Impact of crystalline domains on long-term stability and mechanical performance of anisotropic silk fibroin sponges.

Sponge-like materials made from regenerated silk fibroin biopolymers are a tunable and advantageous platform for in vitro engineered tissue culture and in vivo tissue regeneration. Anisotropic, three-dimensional (3D) silk fibroin sponge-like scaffolds can mimic the architecture of contractile muscle. Herein, we use silk fibroin solution isolated from the cocoons of Bombyx mori silkworms to form aligned sponges via directional ice templating in a custom mold with a slurry of dry ice and ethanol. Hydrated tensile mechanical properties of these aligned sponges were evaluated as a function of silk polymer concentration (3% or 5%), freezing time (50% or 100% ethanol), and post-lyophilization method for inducing crystallinity (autoclaving, water annealing). Hydrated static tensile tests were used to determine Young's modulus and ultimate tensile strength across sponge formulations at two strain rates to evaluate rate dependence in the calculated parameters. Results aligned with previous reports in the literature for isotropic silk fibroin sponge-like scaffolds, where the method by which beta-sheets were formed and level of beta-sheet content (crystallinity) had the greatest impact on static parameters, while polymer concentration and freezing rate did not significantly impact static mechanical properties. We estimated the crystalline organization using molecular dynamics simulations to show that larger crystalline regions may be responsible for strength at low strain amplitudes and brittleness at high strain amplitudes in the autoclaved sponges. Within the parameters evaluated, extensional Young's modulus is tunable in the range of 600-2800 kPa. Dynamic tensile testing revealed the linear viscoelastic region to be between 0% and 10% strain amplitude and 0.2-2 Hz frequencies. Long-term stability was evaluated by hysteresis and fatigue tests. Fatigue tests showed minimal change in the storage and loss modulus of 5% silk fibroin sponges for more than 6000 min of continuous mechanical stimulation in the linear regime at 10% strain amplitude and 1 Hz frequency. Furthermore, we confirmed that these mechanical properties hold when decellularized extracellular matrix is added to the sponges and when the mechanical property assessments were performed in cell culture media. We also used nano-computed tomography (nano-CT) and simulations to explore pore interconnectivity and tortuosity. Overall, these results highlight the potential of anisotropic, sponge-like silk fibroin scaffolds for long-term (>6 weeks) contractile muscle culture with an in vitro bioreactor system that provides routine mechanical stimulation.

Molecular Driving Forces in the Self-Association of Silaffin Peptide R5 from MD Simulations.

The 19-residue silaffin-R5 peptide has been widely studied for its ability to precipitate uniform SiO2 particles through mild temperature and pH pathways, in the absence of any organic solvents. There is consensus that post-translational modification (PTM) of side chains has a large impact on the biomineralization process. Thus, it is imperative to understand the precise mechanisms that dictate the formation of SiO2 from R5 peptide, including the effects of PTM on peptide aggregation and peptide-surface adsorption. In this work, we use molecular dynamics (MD) simulations to study the aggregation of R5 dimer with multiple PTMs, with the presence of different ions in solution. Since this system has strong interactions with deep metastable states, we use parallel bias metadynamics with partitioned families to efficiently sample the different states of the system. We find that peptide aggregation is a prerequisite for biomineralization. We observe that the electrostatic interactions are essential in the R5 dimer aggregation; for wild type R5 that only has positively charged residues, phosphate ions HPO4 2- in the solution form a bridge between two peptides and are essential for peptide aggregation.

PEGylation of Insulin and Lysozyme To Stabilize against Thermal Denaturation: A Molecular Dynamics Simulation Study.

Biologic drugs or "biologics" (proteins derived from living organisms) are one of the fastest-growing classes of FDA-approved therapeutics. These compounds are often fragile and require conjugation to polymers for stabilization, with many proteins too ephemeral for therapeutic use. During storage or administration, proteins tend to unravel and lose their secondary structure due to changes in solution temperature, pH, and other external stressors. To enhance their lifetime, protein drugs currently in the market are conjugated with polyethylene glycol (PEG), owing to its ability to increase the stability, solubility, and pharmacokinetics of protein drugs. Here, we perform all-atom molecular dynamics simulations to study the unfolding process of egg-white lysozyme and insulin at elevated temperatures. We test the validity of two force fields─CHARMM36 and Amber ff99SB-ILDN─in the unfolding process. By calculating global and local properties, we capture residues that deteriorate first─these are the "weak links" in the proteins. Next, we conjugate both proteins with PEG and find that PEG preserves the native structure of the proteins at elevated temperatures by blocking water molecules from entering the hydrophobic core, thereby causing the secondary structure to stabilize.

Hierarchical Self-Assembly Pathways of Peptoid Helices and Sheets.

Peptoids (N-substituted glycines) are a class of tailorable synthetic peptidomic polymers. Amphiphilic diblock peptoids have been engineered to assemble 2D crystalline lattices with applications in catalysis and molecular separations. Assembly is induced in an organic solvent/water mixture by evaporating the organic phase, but the assembly pathways remain uncharacterized. We conduct all-atom molecular dynamics simulations of Nbrpe6Nc6 as a prototypical amphiphilic diblock peptoid comprising an NH2-capped block of six hydrophobic N-((4-bromophenyl)ethyl)glycine residues conjugated to a polar NH3(CH2)5CO tail. We identify a thermodynamically controlled assembly mechanism by which monomers assemble into disordered aggregates that self-order into 1D chiral helical rods then 2D achiral crystalline sheets. We support our computational predictions with experimental observations of 1D rods using small-angle X-ray scattering, circular dichroism, and atomic force microscopy and 2D crystalline sheets using X-ray diffraction and atomic force microscopy. This work establishes a new understanding of hierarchical peptoid assembly and principles for the design of peptoid-based nanomaterials.

MARTINI-Compatible Coarse-Grained Model for the Mesoscale Simulation of Peptoids.

Peptoids (poly-N-substituted glycines) are a class of synthetic polymers that are regioisomers of peptides (poly-C-substituted glycines), in which the point of side-chain connectivity is shifted from the backbone C to the N atom. Peptoids have found diverse applications as peptidomimetic drugs, protein mimetic polymers, surfactants, and catalysts. Computational modeling is valuable in the understanding and design of peptoid-based nanomaterials. In this work, we report the bottom-up parameterization of coarse-grained peptoid force fields based on the MARTINI peptide force field against all-atom peptoid simulation data. Our parameterization pipeline iteratively refits coarse-grained bonded interactions using iterative Boltzmann inversion and nonbonded interactions by matching the potential of mean force for chain extension. We assure good sampling of the amide bond cis/trans isomerizations in the all-atom simulation data using parallel bias metadynamics. We develop coarse-grained models for two representative peptoids-polysarcosine (poly(N-methyl glycine)) and poly(N-((4-bromophenyl)ethyl)glycine)-and show their structural and thermodynamic properties to be in excellent accord with all-atom calculations but up to 25-fold more efficient and compatible with MARTINI force fields. This work establishes a new rigorously parameterized coarse-grained peptoid force field for the understanding and design of peptoid nanomaterials at length and time scales inaccessible to all-atom calculations.

Studies of Dynamic Binding of Amino Acids to TiO2 Nanoparticle Surfaces by Solution NMR and Molecular Dynamics Simulations.

Adsorption of biomolecules onto material surfaces involves a potentially complex mechanism where molecular species interact to varying degrees with a heterogeneous material surface. Surface adsorption studies by atomic force microscopy, sum frequency generation spectroscopy, and solid-state NMR detect the structures and interactions of biomolecular species that are bound to material surfaces, which, in the absence of a solid-liquid interface, do not exchange rapidly between surface-bound forms and free molecular species in bulk solution. Solution NMR has the potential to complement these techniques by detecting and studying transiently bound biomolecules at the liquid-solid interface. Herein, we show that dark-state exchange saturation transfer (DEST) NMR experiments on gel-stabilized TiO2 nanoparticle (NP) samples detect several forms of biomolecular adsorption onto titanium(IV) oxide surfaces. Specifically, we use the DEST approach to study the interaction of amino acids arginine (Arg), lysine (Lys), leucine (Leu), alanine (Ala), and aspartic acid (Asp) with TiO2 rutile NP surfaces. Whereas Leu, Ala, and Asp display only a single weakly interacting form in the presence of TiO2 NPs, Arg and Lys displayed at least two distinct bound forms: a species that is surface bound and retains a degree of reorientational motion and a second more tightly bound form characterized by broadened DEST profiles upon the addition of TiO2 NPs. Molecular dynamics simulations indicate different surface bound states for both Lys and Arg depending on the degree of TiO2 surface hydroxylation but only a single bound state for Asp regardless of the degree of surface hydroxylation, in agreement with results obtained from the analysis of DEST profiles.

Trimethylation of the R5 Silica-Precipitating Peptide Increases Silica Particle Size by Redirecting Orthosilicate Binding.

The unmodified R5 peptide from silaffin in the diatom Cylindrotheca fusiformis rapidly precipitates silica particles from neutral aqueous solutions of orthosilicic acid. A range of post-translational modifications found in R5 contribute toward tailoring silica morphologies in a species-specific manner. We investigated the specific effect of R5 lysine side-chain trimethylation, which adds permanent positive charges, on silica particle formation. Our studies revealed that a doubly trimethylated R5K3,4me3 peptide has reduced maximum activity yet, surprisingly, generates larger silica particles. Molecular dynamics simulations of R5K3,4me3 binding by the precursor orthosilicate anion revealed that orthosilicate preferentially associates with unmodified lysine side-chain amines and the peptide N terminus. Thus, larger silica particles arise from reduced orthosilicate association with trimethylated lysine side chains and their redirection to the N terminus of the R5 peptide.

Molecular Driving Forces in Peptide Adsorption to Metal Oxide Surfaces.

Molecular recognition between peptides and metal oxide surfaces is a fundamental process in biomineralization, self-assembly, and biocompatibility. Yet, the underlying driving forces and dominant mechanisms remain unclear, bringing obstacles to understand and control this process. To elucidate the mechanism of peptide/surface recognition, specifically the role of serine phosphorylation, we employed molecular dynamics simulation and metadynamics-enhanced sampling to study five artificial peptides, DDD, DSS, DpSpS, DpSpSGKK, and DpSKGpSK, interacting with two surfaces: rutile TiO2 and quartz SiO2. On both surfaces, we observe that phosphorylation increases the binding energy. However, the interfacial peptide conformation reveals a distinct binding mechanism on each surface. We also study the impact of peptide sequence to binding free energy and interfacial conformation on both surfaces, specifically the impact on the behavior of phosphorylated serine. Finally, the results are discussed in context of prior studies investigating the role of serine phosphorylation in peptide binding to silica.

Solid-State NMR and MD Study of the Structure of the Statherin Mutant SNa15 on Mineral Surfaces.

Elucidation of the structure and interactions of proteins at native mineral interfaces is key to understanding how biological systems regulate the formation of hard tissue structures. In addition, understanding how these same proteins interact with non-native mineral surfaces has important implications for the design of medical and dental implants, chromatographic supports, diagnostic tools, and a host of other applications. Here, we combine solid-state NMR spectroscopy, isotherm measurements, and molecular dynamics simulations to study how SNa15, a peptide derived from the hydroxyapatite (HAP) recognition domain of the biomineralization protein statherin, interacts with HAP, silica (SiO2), and titania (TiO2) mineral surfaces. Adsorption isotherms are used to characterize the binding affinity of SNa15 to HAP, SiO2, and TiO2. We also apply 1D 13C CP MAS, 1D 15N CP MAS, and 2D 13C-13C DARR experiments to SNa15 samples with uniformly 13C- and 15N-enriched residues to determine backbone and side-chain chemical shifts. Different computational tools, namely TALOS-N and molecular dynamics simulations, are used to deduce secondary structure from backbone and side-chain chemical shift data. Our results show that SNa15 adopts an α-helical conformation when adsorbed to HAP and TiO2, but the helix largely unravels upon adsorption to SiO2. Interactions with HAP are mediated in general by acidic and some basic amino acids, although the specific amino acids involved in direct surface interaction vary with surface. The integrated experimental and computational approach used in this study is able to provide high-resolution insights into adsorption of proteins on interfaces.

Impact of ion content and electric field on mechanical properties of coarse-grained ionomers.

Using a coarse-grained ionomer model for polyethylene-co-methacrylic acid that includes associating acid groups along with pendant anions and unbound counterions, we investigate how ionomer mechanical behavior depends on the acid and ion content. We find that the modulus and yield stress increase as the ion content increases, at all strain rates considered. This is in agreement with prior experimental results. We also apply a very strong external electric field in the melt state and then cool the system to set the aggregate order induced by the field. We find that the application of electric field increases the modulus in the direction parallel to the field, and we postulate that this is related to the observed increase in aggregate ordering in the direction perpendicular to the field.

Influence of a nanoparticle on the structure and dynamics of model ionomer melts.

We simulate a single spherical nanoparticle (NP) surrounded by partially neutralized ionomers. The coarse-grained ionomers consist of a linear backbone of neutral monomer beads with charged pendant beads and counterions, along with pendant 'sticker' beads that represent unneutralized acid groups. Two different NP interactions are considered; one in which the NP interacts uniformly with all beads in the system (neutral NP) and another in which the NP has higher cohesive interactions with ions and stickers (sticky NP). Ions are depleted around the neutral NP relative to the bulk, but are denser around the surface of the sticky NP. The bond vector autocorrelation function was computed as a function of distance from the NP. For the neutral NP, due to the absence of ions, there is an increase in bond rotational dynamics near the surface relative to the bulk, while the reverse trend is observed in the case of the sticky NP. These analyses were done systematically for differing mole content of pendants, levels of neutralization, and NP sizes; lower pendant content causes a significantly larger difference in the bond dynamics near and far from the NP surface.

Impact of ionic aggregate structure on ionomer mechanical properties from coarse-grained molecular dynamics simulations.

Using coarse-grained molecular dynamics simulations, we study ionomers in equilibrium and under uniaxial tensile deformation. The spacing of ions along the chain is varied, allowing us to consider how different ionic aggregate morphologies, from percolated to discrete aggregates, impact the mechanical properties. From the equilibrium simulations, we calculate the stress-stress auto correlation function, showing a distinct deviation from the Rouse relaxation due to ionic associations that depends on ion content. We then quantify the morphology during strain, particularly the degree to which both chains and ionic aggregates tend to align. We also track the location of the ionomer peak in the anisotropic structure factor during strain. The length scale of aggregate order increases in the axial direction and decreases in the transverse direction, in qualitative agreement with prior experimental results.