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.

Correlation between chemical denaturation and the unfolding energetics of Acanthamoeba actophorin.

The actin filament network is in part remodeled by the action of a family of filament severing proteins that are responsible for modulating the ratio between monomeric and filamentous actin. Recent work on the protein actophorin from the amoeba Acanthamoeba castellani identified a series of site-directed mutations that increase the thermal stability of the protein by 22°C. Here, we expand this observation by showing that the mutant protein is also significantly stable to both equilibrium and kinetic chemical denaturation, and employ computer simulations to account for the increase in thermal or chemical stability through an accounting of atomic-level interactions. Specifically, the potential of mean force (PMF) can be obtained from steered molecular dynamics (SMD) simulations in which a protein is unfolded. However, SMD can be inefficient for large proteins as they require large solvent boxes, and computationally expensive as they require increasingly many SMD trajectories to converge the PMF. Adaptive steered molecular dynamics (ASMD) overcomes the second of these limitations by steering the particle in stages, which allows for convergence of the PMF using fewer trajectories compared with SMD. Use of the telescoping water scheme within ASMD partially overcomes the first of these limitations by reducing the number of waters at each stage to only those needed to solvate the structure within a given stage. In the PMFs obtained from ASMD, the work of unfolding Acto-2 was found to be higher than the Acto-WT by approximately 120 kCal/mol and reflects the increased stability seen in the chemical denaturation experiments. The evolution of the average number of hydrogen bonds and number of salt bridges during the pulling process provides a mechanistic view of the structural changes of the actophorin protein as it is unfolded, and how it is affected by the mutation in concert with the energetics reported through the PMF.

Bioplastic degradation by a polyhydroxybutyrate depolymerase from a thermophilic soil bacterium.

As the epidemic of single-use plastic worsens, it has become critical to identify fully renewable plastics such as those that can be degraded using enzymes. Here we describe the structure and biochemistry of an alkaline poly[(R)-3-hydroxybutyric acid] (PHB) depolymerase from the soil thermophile Lihuaxuella thermophila. Like other PHB depolymerases or PHBases, the Lihuaxuella enzyme is active against several different polyhydroxyalkanoates, including homo- and heteropolymers, but L. thermophila PHB depolymerase (LtPHBase) is unique in that it also hydrolyzes polylactic acid and polycaprolactone. LtPHBase exhibits optimal activity at 70°C, and retains 88% of activity upon incubation at 65°C for 3 days. The 1.2 Å resolution crystal structure reveals an α/ß-hydrolase fold typical of PHBases, but with a shallow active site containing the catalytic Ser-His-Asp-triad that appears poised for broad substrate specificity. LtPHBase holds promise for the depolymerization of PHB and related bioplastics at high temperature, as would be required in bioindustrial operations like recycling or landfill management.

Implementation of Telescoping Boxes in Adaptive Steered Molecular Dynamics.

Long-time dynamical processes, such as those involving protein unfolding and ligand interactions, can be accelerated and realized through steered molecular dynamics (SMD). The challenge has been the extraction of information from such simulations that generalize for complex nonequilibrium processes. The use of Jarzynski's equality opened the possibility of determining the free energy along the steered coordinate, but sampling over the nonequilibrium trajectories is slow to converge. Adaptive steered molecular dynamics (ASMD) and other related techniques have been introduced to overcome this challenge through the use of stages. Here, we take advantage of these stages to address the numerical cost that arises from the required use of very large solvent boxes. We introduce telescoping box schemes within adaptive steered molecular dynamics (ASMD) in which we adjust the solvent box between stages and thereby vary (and optimize) the required number of solvent molecules. We have benchmarked the method on a relatively long α-helical peptide, Ala30, with respect to the potential of mean force and hydrogen bonds. We show that the use of telescoping boxes introduces little numerical error while significantly reducing the computational cost.

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.

Structure and activity of a thermally stable mutant of Acanthamoeba actophorin.

Actophorin, which was recently tested for crystallization under microgravity on the International Space Station, was subjected to mutagenesis to identify a construct with improved biophysical properties that were expected to improve the extent of diffraction. First, 20 mutations, including one C-terminal deletion of three residues, were introduced individually into actophorin, resulting in modest increases in thermal stability of between +0.5°C and +2.2°C. All but two of the stabilizing mutants increased both the rates of severing F-actin filaments and of spontaneous polymerization of pyrenyl G-actin in vitro. When the individual mutations were combined into a single actophorin variant, Acto-2, the overall thermal stability was 22°C higher than that of wild-type actophorin. When an inactivating S2P mutation in Acto-2 was restored, Acto-2/P2S was more stable by 20°C but was notably more active than the wild-type protein. The inactivating S2P mutation reaffirms the importance that Ser2 plays in the F-actin-severing reaction. The crystal structure of Acto-2 was solved to 1.7 Šresolution in a monoclinic space group, a first for actophorin. Surprisingly, despite the increase in thermal stability, the extended ß-turn region, which is intimately involved in interactions with F-actin, is disordered in one copy of Acto-2 in the asymmetric unit. These observations emphasize the complex interplay among protein thermal stability, function and dynamics.

Improved resolution crystal structure of Acanthamoeba actophorin reveals structural plasticity not induced by microgravity.

Actophorin, a protein that severs actin filaments isolated from the amoeba Acanthamoeba castellanii, was employed as a test case for crystallization under microgravity. Crystals of purified actophorin were grown under microgravity conditions aboard the International Space Station (ISS) utilizing an interactive crystallization setup between the ISS crew and ground-based experimenters. Crystals grew in conditions similar to those grown on earth. The structure was solved by molecular replacement at a resolution of 1.65 Å. Surprisingly, the structure reveals conformational changes in a remote ß-turn region that were previously associated with actophorin phosphorylated at the terminal residue Ser1. Although crystallization under microgravity did not yield a higher resolution than crystals grown under typical laboratory conditions, the conformation of actophorin obtained from solving the structure suggests greater flexibility in the actophorin ß-turn than previously appreciated and may be beneficial for the binding of actophorin to actin filaments.

Energetics and structure of alanine-rich α-helices via adaptive steered molecular dynamics.

The energetics and hydrogen bonding profiles of the helix-to-coil transition were found to be an additive property and to increase linearly with chain length, respectively, in alanine-rich α-helical peptides. A model system of polyalanine repeats was used to establish this hypothesis for the energetic trends and hydrogen bonding profiles. Numerical measurements of a synthesized polypeptide Ac-Y(AEAAKA)kF-NH2 and a natural α-helical peptide a2N (1-17) provide evidence of the hypothesis's generality. Adaptive steered molecular dynamics was employed to investigate the mechanical unfolding of all of these alanine-rich polypeptides. We found that the helix-to-coil transition is primarily dependent on the breaking of the intramolecular backbone hydrogen bonds and independent of specific side-chain interactions and chain length. The mechanical unfolding of the α-helical peptides results in a turnover mechanism in which a 310-helical structure forms during the unfolding, remaining at a near constant population and thereby maintaining additivity in the free energy. The intermediate partially unfolded structures exhibited polyproline II helical structure as previously seen by others. In summary, we found that the average force required to pull alanine-rich α-helical peptides in between the endpoints-namely the native structure and free coil-is nearly independent of the length or the specific primary structure.

An Oxygen Delivery Polymer Enhances Seed Germination in a Martian-like Environment.

Critical to the success of establishing a sustainable human presence on Mars is the ability to economically grow crop plants. Several environmental factors make it difficult to fully rely on local resources for agriculture. These include nutrient sparse regolith, low and fluctuating temperatures, a high amount of ultraviolet radiation, and water trapped locally in the form of ice or metal oxides. While the 96% CO2 martian atmosphere is ideal to support photosynthesis, high CO2 concentrations inhibit germination. An added difficulty is the fact that a vast majority of crop plants require oxygen for germination. Here, we report the production of a polymer-based oxygen delivery system that supports the germination and growth of cress seeds (Lepidium sativum) in a martian regolith simulant under a martian atmosphere at 101 kPa. The oxygen-donating system is based on a low-density lightly cross-linked polyacrylate that is foamed and converted into a dry powder. It is lightweight, added in low amounts to regolith simulant, and efficiently donates enough oxygen throughout the volume of hydrated regolith simulant to fully support seed germination and plant growth. Germination rates, plant development, and plant mass are nearly identical for L. sativum grown in 100% CO2 in the presence of the oxygen-donating lightly cross-linked polyacrylate compared with plants grown in air. The polymer system also serves to protect root structures and better anchors plants in the regolith simulant.

The relative stability of trpzip1 and its mutants determined by computation and experiment.

Six mutants of the tryptophan zipper peptide trpzip1 have been computationally and experimentally characterized. We determine the varying roles in secondary structure stability of specific residues through a mutation assay. Four of the mutations directly effect the Trp-Trp interactions and two of the mutations target the salt bridge between Glu5 and Lys8. CD spectra and thermal unfolding are used to determine the secondary structure and stability of the mutants compared to the wildtype peptide. Adaptive steered molecular dynamics has been used to obtain the energetics of the unfolding pathways of the mutations. The hydrogen bonding patterns and side-chain interactions over the course of unfolding have also been calculated and compared to wildtype trpzip1. The key finding from this work is the importance of a stabilizing non-native salt bridge pair present in the K8L mutation.

Neonatal Opioid Withdrawal Syndrome: A Developmental Care Approach.

Tens of thousands of infants are impacted yearly by prenatal opioid exposure. The term neonatal opioid withdrawal syndrome (NOWS) is now replacing the more familiar term neonatal abstinence syndrome (NAS). Ongoing debate continues related to standard regimens for treatment of this oftentimes perplexing condition. Historically, treatment has focused on pharmacologic interventions. However, there is limited research that points to nonpharmacologic methods of treatment as viable options, whether alone or in addition to pharmacologic interventions. This article, utilizing a review of pertinent literature, outlines the physical aspects of NOWS, including its pathophysiology and the resulting physical clinical signs. In addition, we present an overview of how age-appropriate, nonpharmacologic interventions, centered on developmental care, may be a valuable approach to organize and prioritize routine care for these infants, their families, and the health care team facing the challenges of NOWS. Finally, the need for further research to better define evidence-based standards of care for these infants and their families is discussed.

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.

Coil geometry effects on scanning single-coil magnetic induction tomography.

Alternative coil designs for single coil magnetic induction tomography are considered in this work, with the intention of improving upon the standard design used previously. In particular, we note that the blind spot associated with this coil type, a portion of space along its axis where eddy current generation can be very weak, has an important effect on performance. The seven designs tested here vary considerably in the size of their blind spot. To provide the most discerning test possible, we use laboratory phantoms containing feature dimensions similar to blind spot size. Furthermore, conductivity contrasts are set higher than what would occur naturally in biological systems, which has the effect of weakening eddy current generation at coil locations that straddle the border between high and low conductivity features. Image reconstruction results for the various coils show that coils with smaller blind spots give markedly better performance, though improvements in signal-to-noise ratio could alter that conclusion.

Metal ion coordination in the E. coli Nudix hydrolase dihydroneopterin triphosphate pyrophosphatase: New clues into catalytic mechanism.

Dihydroneopterin triphosphate pyrophosphatase (DHNTPase), a member of the Mg2+ dependent Nudix hydrolase superfamily, is the recently-discovered enzyme that functions in the second step of the pterin branch of the folate biosynthetic pathway in E. coli. DHNTPase is of interest because inhibition of enzymes in bacterial folate biosynthetic pathways is a strategy for antibiotic development. We determined crystal structures of DHNTPase with and without activating, Mg2+-mimicking metals Co2+ and Ni2+. Four metal ions, identified by anomalous scattering, and stoichiometrically confirmed in solution by isothermal titration calorimetry, are held in place by Glu56 and Glu60 within the Nudix sequence motif, Glu117, waters, and a sulfate ion, of which the latter is further stabilized by a salt bridge with Lys7. In silico docking of the DHNTP substrate reveals a binding mode in which the pterin ring moiety is nestled in a largely hydrophobic pocket, the ß-phosphate activated for nucleophilic attack overlays with the crystallographic sulfate and is in line with an activated water molecule, and remaining phosphate groups are stabilized by all four identified metal ions. The structures and binding data provide new details regarding DHNTPase metal requirements, mechanism, and suggest a strategy for efficient inhibition.

Optically tracked, single-coil, scanning magnetic induction tomography.

Recent work has shown that single-coil, magnetic induction tomography (MIT) is useful for visualizing three-dimensional electrical conductivity distributions within biological targets. Coil-induced eddy currents and the associated secondary field are detected as an inductive loss while the coil is relocated to several unique positions and orientations near a target. Image reconstruction is then accomplished by inversion of a convolution integral that quantitatively maps inductive loss with conductivity. Previously, coil position and orientation had to be established by a template, which required assignment of fixed locations for the coil to visit. Here, our existing device is modified so that coil position and orientation are optically tracked while measuring inductive loss. Optical tracking is accomplished via a set of infrared reflective spheres mounted on the same enclosure that supports the coil. The coil center can be tracked with submillimeter accuracy while orientation angle is known to within a fraction of a degree. This work illustrates the use of single-coil MIT in full, position-orientation-tracked scan mode while imaging laboratory phantoms consisting of features having biologically relevant conductivity.

Precise control of drug loading and release of an NSAID-polymer conjugate for long term osteoarthritis intra-articular drug delivery.

A facile synthesis method of polymer diclofenac conjugates (PDCs) based on biocompatible polyurethane chemistry that provides a high drug loading and offers a high degree of control over diclofenac (DCF) release kinetics is described. DCF incorporating monomer was reacted with ethyl-l-lysine diisocyanate (ELDI) and different amounts of polyethylene glycol (PEG) in a one-step synthesis to yield polymers with pendent diclofenac distributed along the backbone. By adjusting the co-monomers feed ratio, the drug loading could be tailored accordingly to give DCF loading of up to 38 w/w%. The release rate could also be controlled easily by changing the amount of PEG in the backbone. Above 10 w/w% of PEG, the in vitro DCF release studies in physiological conditions showed an apparent zero-order profile without an initial burst effect for up to 120 days. The PDCs described may be suitable for long-term intra-articular (IA) delivery for the treatment of osteoarthritis (OA).

Determining the Energetics of Small ß-Sheet Peptides using Adaptive Steered Molecular Dynamics.

Mechanically driven unfolding is a useful computational tool for extracting the energetics and stretching pathway of peptides. In this work, two representative ß-hairpin peptides, chignolin (PDB: 1UAO ) and trpzip1 (PDB: 1LE0 ), were investigated using an adaptive variant of the original steered molecular dynamics method called adaptive steered molecular dynamics (ASMD). The ASMD method makes it possible to perform energetic calculations on increasingly complex biological systems. Although the two peptides are similar in length and have similar secondary structures, their unfolding energetics are quite different. The hydrogen bonding profile and specific residue pair interaction energies provide insight into the differing stabilities of these peptides and reveal which of the pairs provides the most significant stabilization.

The Hydrolysis of Diclofenac Esters: Synthetic Prodrug Building Blocks for Biodegradable Drug-Polymer Conjugates.

Degradation reactions on diclofenac-monoglycerides (3a,b), diclofenac-(p-hydroxybenzoate)-2-monoglyceride (3c), diclofenac (1), and diclofenac lactam (4) were performed at 37 °C in isotonic buffer solutions (apparent pH range 1-8) containing varying concentrations of acetonitrile (ACN). The concentration remaining of each analyte was measured versus time. Diclofenac-monoglycerides and diclofenac-(p-hydroxybenzoate)-2-monoglyceride (3c) were both found to undergo facile and complete hydrolysis in pH 7.4 isotonic phosphate buffer/10% ACN. Under mildly acidic, neutral or alkaline conditions, diclofenac-(p-hydroxybenzoate)-2-monoglyceride (3c) had the fastest hydrolysis rate (t1/2 = 3.23 h at pH 7.4), with simultaneous formation of diclofenac lactam (4) and diclofenac (1). Diclofenac-monoglycerides (3a,b) hydrolyzed more slowly under the same conditions, to again yield both diclofenac (1) and diclofenac lactam (4). There was also transesterification of diclofenac-2-monoglyceride (3b) to its regioisomer, diclofenac-1-monoglyceride (3a) across the pH range. Diclofenac was shown to be stable in neutral or alkaline conditions but cyclized to form the lactam (4) in acidic conditions. Conversely, the lactam (4) was stable under acidic conditions but was converted to an unknown species under alkaline or neutral conditions.

Constrained Unfolding of a Helical Peptide: Implicit versus Explicit Solvents.

Steered Molecular Dynamics (SMD) has been seen to provide the potential of mean force (PMF) along a peptide unfolding pathway effectively but at significant computational cost, particularly in all-atom solvents. Adaptive steered molecular dynamics (ASMD) has been seen to provide a significant computational advantage by limiting the spread of the trajectories in a staged approach. The contraction of the trajectories at the end of each stage can be performed by taking a structure whose nonequilibrium work is closest to the Jarzynski average (in naive ASMD) or by relaxing the trajectories under a no-work condition (in full-relaxation ASMD--namely, FR-ASMD). Both approaches have been used to determine the energetics and hydrogen-bonding structure along the pathway for unfolding of a benchmark peptide initially constrained as an α-helix in a water environment. The energetics are quite different to those in vacuum, but are found to be similar between implicit and explicit solvents. Surprisingly, the hydrogen-bonding pathways are also similar in the implicit and explicit solvents despite the fact that the solvent contact plays an important role in opening the helix.

Multiple branched adaptive steered molecular dynamics.

Steered molecular dynamics, SMD, [S. Park and K. Schulten, J. Chem. Phys. 120, 5946 (2004)] combined with Jarzynski's equality has been used widely in generating free energy profiles for various biological problems, e.g., protein folding and ligand binding. However, the calculated averages are generally dominated by "rare events" from the ensemble of nonequilibrium trajectories. The recently proposed adaptive steered molecular dynamics, ASMD, introduced a new idea for selecting important events and eliminating the non-contributing trajectories, thus decreasing the overall computation needed. ASMD was shown to reduce the number of trajectories needed by a factor of 10 in a benchmarking study of decaalanine stretching. Here we propose a novel, highly efficient "multiple branching" (MB) version, MB-ASMD, which obtains a more complete enhanced sampling of the important trajectories, while still eliminating non-contributing segments. Compared to selecting a single configuration in ASMD, MB-ASMD offers to select multiple configurations at each segment along the reaction coordinate based on the distribution of work trajectories. We show that MB-ASMD has all benefits of ASMD such as faster convergence of the PMF even when pulling 1000 times faster than the reversible limit while greatly reducing the probability of getting trapped in a non-significant path. We also analyze the hydrogen bond breaking within the decaalanine peptide as we force the helix into a random coil and confirm ASMD results with less noise in the numerical averages.