Free energy simulations for protein ligand binding and stability

Abstract

We summarize several computational techniques to determine relative free energies for condensed-phase systems. The focus is on practical considerations which are capable of making direct contact with experiments. Particular applications include the thermodynamic stability of apo- and holo-myoglobin, insulin dimerization free energy, ligand binding in lysozyme, and ligand diffusion in globular proteins. In addition to provide differential free energies between neighboring states, converged umbrella sampling simulations provide insight into migration barriers and ligand dissociation barriers and analysis of the trajectories yield additional insight into the structural dynamics of fundamental processes. Also, such simulations are useful tools to quantify relative stability changes for situations where experiments are difficult. This is illustrated for NO-bound myoglobin. For the dissociation of benzonitrile from lysozyme it is found that long umbrella sampling simulations are required to approximately converge the free energy profile. Then, however, the resulting differential free energy between the bound and unbound state is in good agreement with estimates from molecular mechanics with generalized Born surface area simulations. Furthermore, comparing the barrier height for ligand escape suggests that ligand dissociation contains a non-equilibrium component.     

Novel polymerizable surfactants with pH-sensitive amphiphilicity and cell membrane disruption for efficient siRNA delivery

Small interfering RNA (siRNA) is a promising new therapeutic modality that can specifically silence disease-related genes. The main challenge for successful clinical development of therapeutic siRNA is the lack of efficient delivery systems. In this study, we have designed and synthesized a small library of novel multifunctional siRNA carriers, polymerizable surfactants with pH-sensitive amphiphilicity based on the hypothesis that pH-sensitive amphiphilicity and environmentally sensitive siRNA release can result in efficient siRNA delivery. The polymerizable surfactants comprise a protonatable amino head group, two cysteine residues, and two lipophilic tails. The surfactants demonstrated pH-sensitive amphiphilic hemolytic activity or cell membrane disruption with rat red blood cells. Most of the surfactants resulted in low hemolysis at pH 7.4 and high hemolysis at reduced pH (6.5 and 5.4). The pH-sensitive cell membrane disruption can facilitate endosomal-lysosomal escape of siRNA delivery systems at the endosomal-lysosomal pH. The surfactants formed compact nanoparticles (160-260 nm) with siRNA at N/P ratios of 8 and 10 via charge complexation with the amino head group, lipophilic condensation, and autoxidative polymerization of dithiols. The siRNA complexes with the surfactants demonstrated low cytotoxicity. The cellular siRNA delivery efficiency and RNAi activity of the surfactants correlated well with their pH-sensitive amphiphilic cell membrane disruption. The surfactants mediated 40-88% silencing of luciferase expression with 100 nM siRNA and 35-75% with 20 nM siRNA in U87-luc cells. Some of the surfactants resulted in similar or higher gene silencing efficiency than TransFast. EHCO with no hemolytic activity at pH 7.4 and 6.5 and high hemolytic activity at pH 5.4 resulted in the best siRNA delivery efficiency. The polymerizable surfactants with pH-sensitive amphiphilicity are promising for efficient siRNA delivery.     

The effect of pH on PAMAM dendrimer-siRNA complexation: endosomal considerations as determined by molecular dynamics simulation

Intracellular degradation of genes, most notably within the endo-lysosomal compartment is considered a significant barrier to (non-viral) gene delivery in vivo. Previous reports based on in vitro studies claim that carriers possessing a mixture of primary, secondary and tertiary amines are able to buffer the acidic environment within the endosome, allowing for timely release of their contents, leading to higher transfection rates. In this report, we adopt an atomistic molecular dynamics (MD) simulation approach, comparing the complexation of 21-bp siRNA with low-generation polyamidoamine (PAMAM) dendrimers (G0 and G1) at both neutral and acidic pHs, the latter of which mimics the degradative environment within maturing 'late-endosomes'. Our simulations reveal that the time taken for the dendrimer-gene complex (dendriplex) to reach equilibrium is appreciably longer at low pH and this is accompanied by more compact packaging of the dendriplex, as compared to simulations performed at neutral pH. We also note larger absolute values of calculated binding free energies of the dendriplex at low pH, indicating a higher dendrimer-nucleic acid affinity in comparison with neutral pH. These novel simulations provide a more detailed understanding of low molecular-weight polymer-siRNA behavior, mimicking the endosomal environment and provide input of direct relevance to the "proton sponge theory", thereby advancing the rational design of non-viral gene delivery systems.     

FRET-based probing to gain direct information on siRNA sustainability in live cells: Asymmetric degradation of siRNA strands

Investigation of the intracellular fate of small interference RNA (siRNA) following their delivery into cells is of great interest to elucidate dynamics of siRNA in cytoplasm. However, its cellular delivery and sustainability should be understood at the molecular level and improved for the successful in vivo application of siRNA. Here we present a fluorescence resonance energy transfer (FRET) based method using oligonucleotide probes to study intracellular dissociation (or melting) and sustainability of siRNAs in live cells. The FRET probes were specifically designed to observe intracellular dissociation (or melting) and degradation of short synthetic RNAs in real-time, thus providing the desired kinetic information in cells. Intracellular FRET analysis shows that siRNA duplex is gradually diffused into cytosol, dissociated, and degraded for a duration of 3.5 h, which is confirmed by confocal microscopy colocalization measurements. In addition, our FRET assays reveal the asymmetric degradation as well as the time-dependent dissociation of each siRNA strand. The application of this FRET technique can allow for direct information on siRNA integrity inside living cells, providing a detection tool for dynamics of biological molecules.     

综述: siRNA脂质纳米输送载体的研究进展

siRNA能高效且特异地阻断内源性同源基因的表达即RNA干涉(RNAi)．RNAi在临床中的应用需要开发安全有效的输送系统,脂质纳米输送载体是一种具有发展潜力的siRNA输送系统．siRNA-脂质复合物的形成主要通过静电相互作用,静电作用必须足够强以至于载体在运输过程中不释放siRNA,而载体到达治疗部位时,解聚释放出siRNA．载体的粒径应小于100 nm,以利于细胞的摄取和透过特定部位的血管开窗．为了减少网状内皮系统(RES)的摄取和延长载体的循环时间,载体的表面由聚乙二醇修饰．本文主要综述了构建siRNA输送载体的基本要求．     

Ligand binding by antibody IgE Lb4: assessment of binding site preferences using microcalorimetry, docking, and free energy simulations.

Antibody IgE Lb4 interacts favorably with a large number of different compounds. To improve the current understanding of the structural basis of this vast cross-reactivity, the binding of three dinitrophenyl (DNP) amino acids (DNP-alanine, DNP-glycine, and DNP-serine) is investigated in detail by means of docking and molecular dynamics free energy simulations. Experimental binding energies obtained by isothermal titration microcalorimetry are used to judge the results of the computational studies. For all three ligands, the docking procedure proposes two plausible subsites within the binding region formed by the antibody CDR loops. By subsequent molecular dynamics simulations and calculations of relative free energies of binding, one of these subsites, a tyrosine-surrounded pocket, is revealed as the preferred point of complexation. For this subsite, results consistent with experimental observations are obtained; DNP-glycine is found to bind better than DNP-serine, and this, in turn, is found to bind better than DNP-alanine. The suggested binding mode makes it possible to explain both the moderate binding affinity and the differences in binding energy among the three ligands.     

Building a foundation for structure‐based cellulosome design for cellulosic ethanol: Insight into cohesin‐dockerin complexation from computer simulation

The organization and assembly of the cellulosome, an extracellular multienzyme complex produced by anaerobic bacteria, is mediated by the high‐affinity interaction of cohesin domains from scaffolding proteins with dockerins of cellulosomal enzymes. We have performed molecular dynamics simulations and free energy calculations on both the wild type (WT) and D39N mutant of the C. thermocellum Type I cohesin‐dockerin complex in aqueous solution. The D39N mutation has been experimentally demonstrated to disrupt cohesin‐dockerin binding. The present MD simulations indicate that the substitution triggers significant protein flexibility and causes a major change of the hydrogen‐bonding network in the recognition strips—the conserved loop regions previously proposed to be involved in binding—through electrostatic and salt‐bridge interactions between β‐strands 3 and 5 of the cohesin and α‐helix 3 of the dockerin. The mutation‐induced subtle disturbance in the local hydrogen‐bond network is accompanied by conformational rearrangements of the protein side chains and bound water molecules. Additional free energy perturbation calculations of the D39N mutation provide differences in the cohesin‐dockerin binding energy, thus offering a direct, quantitative comparison with experiments. The underlying molecular mechanism of cohesin‐dockerin complexation is further investigated through the free energy profile, that is, potential of mean force (PMF) calculations of WT cohesin‐dockerin complex. The PMF shows a high‐free energy barrier against the dissociation and reveals a stepwise pattern involving both the central β‐sheet interface and its adjacent solvent‐exposed loop/turn regions clustered at both ends of the β‐barrel structure.     

Computational studies of the binding mechanisms of fullerenes to human serum albumin

Fullerene and its derivatives show promising prospects for applications in a vast array of biological systems. A key aspect concerning their biomedical applications is how they interact with proteins from molecular levels, which is still poorly understood. In the current study, we investigated the structural and thermodynamic basis of the interactions between two pharmacologically relevant fullerene derivatives and human serum albumin (HSA) using molecular docking, molecular dynamics simulations, and binding free energy calculations. Our results demonstrate that fullerenes steadily bind with HSA at the interfacial cavity formed by subdomains IIA and IIIA. In agreement with available experimental data, our simulations show that the global structure of HSA becomes more compact in the presence of fullerene, while local structural dynamics of the binding cavity behaves diversely depending on the chemical properties of bound fullerenes. Binding free energy calculations confirmed that the interactions between fullerenes and HSA are dominantly stabilized by van der Waals forces and they further allowed the identification of key residues involved in fullerene binding. The structural and energetic insights obtained from this work may help for the development of fullerene-based drug delivery devices and therapeutic agents with improved biological profile.     

Initial Recognition of a Cellodextrin Chain in the Cellulose-Binding Tunnel May Affect Cellobiohydrolase Directional Specificity

Cellobiohydrolases processively hydrolyze glycosidic linkages in individual polymer chains of cellulose microfibrils, and typically exhibit specificity for either the reducing or nonreducing end of cellulose. Here, we conduct molecular dynamics simulations and free energy calculations to examine the initial binding of a cellulose chain into the catalytic tunnel of the reducing-end-specific Family 7 cellobiohydrolase (Cel7A) from Hypocrea jecorina. In unrestrained simulations, the cellulose diffuses into the tunnel from the −7 to the −5 positions, and the associated free energy profiles exhibit no barriers for initial processivity. The comparison of the free energy profiles for different cellulose chain orientations show a thermodynamic preference for the reducing end, suggesting that the preferential initial binding may affect the directional specificity of the enzyme by impeding nonproductive (nonreducing end) binding. Finally, the Trp-40 at the tunnel entrance is shown with free energy calculations to have a significant effect on initial chain complexation in Cel7A.     

Amphiphilic peptides with arginine and valine residues as siRNA carriers

An efficient and safe delivery carrier is required for the therapeutic application of siRNA. In this research, amphiphilic peptides with arginine and valine residues were evaluated as siRNA carriers. The peptides were composed of 1-4 arginine-blocks and 6 valine-blocks. In the aqueous solution, the arginine-valine peptides (RV peptides) formed micelles with hydrophobic cores comprised of a valine block and a cationic surface comprised of an arginine block. In a gel retardation assay, the RV peptides completely retarded siRNA at a 1:10 weight ratio (siRNA:peptide). A heparin competition assay suggested that the RV peptides formed more stable complexes with siRNA than they did with polyethylenimine (25 kDa, PEI25k). In an in vitro silencing assay, a dual luciferase expression (Renilla and firefly luciferases) vector, psiCHECK2, was co-transfected into human embryonic kidney 293 cells with Renilla-siRNA using the RV peptides. The specific silencing effect of Renilla luciferase was analyzed in reference to firefly luciferase. The results showed that the R3V6 peptide was more efficient than the R1V6, R2V6, and R4V6 peptides in silencing Renilla luciferase. In the flow cytometry and in vitro silencing studies, the R3V6 peptide delivered Renila-siRNA as efficiently as PEI25k. The siVEGF/R3V6 peptide also reduced endogenous vascular endothelial growth factor (VEGF) expression in CT27 cells as efficiently as PEI25k. A cytotoxicity assay showed that RV peptides did not cause any cytotoxicity. Therefore, RV peptides may be useful for the development of a safe and efficient delivery carrier of siRNA.     

An Effective Solvation Term Based on Atomic Occupancies for Use in Protein Simulations

Abstract

Several approaches to the treatment of solvent effects based on continuum models are reviewed and a new method based on occupied atomic volumes (occupancies) is proposed and tested. The new method describes protein-water interactions in terms of atomic solvation parameters, which represent the solvation free energy per unit of volume. These parameters were determined for six different atoms types, using experimental free energies of solvation. The method was implemented in the GROMOS and PRESTO molecular simulation program suites. Simulations with the solvation term require 20-50% more CPU time than the corresponding vacuum simulations and are approximately 20 times faster than explicit water simulations. The method and parameters were tested by carrying out 200 ps simulations of BPTI in water, in vacuo, and with the solvation term. The performance of the solvation term was assessed by comparing the structures and energies from the solvation simulations with the equivalent quantities derived from several BPTI crystal structures and from the explicit water and vacuum simulations. The model structures were evaluated in terms of exposed total surface, buried and exposed polar surfaces, secondary structure preservation, number of hydrogen bonds, energy contributions, and positional deviations from BPTI crystal structures. Vacuum simulations produced unrealistic structures with respect to all criteria applied. The structures resulting from the simulations with explicit water were closer to the 5PTI crystal structure, although part of the secondary structure dissolved. The simulations with the effective solvation term produce structures that are normal according to all evaluations and in most respects are remarkably similar to the 5PTI crystal structure despite considerable positional fluctuations during the simulations. The segments where the model and crystal structures differ are known to be flexible and the observed difference may be physically realistic. The effective solvation term based on occupancies is not only very efficient in terms of computer time but also results in meaningful structural properties for BPTI. It may therefore be generally useful in molecular dynamics of macromolecules.     

Slow complexation kinetics for ferric iron and EDTA complexes make EDTA non-biodegradable

Published experimental data on ethylenediaminetetraacetic acid (EDTA) biodegradation in the presence of ferric iron (Fe(III)) showed that rapid biodegradation of EDTA suddenly stopped, leaving a residual of unbiodegraded EDTA that was equal to the concentration of dissolved Fe(III). We hypothesize that slow kinetics for the dissociation of two iron-EDTA complexes - FeEDTA^- and FeOHEDTA^2- – sequestered the EDTA in a form that is biologically unavailable. To evaluate this hypothesis, we added to the biogeochemical model CCBATCH a new sub-model for kinetically controlled complexation. CCBATCH simulations with kinetically controlled complexation for FeEDTA^- and FeOHEDTA^2- and the observed concentration of total dissolved Fe(III) accurately predicted the sudden cessation of EDTA biodegradation at the exact time shown experimentally. Our simulations also correctly predicted the observed residual EDTA concentration and the amounts of biomass and NH₄ ⁺. Alternate explanations for the experimental results – strong equilibrium complexation of ferric iron and EDTA and precipitation of calcium and magnesium solids – could not capture the observed trends. This analysis using CCBATCH's new sub-model for kinetically controlled complexation shows that EDTA, once it becomes complexed with Fe(III), becomes biologically unavailable.     

Photosensitizing carrier proteins for photoinducible RNA interference

RNA interference (RNAi) is being widely explored as a tool in functional genomics and tissue engineering, and in the therapy of intractable diseases, including cancer and neurodegenerative diseases. Recently, we developed a photoinducible RNAi method using photosensitizing carrier proteins, named CLIP-RNAi (CPP-linked RBP-mediated RNA internalization and photoinduced RNAi). Novel carrier proteins were designed for this study to establish a highly efficient delivery system for small interfering RNA (siRNA) or short hairpin RNA (shRNA) and to demonstrate light-dependent gene silencing. In addition, the results suggested that the dissociation of the siRNA (or shRNA) from carrier proteins in the cytoplasm is a critical event in CLIP-RNAi-mediated gene silencing.     

Self-assembly of surfactant aqueous solution confined in a Janus amphiphilic nanotube

Abstract

In recent years, Janus amphiphilic nanotubes with complex surfaces have been synthesized. However, the self-assembly behaviour of surfactant solutions confined in a Janus amphiphilic nanotube has not been investigated so far. We performed molecular simulations to investigate the morphologies and phase diagrams of a surfactant confined in Janus amphiphilic nanotube consisting of both hydrophobic and hydrophilic surfaces. We derived a phase diagram of the representative snapshots of equilibrium morphologies. Morphologies that were not observed in the bulk eventuated in confined systems. Moreover, the self-assembled structures were found to be dependent on the spatial confinement. Furthermore, the self-assembled structures confined in hydroneutral and Janus amphiphilic nanotubes were compared. The results suggested that the self-assembled structures confined in the Janus amphiphilic nanotube resembled that confined in the hydroneutral nanotube owing to a strong confinement effect. Further developments in controlling the morphologies and self-assemblies will greatly advance their applications of these materials in nanofluidic devices, or for nanopatterning.     

Ligand-release pathways in the pheromone-binding protein of Bombyx mori

Pheromone-binding proteins (PBP) supply olfactory neuron cells with pheromones by binding the ligands they are tailored for and carrying them to their receptor. The function of a PBP as an efficient carrier requires fast ligand uptake and release. The molecular basis of the ligand-binding mechanism was addressed here for the intriguing case of the PBP of the silk moth Bombyx mori. This PBP completely encapsulates its ligand bombykol without displaying any obvious ligand entrance/exit sites. Here, two opposite dissociation routes were identified as the most likely entrance/exit paths by replica-exchange molecular dynamics, essential dynamics, and force-probe molecular dynamics simulations. One of the paths runs along a flexible front lid; the other along the termini at the back. Calculated forces and energies suggest that both routes are physiologically relevant. The multiplicity of pathways may reduce or tune the entropic barrier for ligand binding.     

Development of a specific siRNA delivery system into HeLa cells using an IgG-binding fusion protein

Stable carriers are required for delivering siRNA to cells. The use of polyethyleneimine (PEI) as gene carrier has been researched extensively; however, it does not provide sufficient protection from RNase degradation and is not suitable for targeted siRNA delivery to specific cells. In this study, two repeats of Fc binding domain of protein G (C2) were used to introduce a specific antibody to PEI-based carrier of siRNA. In addition, we used the double-stranded RNA binding domain (DRBD) that can bind to siRNA. The complex, consisting of PEI, siRNA and constructed fusion protein, TrxC2DRBD including C2 and DRBD domains, could protect siRNA from RNase degradation. Furthermore, cell specific siRNA delivery into HeLa cells could be performed by the complex fusion with specific antibodies via C2 domain.     

Molecular mechanism of transdermal co-delivery of interferon-alpha protein with gold nanoparticle – a molecular dynamics study

The transdermal route provides numerous advantages over conventional drug delivery routes. However, passive delivery of large molecules such as proteins through the skin is challenging due to its barrier function. Therefore, to design a successful formulation, molecular interaction of these proteins with constituent molecules present in the skin responsible for its barrier function, is necessary. In this study, we have shown through extensive computer simulations that the therapeutic protein, interferon alpha (INF), can be co-delivered through the skin using the gold nanoparticle. We carried out both steered (umbrella sampling) and unrestrained coarse-grained molecular dynamics simulation to show the molecular mechanism of absorption/permeation of protein on/through skin layer in the absence/presence of gold nanoparticle. According to the steered simulations, when INF was taken alone, the free energy minimum was observed at the head group of the skin layer, whereas, when co-delivered with AuNP, it was observed in the interior of the bilayer. Unrestrained simulations have also shown that INF was adsorbed on the skin lipid bilayer head group, while in presence of AuNP, it first complexed with the AuNP and then breached the barrier. The MD simulations thus established the transdermal delivery as a possible pathway for delivery of INF protein.     

Simulation of diffusion of O 2 and CO 2 in amorphous poly(ethylene terephthalate) and related alkylene and isomeric polyesters

The diffusion of small molecules through polymers is important in many areas of polymer science, such as gas barrier and separation membrane materials, polymeric foams, and in the processing and properties of polymers. Molecular simulation techniques have been applied to study the diffusion of oxygen and carbon dioxide as small molecule penetrants in models of bulk amorphous poly(ethylene terephthalate) (PET) and related alkylene and isomeric polyesters. A bulk amorphous configuration with periodic boundary conditions made into a unit cell whose dimensions were determined for each of the simulated polyesters in the cell having the experimental density. The diffusion coefficients for O 2 and CO 2 were determined via NVE molecular dynamics simulations using the Dreiding 2.21 molecular mechanics force field over a range of temperatures (300, 500 and 600 K) using up to 3 ns simulation time. We have focussed on the influence of the temperature, polymer dynamics, number of CH 2 groups, density and free volume distribution on the diffusion properties. Correlation of diffusion coefficients with free volume and number of CH 2 groups was found.     

Molecular Dynamics Simulations of a Nucleosome and Free DNA

Abstract

All atom molecular dynamics simulations (10ns) of a nucleosome and of its 146 basepairs of DNA free in solution have been conducted. DNA helical parameters (Roll, Tilt, Twist, Shift, Slide, Rise) were extracted from each trajectory to compare the conformation, effective force constants, persistence length measures, and fluctuations of nucleosomal DNA to free DNA. The conformation of DNA in the nucleosome, as determined by helical parameters, is found to be largely within the range of thermally accessible values obtained for free DNA. DNA is found to be less flexible on the nucleosome than when free in solution, however such measures are length scale dependent. A method for disassembling and reconstructing the conformation and dynamics of the nucleosome using Fourier analysis is presented. Long length variations in the conformation of nucleosomal DNA are identified other than those associated with helix repeat. These variations are required to create a proposed tetrasome conformation or to qualitatively reconstruct the 1.75 turns of the nucleosome's superhelix. Reconstruction of free DNA using selected long wavelength variations in conformation can produce either a left-handed or a right-handed superhelix. The long wavelength variations suggest 146 basepairs is a natural length of DNA to wrap around the histone core.