Tertiary Plasticity Drives the Efficiency of Enterocin 7B Interactions with Lipid Membranes.

The ability of antimicrobial peptides to efficiently kill their bacterial targets depends on the efficiency of their binding to the microbial membrane. In the case of enterocins, there is a three-part interaction: initial binding, unpacking of helices on the membrane surface, and permeation of the lipid bilayer. Helical unpacking is driven by disruption of the peptide hydrophobic core when in contact with membranes. Enterocin 7B is a leaderless enterocin antimicrobial peptide produced from Enterococcus faecalis that functions alone, or with its cognate partner enterocin 7A, to efficiently kill a wide variety of Gram-stain positive bacteria. To better characterize the role that tertiary structural plasticity plays in the ability of enterocin 7B to interact with the membranes, a series of arginine single-site mutants were constructed that destabilize the hydrophobic core to varying degrees. A series of experimental measures of structure, stability, and function, including CD spectra, far UV CD melting profiles, minimal inhibitory concentrations analysis, and release kinetics of calcein, show that decreased stabilization of the hydrophobic core is correlated with increased efficiency of a peptide to permeate membranes and in killing bacteria. Finally, using the computational technique of adaptive steered molecular dynamics, we found that the atomistic/energetic landscape of peptide mechanical unfolding leads to free energy differences between the wild type and its mutants, whose trends correlate well with our experiment.

Benchmarking Adaptive Steered Molecular Dynamics (ASMD) on CHARMM Force Fields.

The potentials of mean force (PMFs) along the end-to-end distance of two different helical peptides have been obtained and benchmarked using the adaptive steered molecular dynamics (ASMD) method. The results depend strongly on the choice of force field driving the underlying all-atom molecular dynamics, and are reported with respect to the three most popular CHARMM force field versions: c22, c27 and c36. Two small peptides, ALA 10 and 1PEF, serve as the particular case studies. The comparisons between the versions of the CHARMM force fields provides both a qualitative and quantitative look at their performance in forced unfolding simulations in which peptides undergo large changes in structural conformations. We find that ASMD with the underlying c36 force field provides the most robust results for the selected benchmark peptides.

Surface Coating Structure and Its Interaction with Cytochrome c in EG6-Coated Nanoparticles Varies with Surface Curvature.

The composition, orientation, and conformation of proteins in biomolecular coronas acquired by nanoparticles in biological media contribute to how they are identified by a cell. While numerous studies have investigated protein composition in biomolecular coronas, relatively little detail is known about how the nanoparticle surface influences the orientation and conformation of the proteins associated with them. We previously showed that the peripheral membrane protein cytochrome c adopts preferred poses relative to negatively charged 3-mercaptopropionic acid (MPA)-gold nanoparticles (AuNPs). Here, we employ molecular dynamics simulations and complementary experiments to establish that cytochrome c also assumes preferred poses upon association with nanoparticles functionalized with an uncharged ligand, specifically ω-(1-mercaptounde-11-cyl)hexa(ethylene glycol) (EG6). We find that the display of the EG6 ligands is sensitive to the curvature of the surface-and, consequently, the effective diameter of the nearly spherical nanoparticle core-which in turn affects the preferred poses of cytochrome c.

Multi-cellular engineered living systems: building a community around responsible research on emergence.

Ranging from miniaturized biological robots to organoids, multi-cellular engineered living systems (M-CELS) pose complex ethical and societal challenges. Some of these challenges, such as how to best distribute risks and benefits, are likely to arise in the development of any new technology. Other challenges arise specifically because of the particular characteristics of M-CELS. For example, as an engineered living system becomes increasingly complex, it may provoke societal debate about its moral considerability, perhaps necessitating protection from harm or recognition of positive moral and legal rights, particularly if derived from cells of human origin. The use of emergence-based principles in M-CELS development may also create unique challenges, making the technology difficult to fully control or predict in the laboratory as well as in applied medical or environmental settings. In response to these challenges, we argue that the M-CELS community has an obligation to systematically address the ethical and societal aspects of research and to seek input from and accountability to a broad range of stakeholders and publics. As a newly developing field, M-CELS has a significant opportunity to integrate ethically responsible norms and standards into its research and development practices from the start. With the aim of seizing this opportunity, we identify two general kinds of salient ethical issues arising from M-CELS research, and then present a set of commitments to and strategies for addressing these issues. If adopted, these commitments and strategies would help define M-CELS as not only an innovative field, but also as a model for responsible research and engineering.

Preferential Binding of Cytochrome c to Anionic Ligand-Coated Gold Nanoparticles: A Complementary Computational and Experimental Approach.

Membrane-bound proteins can play a role in the binding of anionic gold nanoparticles (AuNPs) to model bilayers; however, the mechanism for this binding remains unresolved. In this work, we determine the relative orientation of the peripheral membrane protein cytochrome c in binding to a mercaptopropionic acid-functionalized AuNP (MPA-AuNP). As this is nonrigid binding, traditional methods involving crystallographic or rigid molecular docking techniques are ineffective at resolving the question. Instead, we have implemented a computational assay technique using a cross-correlation of a small ensemble of 200 ns long molecular dynamics trajectories to identify a preferred nonrigid binding orientation or pose of cytochrome c on MPA-AuNPs. We have also employed a mass spectrometry-based footprinting method that enables the characterization of the stable protein corona that forms at long time-scales in solution but remains in a dynamic state. Through the combination of these computational and experimental primary results, we have established a consensus result establishing the identity of the exposed regions of cytochrome c in proximity to MPA-AuNPs and its complementary pose(s) with amino-acid specificity. Moreover, the tandem use of the two methods can be applied broadly to determine the accessibility of membrane-binding sites for peripheral membrane proteins upon adsorption to AuNPs or to determine the exposed amino-acid residues of the hard corona that drive the acquisition of dynamic soft coronas. We anticipate that the combined use of simulation and experimental methods to characterize biomolecule-nanoparticle interactions, as demonstrated here, will become increasingly necessary as the complexity of such target systems grows.

Peripheral Membrane Proteins Facilitate Nanoparticle Binding at Lipid Bilayer Interfaces.

Molecular understanding of the impact of nanomaterials on cell membranes is critical for the prediction of effects that span environmental exposures to nanoenabled therapies. Experimental and computational studies employing phospholipid bilayers as model systems for membranes have yielded important insights but lack the biomolecular complexity of actual membranes. Here, we increase model membrane complexity by incorporating the peripheral membrane protein cytochrome c and studying the interactions of the resulting membrane systems with two types of anionic nanoparticles. Experimental and computational studies reveal that the extent of cytochrome c binding to supported lipid bilayers depends on anionic phospholipid number density and headgroup chemistry. Gold nanoparticles functionalized with short, anionic ligands or wrapped with an anionic polymer do not interact with silica-supported bilayers composed solely of phospholipids. Strikingly, when cytochrome c was bound to these bilayers, nanoparticles functionalized with short anionic ligands attached to model biomembranes in amounts proportional to the number of bound cytochrome c molecules. In contrast, anionic polymer-wrapped gold nanoparticles appeared to remove cytochrome c from supported lipid bilayers in a manner inversely proportional to the strength of cytochrome c binding to the bilayer; this reflects the removal of a weakly bound pool of cytochrome c, as suggested by molecular dynamics simulations. These results highlight the importance of the surface chemistry of both the nanoparticle and the membrane in predicting nano-bio interactions.

Mutational Analysis of Neuropeptide Y Reveals Unusual Thermal Stability Linked to Higher-Order Self-Association.

Neuropeptide Y (NPY) is a 36-residue peptide, abundant in the central and peripheral nervous system. The peptide interacts with membrane-bound receptors to control processes such as food intake, vasoconstriction, and memory retention. The N-terminal polyproline sequence of NPY folds back onto a C-terminal α-helix to form a hairpin structure. The hairpin undergoes transient unfolding to allow the monomer to interact with its target membranes and receptors and to form reversible dimers in solution. Using computational, functional, and biophysical approaches, we characterized the role of two conserved tyrosines (Y20 and Y27) located within the hydrophobic core of the hairpin fold. Successive mutation of the tyrosines to more hydrophobic phenylalanines increased the thermal stability of NPY and reduced functional activity, consistent with computational studies predicting a more stable hairpin structure. However, mutant stability was high relative to wild-type: melting temperatures increased by approximately 20 °C for the single mutants (Y20F and Y27F) and by 30 °C for the double mutant (Y20F + Y27F). These findings suggested that the mutations were not just simply enhancing hairpin structure stability, but might also be driving self-association to dimer. Using analytical ultracentrifugation, we determined that the mutations indeed increased self-association, but shifted the equilibrium toward hexamer-like species. Notably, these latter species were not unique to the NPY mutants, but were found to preexist at low levels in the wild-type population. Collectively, the findings indicate that NPY self-association is more complex than previously recognized and that the ensemble of NPY quaternary states is tunable by modulating hairpin hydrophobicity.

Examining Ionic Liquid Effects on Mononuclear Rearrangement of Heterocycles Using QM/MM Simulations.

The mononuclear rearrangement of heterocycles (MRH) reaction of the Z-phenylhydrazone of 3-benzoyl-5-phenyl-1,2,4-oxadiazole into 4-benzoylamino-2,5-diphenyl-1,2,3-triazole derives a sizable rate enhancement in the 1-butyl-3-methylimidazolium tetrafluoroborate [BMIM][BF4] ionic liquid as compared to the hexafluorophosphate-based [BMIM][PF6] and conventional organic solvents. However, the origin of the rate difference between [BMIM][BF4] and [BMIM][PF6] has proven difficult to rationalize as no experimental trend relates the physical properties of the solvents, e.g., polarity and viscosity, to the rates of reaction. QM/MM calculations in combination with free-energy perturbation theory and Monte Carlo sampling have been carried out for the MRH reaction to elucidate the disparities in rates when using ionic liquids, methanol, and acetonitrile. Activation barriers and solute-solvent interactions have been computed for both an uncatalyzed and a specific base-catalyzed mechanism. Energetic and structural analyses determined that favorable π+-π interactions between the BMIM cation, the substrate phenyl rings, and the bicyclic quasi-aromatic 10π oxadiazole/triazole transition state region imposed a preordered geometric arrangement that enhanced the rate of reaction. An ionic liquid clathrate formation enforced a coplanar orientation of the phenyl rings that maximized the electronic effects exerted on the reaction route. In addition, site-specific electrostatic stabilization between the ions and the MRH substrate was more prevalent in [BMIM][BF4] as compared to [BMIM][PF6].

Ionic liquid effects on nucleophilic aromatic substitution reactions from QM/MM simulations.

Nucleophilic aromatic substitution (SNAr) reactions are particularly sensitive to medium effects and have been reported to benefit from ionic liquids. The SNAr reaction between cyclic secondary amines (i.e., piperidine, pyrrolidine, and morpholine) and the 2-L-5-nitrothiophene (para-like) and 2-L-3-nitrothiophene (ortho-like) isomers, where L = bromo, methoxy, phenoxy, and 4-nitrophenoxy, has been computationally investigated in 1-butyl-3-methylimidazolium tetrafluoroborate and hexafluorophosphate [BMIM][BF4] and [BMIM][PF6], respectively. QM/MM Monte Carlo simulations utilizing free-energy perturbation theory were used to characterize the solute-solvent interactions over the addition-elimination reaction pathway. Energetic and structural analyses determined that the improved SNAr reactivity in [BMIM][BF4] and [BMIM][PF6] can be attributed to (1) an enhanced nucleophilicity of the cyclic amines in the ionic liquids with an order of Pyr ≥ Pip > Mor, (2) beneficial π(+)-π interactions between the BMIM cations and the aromatic rings present on the substrate that enhanced coplanarity between the thiophene ring and the aromatic substituents, resulting in a larger positive charge on the reacting ipso carbon, and (3) a highly ordered ionic liquid clathrate formation that, despite an entropy penalty, provided reduced activation free-energy barriers derived from an increasing number of solvent ions favorably interacting with the emerging charge separation at the rate-limiting addition step.

An ionic liquid dependent mechanism for base catalyzed ß-elimination reactions from QM/MM simulations.

Ionic liquids have been proposed to induce a mechanistic change in the reaction pathway for the fundamentally important base-induced ß-elimination class compared to conventional solvents. The role of the reaction medium in the elimination of 1,1,1-tribromo-2,2-bis(3,4-dimethoxyphenyl)ethane via two bases, piperidine and pyrrolidine, has been computationally investigated using methanol and the ionic liquids 1-butyl-3-methylimidazolium tetrafluoroborate and hexafluorophosphate [BMIM][BF(4)] and [BMIM][PF(6)], respectively. QM/MM Monte Carlo simulations utilizing free-energy perturbation theory found the ionic liquids did produce a reaction pathway change from an E1cB-like mechanism in methanol to a pure E2 route that is consistent with experimental observations. The origin of the ionic liquid effect has been found as: (1) a combination of favorable electrostatic interactions, for example, bromine-imidazolium ion, and (2) π-π interactions that enhance the coplanarity between aromatic rings maximizing the electronic effects exerted on the reaction route. Solute-solvent interaction energies have been analyzed and show that liquid clathrate solvation of the transition state is primarily responsible for the observed mechanistic changes. This work provides the first theoretical evidence of an ionic liquid dependent mechanism and elucidates the interplay between sterics and electrostatics crucial to understanding the effect of these unique solvents upon chemical reactions.