K. G. Sprenger

The General AMBER Force Field (GAFF) Can Accurately Predict Thermodynamic and Transport Properties of Many Ionic Liquids

We have applied molecular dynamics to calculate thermodynamic and transport properties of a set of 19 room-temperature ionic liquids. Since accurately simulating the thermophysical properties of solvents strongly depends upon the force field of choice, we tested the accuracy of the general AMBER force field, without refinement, for the case of ionic liquids. Electrostatic point charges were developed using ab initio calculations and a charge scaling factor of 0.8 to more accurately predict dynamic properties. The density, heat capacity, molar enthalpy of vaporization, self-diffusivity, and shear viscosity of the ionic liquids were computed and compared to experimentally available data, and good agreement across a wide range of cation and anion types was observed. Results show that, for a wide range of ionic liquids, the general AMBER force field, with no tuning of parameters, can reproduce a variety of thermodynamic and transport properties with similar accuracy to that of other published, often IL-specific, force fields.

Lytic Polysaccharide Monooxygenases ScLPMO10B and ScLPMO10C Are Stable in Ionic Liquids As Determined by Molecular Simulations

Lytic polysaccharide monooxygenases (LPMOs) are a newly discovered family of enzymes proposed to work synergistically with cellulases and aid in the decomposition of cellulose for the creation of environmentally friendly fuels and chemicals. To our knowledge, evaluation of the stability of LPMOs in ionic liquid (IL) solvents at relevant biomass processing conditions has not been explored. Herein, molecular dynamics simulations of ScLPMO10B and ScLPMO10C in three ILs at 10 and 20 wt% in water and in pure water have been performed. Enzyme stability was predicted to be high on the basis of structural and dynamic analyses we performed. We used the simulations to identify key areas that deviate from the crystal structures as a starting place for surface charge modifications to increase stability in ILs. Results show that, in general, both enzymes have a high degree of stability across the range of IL solutions tested. For each enzyme, two regions were identified that showed notable deviations from the crystal structure. In addition to providing a basis for future rational design efforts, this work represents a first step toward engineering LPMOs to function efficiently in enzyme cocktails for use in industrial biomass processing applications with ILs.

Strong Electrostatic Interactions Lead to Entropically Favorable Binding of Peptides to Charged Surfaces

Thermodynamic analyses can provide key insights into the origins of protein self-assembly on surfaces, protein function, and protein stability. However, obtaining quantitative measurements of thermodynamic observables from unbiased classical simulations of peptide or protein adsorption is challenging because of sampling limitations brought on by strong biomolecule/surface binding forces as well as time scale limitations. We used the parallel tempering metadynamics in the well-tempered ensemble (PTMetaD-WTE) enhanced sampling method to study the adsorption behavior and thermodynamics of several explicitly solvated model peptide adsorption systems, providing new molecular-level insight into the biomolecule adsorption process. Specifically studied were peptides LKα14 and LKβ15 and trpcage miniprotein adsorbing onto a charged, hydrophilic self-assembled monolayer surface functionalized with a carboxylic acid/carboxylate headgroup and a neutral, hydrophobic methyl-terminated self-assembled monolayer surface. Binding free energies were calculated as a function of temperature for each system and decomposed into their respective energetic and entropic contributions. We investigated how specific interfacial features such as peptide/surface electrostatic interactions and surface-bound ion content affect the thermodynamic landscape of adsorption and lead to differences in surface-bound conformations of the peptides. Results show that upon adsorption to the charged surface, configurational entropy gains of the released solvent molecules dominate the configurational entropy losses of the bound peptide. This behavior leads to an apparent increase in overall system entropy upon binding and therefore to the surprising and seemingly nonphysical result of an apparent increased binding free energy at elevated temperatures. Opposite effects and conclusions are found for the neutral surface. Additional simulations demonstrate that by adjusting the ionic strength of the solution, results that show the expected physical behavior, i.e., peptide binding strength that decreases with increasing temperature or is independent of temperature altogether, can be recovered on the charged surface. On the basis of this analysis, an overall free energy for the entire thermodynamic cycle for peptide adsorption on charged surfaces is constructed and validated with independent simulations.

Mechanism of Competitive Inhibition and Destabilization of Acidothermus Cellulolyticus Endoglucanase 1 by Ionic Liquids

The ability of ionic liquids (ILs) to solubilize cellulose has sparked interest in their use for enzymatic biomass processing. However, this potential is yet to be realized, primarily because ILs inactivate requisite cellulases by mechanisms that are yet to be identified. We used a combination of enzymology, circular dichroism (CD), nuclear magnetic resonance (NMR), and molecular dynamics (MD) methods to investigate the molecular basis for the inactivation of the endocellulase 1 (E1) from Acidothermus cellulolyticus by the imidazolium IL 1-butyl-3-methylimidazolium chloride ([BMIM][Cl]). Enzymatic studies revealed that [BMIM][Cl] inactivates E1 in a biphasic manner that involves rapid, reversible inhibition, followed by slow, irreversible deactivation. Backbone NMR signals of the 40.5 kDa E1 were assigned by triple resonance NMR methods, enabling monitoring of residue-specific perturbations. 1H-15N NMR titration experiments revealed that [BMIM][Cl] binds reversibly to the E1 active site, indicating that reversible deactivation is due to competitive inhibition of substrate binding. Prolonged incubation with [BMIM][Cl] led to substantial global changes in the 1H-15N heteronuclear single quantum coherence NMR and CD spectra of E1 indicative of protein denaturation. Notably, weak interactions between [BMIM][Cl] and residues at the termini of several helices were also observed, which, together with MD simulations, suggest that E1 denaturation is promoted by [BMIM][Cl]-induced destabilization of helix capping structures. In addition to identifying determinants of E1 inactivation, our findings establish a molecular framework for engineering cellulases with improved IL compatibility.

Determining dominant driving forces affecting controlled protein release from polymeric nanoparticles

Enzymes play a critical role in many applications in biology and medicine as potential therapeutics. One specific area of interest is enzyme encapsulation in polymer nanostructures, which have applications in drug delivery and catalysis. A detailed understanding of the mechanisms governing protein/polymer interactions is crucial for optimizing the performance of these complex systems for different applications. Using a combined computational and experimental approach, this study aims to quantify the relative importance of molecular and mesoscale driving forces to protein release from polymeric nanoparticles. Classical molecular dynamics (MD) simulations have been performed on bovine serum albumin (BSA) in aqueous solutions with oligomeric surrogates of poly(lactic-co-glycolic acid) copolymer, poly(styrene)-poly(lactic acid) copolymer, and poly(lactic acid). The simulated strength and location of polymer surrogate binding to the surface of BSA have been compared to experimental BSA release rates from nanoparticles formulated with these same polymers. Results indicate that the self-interaction tendencies of the polymer surrogates and other macroscale properties may play governing roles in protein release. Additional MD simulations of BSA in solution with poly(styrene)-acrylate copolymer reveal the possibility of enhanced control over the enzyme encapsulation process by tuning polymer self-interaction. Last, the authors find consistent protein surface binding preferences across simulations performed with polymer surrogates of varying lengths, demonstrating that protein/polymer interactions can be understood in part by studying the interactions and affinity of proteins with small polymer surrogates in solution.

Essential slow degrees of freedom in protein-surface simulations: A metadynamics investigation

Many proteins exhibit strong binding affinities to surfaces, with binding energies much greater than thermal fluctuations. When modelling these protein-surface systems with classical molecular dynamics (MD) simulations, the large forces that exist at the protein/surface interface generally confine the system to a single free energy minimum. Exploring the full conformational space of the protein, especially finding other stable structures, becomes prohibitively expensive. Coupling MD simulations with metadynamics (enhanced sampling) has fast become a common method for sampling the adsorption of such proteins. In this paper, we compare three different flavors of metadynamics, specifically well-tempered, parallel-bias, and parallel-tempering in the well-tempered ensemble, to exhaustively sample the conformational surface-binding landscape of model peptide GGKGG. We investigate the effect of mobile ions and ion charge, as well as the choice of collective variable (CV), on the binding free energy of the peptide. We make the case for explicitly biasing ions to sample the true binding free energy of biomolecules when the ion concentration is high and the binding free energies of the solute and ions are similar. We also make the case for choosing CVs that apply bias to all atoms of the solute to speed up calculations and obtain the maximum possible amount of information about the system.

Probing How Defects in Self-assembled Monolayers Affect Peptide Adsorption with Molecular Simulation

Due to their flexible chemical functionality and simple formulation, self-assembled monolayer (SAM) surfaces have become an ideal choice for a multitude of wide-ranging applications. However, a major issue in the preparation of SAM surfaces is naturally occurring defects that manifest in a number of different ways, including depressions in the underlying gold substrate that cause surface roughness or through incorrect self-assembly of the chains that causes domain boundary effects. Molecular simulations can provide valuable insight into the origins of these defects and the effect they have on biological and other processes. Molecular dynamics (MD) simulations have been performed on a SAM surface with a carboxylic acid/carboxylate terminal functionality and induced with two types of experimentally observed defects. The enhanced sampling method PTMetaD-WTE has been used to model the adsorption of LKα14 onto the two types of defective SAM surfaces and onto a control SAM surface with no defective chains. An advanced clustering algorithm has been used to elucidate the effect of the surface defects on the conformations of the adsorbed peptide. Results show significant structural differences arise as a result of the defects. Specifically, both types of defects lead to a near-complete loss of secondary structure of the adsorbed peptide as compared to the control simulation, in which LKα14 adopts a perfect helical structure at the SAM/water interface. On the surface with domain boundary effects, extended conformations of the peptide are stabilized, whereas on the SAM with surface roughness (i.e., chains of various lengths), random coil conformations dominate the ensemble of surface-bound structures.

Investigating the Role of Phosphorylation in the Binding of Silaffin Peptide R5 to Silica with Molecular Dynamics Simulations

Biomimetic silica formation, a process that is largely driven by proteins, has garnered considerable interest in recent years due to its role in the development of new biotechnologies. However, much remains unknown of the molecular-scale mechanisms underlying the binding of proteins to biomineral surfaces such as silica, or even of the key residue-level interactions between such proteins and surfaces. In this study, we employ molecular dynamics (MD) simulations to study the binding of R5-a 19-residue segment of a native silaffin peptide used for in vitro silica formation-to a silica surface. The metadynamics enhanced sampling method is used to converge the binding behavior of R5 on silica at both neutral (pH 7.5) and acidic (pH 5) conditions. The results show fundamental differences in the mechanism of binding between the two cases, providing unique insight into the pH-dependent ability of R5 and native silaffin to precipitate silica. We also study the effect of phosphorylation of serine residues in R5 on both the binding free energy to silica and the interfacial conformation of the peptide. Results indicate that phosphorylation drastically decreases the binding free energy and changes the structure of silica-adsorbed R5 through the introduction of charge and steric repulsion. New mechanistic insights from this work could inform rational design of new biomaterials and biotechnologies.