Predicting RNA Duplex Dimerization Free-Energy Changes upon Mutations Using Molecular Dynamics Simulations

The dimerization free energies of RNA–RNA duplexes are fundamental values that represent the structural stability of RNA complexes. We report a comparative analysis of RNA–RNA duplex dimerization free-energy changes upon mutations, estimated from a molecular dynamics simulation and experiments. A linear regression for nine pairs of double-stranded RNA sequences, six base pairs each, yielded a mean absolute deviation of 0.55 kcal/mol and an <i>R</i>² value of 0.97, indicating quantitative agreement between simulations and experimental data. The observed accuracy indicates that the molecular dynamics simulation with the current molecular force field is capable of estimating the thermodynamic properties of RNA molecules

Improved Accuracy in RNA–Protein Rigid Body Docking by Incorporating Force Field for Molecular Dynamics Simulation into the Scoring Function

RNA–protein interactions play fundamental roles in many biological processes. To understand these interactions, it is necessary to know the three-dimensional structures of RNA–protein complexes. However, determining the tertiary structure of these complexes is often difficult, suggesting that an accurate rigid body docking for RNA–protein complexes is needed. In general, the rigid body docking process is divided into two steps: generating candidate structures from the individual RNA and protein structures and then narrowing down the candidates. In this study, we focus on the former problem to improve the prediction accuracy in RNA–protein docking. Our method is based on the integration of physicochemical information about RNA into ZDOCK, which is known as one of the most successful computer programs for protein–protein docking. Because recent studies showed the current force field for molecular dynamics simulation of protein and nucleic acids is quite accurate, we modeled the physicochemical information about RNA by force fields such as AMBER and CHARMM. A comprehensive benchmark of RNA–protein docking, using three recently developed data sets, reveals the remarkable prediction accuracy of the proposed method compared with existing programs for docking: the highest success rate is 34.7% for the predicted structure of the RNA–protein complex with the best score and 79.2% for 3,600 predicted ones. Three full atomistic force fields for RNA (AMBER94, AMBER99, and CHARMM22) produced almost the same accurate result, which showed current force fields for nucleic acids are quite accurate. In addition, we found that the electrostatic interaction and the representation of shape complementary between protein and RNA plays the important roles for accurate prediction of the native structures of RNA–protein complexes

Noncovalent PEGylation through Protein–Polyelectrolyte Interaction: Kinetic Experiment and Molecular Dynamics Simulation

Noncovalent binding of polyethylene glycol (PEG) to a protein surface is a unique protein handling technique to control protein function and stability. A diblock copolymer containing PEG and polyelectrolyte chains (PEGylated polyelectrolyte) is a promising candidate for noncovalent attachment of PEG to a protein surface because of the binding through multiple electrostatic interactions without protein denaturation. To obtain a deeper understanding of protein–polyelectrolyte interaction at the molecular level, we investigated the manner in which cationic PEGylated polyelectrolyte binds to anionic α-amylase in enzyme kinetic experiments and molecular dynamics (MD) simulations. Cationic PEG-<i>block</i>-poly­(<i>N</i>,<i>N</i>-dimethylaminoethyl) (PEG-<i>b</i>-PAMA) inhibited the enzyme activity of anionic α-amylase due to binding of PAMA chains. Enzyme kinetics revealed that the inhibition of α-amylase activity by PEG-<i>b</i>-PAMA is noncompetitive inhibition manner. In MD simulations, the PEG-<i>b</i>-PAMA molecule was initially located at six different placements of the <i>x</i>-, <i>y</i>-, and <i>z</i>-axis ±20 Å from the center of α-amylase, which showed that the PEG-<i>b</i>-PAMA nonspecifically bound to the α-amylase surface, corresponding to the noncompetitive inhibition manner that stems from the polymer binding to an enzyme surface other than the active site. In addition, the enzyme activity of α-amylase in the presence of PEG-<i>b</i>-PAMA was not inhibited by increasing the ionic strength, consistent with the MD simulation; i.e., PEG-<i>b</i>-PAMA did not interact with α-amylase in high ionic strength conditions. The results reported in this paper suggest that enzyme inhibition by PEGylated polyelectrolyte can be attributed to the random electrostatic interaction between protein and polyelectrolyte

Molecular Dynamics Simulation of the Arginine-Assisted Solubilization of Caffeic Acid: Intervention in the Interaction

We have previously demonstrated that arginine increases the solubility of aromatic compounds that have poor water solubility, an effect referred to as the “arginine-assisted solubilization system (AASS)”. In the current study, we utilized a molecular dynamics simulation to examine the solubilization effects of arginine on caffeic acid, which has a tendency to aggregate in aqueous solution. Caffeic acid has a hydrophobic moiety containing a π-conjugated system that includes an aromatic ring and a hydrophilic moiety with hydroxyl groups and a carboxyl group. While its solubility increases at higher pH values due to the acquisition of a negative charge, the solubility was greatly enhanced by the addition of 1 M arginine hydrochloride at any pH. The results of the simulation indicated that the caffeic acid aggregates were dissociated by the arginine hydrochloride, which is consistent with the experimental data. The binding free energy calculation for two caffeic acid molecules in an aqueous 1 M arginine hydrochloride solution indicated that arginine stabilized the dissociated state due to the interaction between its guanidinium group and the π-conjugated system of the caffeic acid. The binding free energy of two caffeic acid molecules in the arginine hydrochloride solution exhibited a local minimum at approximately 8 Å, at which the arginine intervened between the caffeic acid molecules, causing a stabilization of the dissociated state of caffeic acid. Such stabilization by arginine likely led to the caffeic acid solubilization, as observed in both the experiment and the MD simulation. The results reported in this paper suggest that AASS can be attributed to the stabilization resulting from the intervention of arginine in the interaction between the aromatic compounds

Molecular Dynamics Simulation of the Arginine-Assisted Solubilization of Caffeic Acid: Intervention in the Interaction

We have previously demonstrated that arginine increases the solubility of aromatic compounds that have poor water solubility, an effect referred to as the “arginine-assisted solubilization system (AASS)”. In the current study, we utilized a molecular dynamics simulation to examine the solubilization effects of arginine on caffeic acid, which has a tendency to aggregate in aqueous solution. Caffeic acid has a hydrophobic moiety containing a π-conjugated system that includes an aromatic ring and a hydrophilic moiety with hydroxyl groups and a carboxyl group. While its solubility increases at higher pH values due to the acquisition of a negative charge, the solubility was greatly enhanced by the addition of 1 M arginine hydrochloride at any pH. The results of the simulation indicated that the caffeic acid aggregates were dissociated by the arginine hydrochloride, which is consistent with the experimental data. The binding free energy calculation for two caffeic acid molecules in an aqueous 1 M arginine hydrochloride solution indicated that arginine stabilized the dissociated state due to the interaction between its guanidinium group and the π-conjugated system of the caffeic acid. The binding free energy of two caffeic acid molecules in the arginine hydrochloride solution exhibited a local minimum at approximately 8 Å, at which the arginine intervened between the caffeic acid molecules, causing a stabilization of the dissociated state of caffeic acid. Such stabilization by arginine likely led to the caffeic acid solubilization, as observed in both the experiment and the MD simulation. The results reported in this paper suggest that AASS can be attributed to the stabilization resulting from the intervention of arginine in the interaction between the aromatic compounds

Carbon Nanotubes Facilitate Oxidation of Cysteine Residues of Proteins

The adsorption of proteins onto nanoparticles such as carbon nanotubes (CNTs) governs the early stages of nanoparticle uptake into biological systems. Previous studies regarding these adsorption processes have primarily focused on the physical interactions between proteins and nanoparticles. In this study, using reduced lysozyme and intact human serum albumin in aqueous solutions, we demonstrated that CNTs interact chemically with proteins. The CNTs induce the oxidation of cysteine residues of the proteins, which is accounted for by charge transfer from the sulfhydryl groups of the cysteine residues to the CNTs. The redox reaction simultaneously suppresses the intermolecular association of proteins via disulfide bonds. These results suggest that CNTs can affect the folding and oxidation degree of proteins in biological systems such as blood and cytosol

Solubilization of Single-Walled Carbon Nanotubes Using a Peptide Aptamer in Water below the Critical Micelle Concentration

The solubilizing ability of single-walled carbon nanotubes (SWCNTs) in water with several dispersants was investigated. Among the dispersants, including low-molecular-weight surfactants, peptides, DNA, and a water-soluble polymer, the peptide aptamer, A2 (IFRLSWGTYFS), exhibited the highest dispersion capability below the critical micelle concentration at a concentration of 0.02 w/v%. The dispersion of supernatant aqueous solution of SWCNTs containing aptamer A2 was essentially unchanged for several months after high-speed ultracentrifugation and gave rise to an efficient and stable dispersion of the SWCNTs in water. From the results of isothermal titration calorimetry and molecular dynamics simulations, the effective binding capability of A2 was due to π–π interaction between aromatic groups in the peptide aptamer and the side walls of SWCNTs. Interestingly, the peptide aptamer showed the possibility of diameter separation of semiconducting SWCNTs using a uniform density gradient ultracentrifuge. These phenomena are encouraging results toward an effective approach to the dispersion and separation of SWCNTs

Use of a Compact Tripodal Tris(bipyridine) Ligand to Stabilize a Single-Metal-Centered Chirality: Stereoselective Coordination of Iron(II) and Ruthenium(II) on a Semirigid Hexapeptide Macrocycle

Fe­(II)-coordinating hexapeptides containing three 2,2′-bipyridine moieties as side chains were designed and synthesized. A cyclic hexapeptide having three [(2,2′-bipyridin)-5-yl]-d-alanine (d-Bpa5) residues, in which d-Bpa5 and Gly are alternately arranged with 3-fold rotational symmetry, coordinated with Fe­(II) to form a 1:1 octahedral Fe­(II)–peptide complex with a single <i>facial</i>-Λ configuration of the metal-centered chirality. NMR spectroscopy and molecular dynamics simulations revealed that the Fe­(II)–peptide complex has an apparent <i>C</i>₃-symmetric conformations on the NMR time scale, while the peptide backbone is subject to dynamic conformational exchange between three asymmetric β/γ conformations and one <i>C</i>₃-symmetric γ/γ/γ conformation. The semirigid cyclic hexapeptide preferentially arranged these conformations of the small octahedral Fe­(II)–bipyridine complex, as well as the Ru­(II) congener, to underpin the single configuration of the metal-centered chirality

Close Identity between Alternatively Folded State N₂ of Ubiquitin and the Conformation of the Protein Bound to the Ubiquitin-Activating Enzyme

We present the nuclear Overhauser effect-based structure determination of the Q41N variant of ubiquitin at 2500 bar, where the alternatively folded N₂ state is 97% populated. This allows us to characterize the structure of the “pure” N₂ state of ubiquitin. The N₂ state shows a substantial change in the orientation of strand β₅ compared to that of the normal folded N₁ state, which matches the changes seen upon binding of ubiquitin to ubiquitin-activating enzyme E1. The recognition of E1 by ubiquitin is therefore best explained by conformational selection rather than induced-fit motion

Solution Structure of the Q41N Variant of Ubiquitin as a Model for the Alternatively Folded N₂ State of Ubiquitin

It is becoming increasingly clear that proteins transiently populate high-energy excited states as a necessary requirement for function. Here, we demonstrate that rational mutation based on the characteristics of the structure and dynamics of proteins obtained from pressure experiments is a new strategy for amplifying particular fluctuations in proteins. We have previously shown that ubiquitin populates a high-energy conformer, N₂, at high pressures. Here, we show that the Q41N mutation favors N₂: high-pressure nuclear magnetic resonance (NMR) shows that N₂ is ∼70% populated in Q41N but only ∼20% populated in the wild type at ambient pressure. This allows us to characterize the structure of N₂, in which α₁-helix, the following loop, β₃-strand, and β₅-strand change their orientations relative to the remaining regions. Conformational fluctuation on the microsecond time scale, characterized by ¹⁵N spin relaxation NMR analysis, is markedly increased for these regions of the mutant. The N₂ conformers produced by high pressure and by the Q41N mutation are quite similar in both structure and dynamics. The conformational change to produce N₂ is proposed to be a novel dynamic feature beyond the known recognition dynamics of the protein. Indeed, it is orthogonal to that seen when proteins containing a ubiquitin-interacting motif bind at the hydrophobic patch of ubiquitin but matches changes seen on binding to the E2 conjugating enzyme. More generally, structural and dynamic effects of hydrodynamic pressure are shown to be useful for characterizing functionally important intermediates