocomposites for High-Throughput Manufacturing of Transparent Ceramics.

Transparent ceramics like magnesium aluminate spinel (MAS) are considered the next step in material evolution showing unmatched mechanical, chemical and physical resistance combined with high optical transparency. Unfortunately, transparent ceramics are notoriously difficult to shape, especially on the microscale. Therefore, a thermoplastic MAS nanocomposite is developed that can be shaped by polymer injection molding at high speed and precision. The nanocomposite is converted to dense MAS by debinding, pre-sintering, and hot isostatic pressing yielding transparent ceramics with high optical transmission up to 84 % and high mechanical strength. A transparent macroscopic MAS components with wall thicknesses up to 4 mm as well as microstructured components with single micrometer resolution are shown. This work makes transparent MAS ceramics accessible to modern high-throughput polymer processing techniques for fast and cost-efficient manufacturing of macroscopic and microstructured components enabling a plethora of potential applications from optics and photonics, medicine to scratch and break-resistant transparent windows for consumer electronics.

High Resolution Patterning of an Organic-Inorganic Photoresin for the Fabrication of Platinum Microstructures.

Platinum (Pt) is an interesting material for many applications due to its high chemical resilience, outstanding catalytic activity, high electrical conductivity, and high melting point. However, microstructuring and especially 3D microstructuring of platinum is a complex process, based on expensive and specialized equipment often suffering from very slow processing speeds. In this work, organic-inorganic photoresins, which can be structured using direct optical lithography as well as two-photon lithography (TPL) with submicrometer resolution and high-throughput is presented. The printed structures are subsequently converted to high-purity platinum using thermal debinding of the binder and reduction of the salt. With this technique, complex 3D structures with a 3D resolution of 300 nm were fabricated. At a layer thickness of 35 nm, the patterns reach a high conductivity of 67% compared to bulk platinum. Microheaters, thermocouple sensors as well as a Lab-on-a-Chip system are presented as exemplary applications. This technology will enable a broad range of application from electronics, sensing and heating elements to 3D photonics and metamaterials.

Molecular Dynamics Analysis of 4E-BP2 Protein Fold Stabilization Induced by Phosphorylation.

Protein phosphorylation can affect the interaction with partner proteins but can also induce conformational transitions. In case of the eukaryotic translation initiation factor 4E-binding protein 2 (4E-BP2) threonine (Thr) phosphorylation at two turn motifs results in transition from a disordered to a folded structure. In order to elucidate the stabilizing mechanism we employed comparative molecular dynamics (MD) free energy simulations on the turn motifs indicating that Thr-phosphorylation favors a folded whereas dephosphorylation or substitution by Glu residues destabilizes the turn structure. In multiple unrestrained MD simulations at elevated temperature of the 4E-BP2 domain only the double phosphorylated variant remained close to the folded structure in agreement with experiment. Three surface Arg residues were identified as additional key elements for the tertiary structure stabilization of the whole phosphorylated domain. In addition to the local turn structure double phosphorylation also leads to an overall electrostatic stabilization of the folded form compared to wild type and other investigated variants of 4E-BP2. The principles of phosphorylation mediated fold stabilization identified in the present study may also be helpful for identifying other structural motifs that can be affected by phosphorylation or provide a route to design such motifs.

Multiscale Simulation of Receptor-Drug Association Kinetics: Application to Neuraminidase Inhibitors.

A detailed understanding of the drug-receptor association process is of fundamental importance for drug design. Due to the long time scales of typical binding kinetics, the atomistic simulation of the ligand traveling from bulk solution into the binding site is still computationally challenging. In this work, we apply a multiscale approach of combined Molecular Dynamics (MD) and Brownian Dynamics (BD) simulations to investigate association pathway ensembles for the two prominent H1N1 neuraminidase inhibitors oseltamivir and zanamivir. Including knowledge of the approximate binding site location allows for the selective confinement of detailed but expensive MD simulations and application of less demanding BD simulations for the diffusion controlled part of the association pathway. We evaluate a binding criterion based on the residence time of the inhibitor in the binding pocket and compare it to geometric criteria that require prior knowledge about the binding mechanism. The method ranks the association rates of both inhibitors in qualitative agreement with experiment and yields reasonable absolute values depending, however, on the reaction criteria. The simulated association pathway ensembles reveal that, first, ligands are oriented in the electrostatic field of the receptor. Subsequently, a salt bridge is formed between the inhibitor's carboxyl group and neuraminidase residue Arg368, followed by adopting the native binding mode. Unexpectedly, despite oseltamivir's higher overall association rate, the rate into the intermediate salt-bridge state was found to be higher for zanamivir. The present methodology is intrinsically parallelizable and, although computationally demanding, allows systematic binding rate calculation on selected sets of potential drug molecules.

Transient helicity in intrinsically disordered Axin-1 studied by NMR spectroscopy and molecular dynamics simulations.

Many natural proteins are, as a whole or in part, intrinsically disordered. Frequently, such intrinsically disordered regions (IDRs) undergo a transition to a defined and often helical conformation upon binding to partner molecules. The intrinsic propensity of an IDR sequence to fold into a helical conformation already in the absence of a binding partner can have a decisive influence on the binding process and affinity. Using a combination of NMR spectroscopy and molecular dynamics (MD) simulations we have investigated the tendency of regions of Axin-1, an intrinsically disordered scaffolding protein of the WNT signaling pathway, to form helices in segments interacting with binding partners. Secondary chemical shifts from NMR measurements show an increased helical population in these regions. Systematic application of MD advanced sampling approaches on peptide segments of Axin-1 reproduces the experimentally observed tendency and allows insights into the distribution of segment conformations and free energies of helix formation. The results, however, were found to dependent on the force field water model. Recent water models specifically designed for IDRs significantly reduce the predicted helical content and do not improve the agreement with experiment.

Covalent dye attachment influences the dynamics and conformational properties of flexible peptides.

Fluorescence spectroscopy techniques like Förster resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) have become important tools for the in vitro and in vivo investigation of conformational dynamics in biomolecules. These methods rely on the distance-dependent quenching of the fluorescence signal of a donor fluorophore either by a fluorescent acceptor fluorophore (FRET) or a non-fluorescent quencher, as used in FCS with photoinduced electron transfer (PET). The attachment of fluorophores to the molecule of interest can potentially alter the molecular properties and may affect the relevant conformational states and dynamics especially of flexible biomolecules like intrinsically disordered proteins (IDP). Using the intrinsically disordered S-peptide as a model system, we investigate the impact of terminal fluorescence labeling on the molecular properties. We perform extensive molecular dynamics simulations on the labeled and unlabeled peptide and compare the results with in vitro PET-FCS measurements. Experimental and simulated timescales of end-to-end fluctuations were found in excellent agreement. Comparison between simulations with and without labels reveal that the π-stacking interaction between the fluorophore labels traps the conformation of S-peptide in a single dominant state, while the unlabeled peptide undergoes continuous conformational rearrangements. Furthermore, we find that the open to closed transition rate of S-peptide is decreased by at least one order of magnitude by the fluorophore attachment. Our approach combining experimental and in silico methods provides a benchmark for the simulations and reveals the significant effect that fluorescence labeling can have on the conformational dynamics of small biomolecules, at least for inherently flexible short peptides. The presented protocol is not only useful for comparing PET-FCS experiments with simulation results but provides a strategy to minimize the influence on molecular properties when chosing labeling positions for fluorescence experiments.

Mechanism of pKID/KIX Association Studied by Molecular Dynamics Free Energy Simulations.

The phosphorylated kinase-inducible domain (pKID) associates with the kinase interacting domain (KIX) via a coupled folding and binding mechanism. The pKID domain is intrinsically disordered when unbound and upon phosphorylation at Ser133 binds to the KIX domain adopting a well-defined kinked two-helix structure. In order to identify putative hot spot residues of binding that could serve as an initial stable anchor, we performed in silico alanine scanning free energy simulations. The simulations indicate that charged residues including the phosphorylated central Ser133 of pKID make significant contributions to binding. However, these are of slightly smaller magnitude compared to several hydrophobic side chains not defining a single dominant binding hot spot. Both continuous molecular dynamics (MD) simulations and free energy analysis demonstrate that phosphorylation significantly stabilizes the central kinked motif around Ser133 of pKID and shifts the conformational equilibrium toward the bound conformation already in the absence of KIX. This result supports a view that pKID/KIX association follows in part a conformational selection process. During a 1.5 µs explicit solvent MD simulation, folding of pKID on the surface of KIX was observed after an initial contact at the bound position of the phosphorylation site was enforced following a sequential process of αA helix association and a stepwise association and folding of the second αB helix compatible with available experimental results.

Adenylylation of Tyr77 stabilizes Rab1b GTPase in an active state: A molecular dynamics simulation analysis.

The pathogenic pathway of Legionella pneumophila exploits the intercellular vesicle transport system via the posttranslational attachment of adenosine monophosphate (AMP) to the Tyr77 sidechain of human Ras like GTPase Rab1b. The modification, termed adenylylation, is performed by the bacterial enzyme DrrA/SidM, however the effect on conformational properties of the molecular switch mechanism of Rab1b remained unresolved. In this study we find that the adenylylation of Tyr77 stabilizes the active Rab1b state by locking the switch in the active signaling conformation independent of bound GTP or GDP and that electrostatic interactions due to the additional negative charge in the switch region make significant contributions. The stacking interaction between adenine and Phe45 however, seems to have only minor influence on this stabilisation. The results may also have implications for the mechanistic understanding of conformational switching in other signaling proteins.

Exploring biomolecular dynamics and interactions using advanced sampling methods.

Molecular dynamics (MD) and Monte Carlo (MC) simulations have emerged as a valuable tool to investigate statistical mechanics and kinetics of biomolecules and synthetic soft matter materials. However, major limitations for routine applications are due to the accuracy of the molecular mechanics force field and due to the maximum simulation time that can be achieved in current simulations studies. For improving the sampling a number of advanced sampling approaches have been designed in recent years. In particular, variants of the parallel tempering replica-exchange methodology are widely used in many simulation studies. Recent methodological advancements and a discussion of specific aims and advantages are given. This includes improved free energy simulation approaches and conformational search applications.