Internal Binding Research Articles

Gold nanoparticle design for RNA compaction.

RNA-based therapeutics hold a great promise in treating a variety of diseases. However, double-stranded RNAs (dsRNAs) are inherently unstable, highly charged, and stiff macromolecules that require a delivery vehicle. Cationic ligand functionalized gold nanoparticles (AuNPs) are able to compact nucleic acids and assist in RNA delivery. Here, we use large-scale all-atom molecular dynamics simulations to show that correlations between ligand length, metal core size, and ligand excess free volume control the ability of nanoparticles to bend dsRNA far below its persistence length. The analysis of ammonium binding sites showed that longer ligands that bind deep within the major groove did not cause bending. By limiting ligand length and, thus, excess free volume, we have designed nanoparticles with controlled internal binding to RNA's major groove. NPs that are able to induce RNA bending cause a periodic variation in RNA's major groove width. Density functional theory studies on smaller models support large-scale simulations. Our results are expected to have significant implications in packaging of nucleic acids for their applications in nanotechnology and gene delivery.

Multiple Timescale Dynamic Analysis of Functionally-Impairing Mutations in Human Ileal Bile Acid-Binding Protein.

Human ileal bile acid-binding protein (hI-BABP) has a key role in the enterohepatic circulation of bile salts. Its two internal binding sites exhibit positive cooperativity accompanied by a site-selectivity of glycocholate (GCA) and glycochenodeoxycholate (GCDA), the two most abundant bile salts in humans. To improve our understanding of the role of dynamics in ligand binding, we introduced functionally impairing single-residue mutations at two key regions of the protein and subjected the mutants to NMR relaxation analysis and MD simulations. According to our results, mutation in both the vicinity of the C/D (Q51A) and the G/H (Q99A) turns results in a redistribution of motional freedom in apo hI-BABP. Mutation Q51A, deteriorating the site-selectivity of GCA and GCDA, results in the channeling of ms fluctuations into faster motions in the binding pocket hampering the realization of key side chain interactions. Mutation Q99A, abolishing positive binding cooperativity for GCDA, leaves ms motions in the C-terminal half unchanged but by decoupling βD from a dynamic cluster of the N-terminal half displays an increased flexibility in the vicinity of site 1. MD simulations of the variants indicate structural differences in the portal region and mutation-induced changes in dynamics, which depend on the protonation state of histidines. A dynamic coupling between the EFGH portal, the C/D-region, and the helical cap is evidenced highlighting the interplay of structural and dynamic effects in bile salt recognition in hI-BABP.

Computational design of a sensitive, selective phase-changing sensor protein for the VX nerve agent.

The VX nerve agent is one of the deadliest chemical warfare agents. Specific, sensitive, real-time detection methods for this neurotoxin have not been reported. The creation of proteins that use biological recognition to fulfill these requirements using directed evolution or library screening methods has been hampered because its toxicity makes laboratory experimentation extraordinarily expensive. A pair of VX-binding proteins were designed using a supercharged scaffold that couples a large-scale phase change from unstructured to folded upon ligand binding, enabling fully internal binding sites that present the maximum surface area possible for high affinity and specificity in target recognition. Binding site residues were chosen using a new distributed evolutionary algorithm implementation in protCAD. Both designs detect VX at parts per billion concentrations with high specificity. Computational design of fully buried molecular recognition sites, in combination with supercharged phase-changing chassis proteins, enables the ready development of a new generation of small-molecule biosensors.

Binding Networks Identify Targetable Protein Pockets for Mechanism-Based Drug Design.

The human genome codes only a few thousand druggable proteins, mainly receptors and enzymes. While this pool of available drug targets is limited, there is an untapped potential for discovering new drug-binding mechanisms and modes. For example, enzymes with long binding cavities offer numerous prerequisite binding sites that may be visited by an inhibitor during migration from a bulk solution to the destination site. Drug design can use these prerequisite sites as new structural targets. However, identifying these ephemeral sites is challenging. Here, we introduce a new method called NetBinder for the systematic identification and classification of prerequisite binding sites at atomic resolution. NetBinder is based on atomistic simulations of the full inhibitor binding process and provides a networking framework on which to select the most important binding modes and uncover the entire binding mechanism, including previously undiscovered events. NetBinder was validated by a study of the binding mechanism of blebbistatin (a potent inhibitor) to myosin 2 (a promising target for cancer chemotherapy). Myosin 2 is a good test enzyme because, like other potential targets, it has a long internal binding cavity that provides blebbistatin with numerous potential prerequisite binding sites. The mechanism proposed by NetBinder of myosin 2 structural changes during blebbistatin binding shows excellent agreement with experimentally determined binding sites and structural changes. While NetBinder was tested on myosin 2, it may easily be adopted to other proteins with long internal cavities, such as G-protein-coupled receptors or ion channels, the most popular current drug targets. NetBinder provides a new paradigm for drug design by a network-based elucidation of binding mechanisms at an atomic resolution.

Toward Improved Charge Separation through Conformational Control in Copper Coordination Complexes.

The continued development of solar energy as a renewable resource necessitates new approaches to sustaining photodriven charge separation (CS). We present a bioinspired approach in which photoinduced conformational rearrangements at a ligand are translated into changes in coordination geometry and environment about a bound metal ion. Taking advantage of the differential coordination properties of CuI and CuII, these dynamics aim to facilitate intramolecular electron transfer (ET) from CuI to the ligand to create a CS state. The synthesis and photophysical characterization of CuCl(dpaaR) (dpaa = dipicolylaminoacetophenone, with R = H and OMe) are presented. These ligands incorporate a fluorophore that gives rise to a twisted intramolecular charge transfer (TICT) excited state. Excited-state ligand twisting provides a tetragonal coordination geometry capable of capturing CuII when an internal ortho-OMe binding site is present. NMR, IR, electron paramagnetic resonance (EPR), and optical spectroscopies, X-ray diffraction, and electrochemical methods establish the ground-state properties of these CuI and CuII complexes. The photophysical dynamics of the CuI complexes are explored by time-resolved photoluminescence and optical transient absorption spectroscopies. Relative to control complexes lacking a TICT-active ligand, the lifetimes of CS states are enhanced ∼1000-fold. Further, the presence of the ortho-OMe substituent greatly enhances the lifetime of the TICT* state and biases the coordination environment toward CuII. The presence of CuI decreases photoinduced degradation from 14 to <2% but does not result in significant quenching via ET. Factors affecting CS in these systems are discussed, laying the groundwork for our strategy toward solar energy conversion.

Low potency inhibition of NaV1.7 by externally applied QX-314 via a depolarizing shift in the voltage-dependence of activation

QX-314 is a quaternary permanently charged lidocaine derivative that inhibits voltage-gated sodium channels (NaV). As it is membrane impermeable, it is generally considered that QX-314 applied externally is inactive, unless it can gain access to the internal local anesthetic binding site via another entry pathway. Here, we characterized the electrophysiological effects of QX-314 on NaV1.7 heterologously expressed in HEK293 cells, and found that at high concentrations, external QX-314 inhibited NaV1.7 current (IC50 2.0 ± 0.3 mM) and shifted the voltage-dependence to more depolarized potentials (ΔV50 +10.6 mV). Unlike lidocaine, the activity of external QX-314 was not state- or use-dependent. The effect of externally applied QX-314 on NaV1.7 channel biophysics differed to that of internally applied QX-314, suggesting QX-314 has an additional externally accessible site of action. In line with this hypothesis, disruption of the local anesthetic binding site in a [F1748A]NaV1.7 mutant reduced the potency of lidocaine by 40-fold, but had no effect on the potency or activity of externally applied QX-314. Therefore, we conclude, using an expression system where QX-314 was unable to cross the membrane, that externally applied QX-314 is able to inhibit NaV1.7 peak current at low millimolar concentrations.

An Unconventional Ligand for Scribble PDZ-4 Domain Mediates Its Interaction with Dusp26

PDZ domains are involved in many cellular processes and are key regulators of the cell physiology. A huge number of studies have investigated the binding specificity of PDZ domains to the carboxyl-terminal sequence of target proteins, while the molecular mechanisms that mediate the recognition of internal binding regions are largely unexplored. In the present study, we describe a ligand motif located in the catalytic domain of the phosphatase Dusp26 which mediates its binding to the PDZ-4 of Scribble. Site-directed mutagenesis identified a conserved tyrosine residue as relevant for the binding. The interaction with the PDZ domain could help the phosphatase to recruit its specific targets.

The retinal binding protein family: a powerful template for design and discovery

Title: The retinal binding protein family: a powerful template for design and discovery.The field of protein design has incredible potential for the development of myriad new andimproved chemical and biochemical function, from the development of powerful new catalyststo new biological tools for the detection and treatment of disease. It can also be a valuable toolfor understanding the structure, function and biophysics of proteins. We have focused on usingmembers of the intracellular lipid binding protein (iLBP) family as templates for protein design,due to their small size, relative stability to mutation, ease of structural study and comparativelylarge internal binding cavity, suitable to binding a wide range of large hydrophobic ligands.

A singular inter-particle force in Cucker-Smale model to avoid collisions

In this paper, by introducing a singular inter-particle force into Cucker-Smale model, the result of collision-avoidance is obtained. The inter-particle force can switch between attractive and repelling with the change of the inner product of relative velocities and positions, which is different from the force repelling only and more in line with the internal binding force of multi-agent systems. Through the establishing of Lyapunov functions, we show that the system can avoid collisions when initial positions are non-collisional. Moreover, for Cucker-Smale model with the singular inter-particle force, results about flocking behaviour are presented.

Tweezer-type binding cavity formed by the helical folding of a carbazole-pyridine oligomer.

We have synthesised a new aromatic foldamer based on the carbazole-pyridine oligomers that adopt helical conformations via dipole-dipole interactions and π-stacking between two ethynyl bond-linked monomers. This foldamer scaffold has been further modified into a synthetic receptor with a tweezer-type binding cavity outside the helical backbone upon folding, in contrast to most aromatic foldamers with internal binding cavities. The tweezer-type cavity is composed of two parallel pyrenyl planes, allowing for the intercalation of a naphthalenediimide guest via π-stacking and CH⋯O interactions, as demonstrated using its 1H NMR spectra and X-ray crystal structure.

Oscillations and potentials of mesons and baryons

In the following, the oscillations and potentials of mesons and baryons are examined and analyzed in detail. The oscillations result from a simple formula that describes the resonance energy at which the corresponding particle can absorb energy and thus appear. The potentials describe three mechanisms that describe the fine splitting of the masses of the elementary particles. These potentials can be read off and derived from the experimentally determined masses of the elementary particles as coefficients. The three mechanisms are internal mass charge binding energy, external mass charge binding energy, and Coulomb interaction.

Capability of Organically Modified Montmorillonite Nanoclay as a Carrier for Imidacloprid Delivery

Organically modified clays have attracted increasing research attention for their various commercial and industrial applications, such as being carriers for pesticide delivery. Besides, the suitability and performance of commercially available organoclays could further promote their applicability. Hence, this study investigated the potential application of a commercially available alkylamine-modified montmorillonite (MMT) nanoclay as a carrier for a widely used insecticide, imidacloprid. X-ray diffraction and thermogravimetric analysis were employed to illustrate the arrangement, orientation, and conformation of surface-modifying agents (SMAs) on MMT nanoclay. It was observed that the clay was modified at an ∼1.0 cation exchange capacity, with the SMAs, especially octadecylamine, arranged in the MMT nanoclay as a bilayer to a pseudo-trilayer or a paraffin monolayer with a tilting angle of ∼25°, which indicated the nanoclay’s ability to adsorb a large amount of imidacloprid. The adsorption–desorption of imidacloprid to MMT nanoclay further confirmed this applicability. A high adsorption capacity (∼85 mg g–1) was observed, with a high reversibility in desorption, showing a hysteresis value of 0.75. Further, the adsorption kinetics and response of the nanoclay to imidacloprid revealed that, initially, a rapid sorption occurred due to a hydrophobic interaction. This was followed by a slower diffusion-controlled sorption due to hydrogen bonding to the internal binding sites. The releasing pattern of imidacloprid from the MMT nanoclay indicated its potential for the preparation of a slow-releasing pesticide formulation where the nanoclay will reduce the instantaneous release of the total amount of pesticide.

Biomimetic nitrogen-rich photocatalyst based on cadmium sulfide for photocatalytic hydrogen evolution

A novel N-rich sugarcane-like photocatalyst CdS/C3N5 (CCN) was prepared by a thermal polymerization method and tested for generating H2 and realizing antiphotocorrosive performance. The best photocatalytic H2 evolution is obtained for a CdS to C3N5 mass ratio of 1:1 (CCN3), which is nearly 33 and 3 times higher than that of pure C3N5 and CdS, respectively. CCN3 can be used to effectively reduce CdS photocorrosion and increase stability because of its N-rich performance and sugarcane-like structure, which can affect electron transport and enhance the internal binding force, respectively. CCN3 can maintain a high H2 evolution ability after 5 cycles, while still maintaining the original sugarcane-like shape, which has an anti-photocorrosive ability.

Pythagorean numbers and the calculation of the masses of the elementary particles

We attempt here to calculate the particle masses for all known elementary particles starting from the Rydberg equation and from the Sommerfeld fine structure constant. Remarkably, this is possible. Next, we try to explain why this is possible and what the meaning of the approach seems to be. Thereby, we find some interesting connections. In addition, we realize that there are two different kinds of mass-charge binding energies in an elementary particle: The internal mass-charge binding energy and the external mass-charge binding energy. These two kinds of mass-charge binding energies can explain the higher masses of the highly charged brother particles in some of the heavier particle triplets (such as the charmed sigma particles).

Halide Size-Selective Binding by Cucurbit[5]uril-Alkali Cation Complexes in the Gas Phase.

We report data that suggest complexes with alkali cations capping the portals of cucurbit[5]uril (CB[5]) bind halide anions size-selectively as observed in the gas phase: Cl- binds inside the CB[5] cavity, Br- is observed both inside and outside, and I- binds weakly outside. This is reflected in sustained off-resonance irradiation collision-induced dissociation (SORI-CID) experiments: all detected Cl- complexes dissociate at higher energies, and Br- complexes exhibit unusual bimodal dissociation behavior, with part of the ion population dissociating at very low energies and the remainder dissociating at significantly higher energies comparable to those observed for Cl-. Decoherence cross sections measured in SF6 using cross-sectional areas by Fourier transform ion cyclotron resonance techniques for [CB[5] + M2X]+ (M = Na, X = Cl or Br) are comparable to or less than that of [CB[5] + Na]+ over a wide energy range, suggesting that Cl- or Br- in these complexes are bound inside the CB[5] cavity. In contrast, [CB[5] + K2Br]+ has a cross section measured about 20% larger than that of [CB[5] + Na]+, suggesting external binding that may correspond with the weakly bound component seen in SORI. While I- complexes with alkali cation caps were not observed, alkaline earth iodides with CB[5] yielded complexes with cross sections 5-10% larger than that of [CB[5] + Na]+, suggesting externally bound iodide. Geometry optimization at the M06-2X/6-31+G* level of ab initio theory suggests that internal anion binding is energetically favored by approximately 50-200 kJ mol-1 over external binding; thus, the externally bound complexes observed experimentally must be due to large energetic barriers hindering the passing of large anions through the CB[5] portal, preventing access to the interior. Calculation of the barriers to anion egress using MMFF//M06-2X/6-31+G* theory supports this idea and suggests that the size-selective binding we observe is due to anion size-dependent differences in the barriers.

Yeast Ssd1 is a non-enzymatic member of the RNase II family with an alternative RNA recognition site.

Ssd1, a conserved fungal RNA-binding protein, is important in stress responses, cell division and virulence. Ssd1 is closely related to Dis3L2 of the RNase II family of nucleases, but lacks catalytic activity and likely suppresses translation of bound mRNAs. Previous studies identified RNA motifs enriched in Ssd1-associated transcripts, yet the sequence requirements for Ssd1 binding are not defined. Here, we identify precise binding sites of Ssd1 on RNA using in vivo cross-linking and cDNA analysis. These sites are enriched in 5′ untranslated regions of a subset of mRNAs encoding cell wall proteins. We identified a conserved bipartite motif that binds Ssd1 with high affinity in vitro. Active RNase II enzymes have a characteristic, internal RNA binding path; the Ssd1 crystal structure at 1.9 Å resolution shows that remnants of regulatory sequences block this path. Instead, RNA binding activity has relocated to a conserved patch on the surface of the protein. Structure-guided mutations of this surface prevent Ssd1 from binding RNA in vitro and phenocopy Ssd1 deletion in vivo. These studies provide a new framework for understanding the function of a pleiotropic post-transcriptional regulator of gene expression and give insights into the evolution of regulatory and binding elements in the RNase II family.

Flyash/polymer composite electrolyte with internal binding interaction enables highly-stable extrinsic-interfaces of all-solid-state lithium batteries

Satisfactory electrochemical stability of extrinsic-interfaces between electrodes and electrolyte is the prerequisite for the safe and durable applications of all-solid-state Lithium batteries (ALBs). Unfortunately, the oxidative decomposition of anions from Li-salt at the extrinsic-interfaces of cathode side leads to the electrochemical instability during charge process. Here, a novel composite polymer electrolyte (CPE) containing flyash-treated (FA) submicro/micron particles and poly(ethylene oxide) (PEO) matrix is reported. After the addition of FA filler, Li+ transference number increases from 0.21 to 0.37 and electrochemical stability window reaches 5.2 V. FA as filler can efficiently immobilize (trifluoromethanesulfonyl) imide (TFSI–) anions and mitigate their irreversible oxidative decomposition at the extrinsic-interface of cathode||CPE due to the internal binding interaction between TFSI– anions and FA particles. ALBs using CPE with FA can not only provide stable extrinsic-interfaces for uniform Li deposition, but also tolerate strong electrochemical polarization with excellent cycling performance.

Microaerophilia enhances heavy metal biosorption and internal binding by polyphosphates in photosynthetic Euglena gracilis

Euglena gracilis is a photosynthetic protist with proved ability to resist and remove toxic single heavy metals under different laboratory conditions. To assess these abilities under more realistic conditions, cells subjected to combinations of heavy metals in a defined culture medium as well as in field polluted enriched medium were analyzed. Cells were cultured in the presence of Cd2+plus Cu2+ or Cr(VI) plus Fe3+ under aerobiosis or microaerophilia. Under aerobic growth, Cd2+plus Cu2+ (50 + 80 μM) and Cr(VI) plus Fe3+ (2 + 1 mM) strongly inhibited cell growth by inducing severe oxidative stress. In contrast, under microaerophilia, higher resistance and heavy metal removal were attained. This was indicated by (i) substantial biomass production; (ii) greater activities of glutathione reductase, glutathione peroxidase and ascorbate peroxidase as well as greater amounts of intracellular glutathione and ascorbate; (iii) increased metal removal by biosorption (i.e. greater than 80% for Cd, Cr, and Fe) due to an enhanced biofilm synthesis. The fraction of intracellularly heavy metal accumulated was bound mostly by polyphosphates (polyP). Cells cultured under microaerophilia in heavy metal polluted wastewater and sediments media were also able to grow and remove Fe, Cr, Cu and Cd. It is concluded that microaerophilia favors, the resistance of photosynthetic Euglena gracilis to heavy metal toxicity, and its ability to remove such metals from polluted water and solid samples. This work provides the biochemical basis for designing genetically engineered microorganisms with enhanced efficiency in metal removal. Moreover, it brings forward the combination of physicochemical and biological techniques to profit recovery.

A Self-Assembled Cage for Wide-Scope Chiral Recognition in Water.

Herein, we report the self-assembly of an anionic homochiral octahedral cage by condensing six Ga3+ cations and four trisacylhydrazone ligands. The robust nature of the hydrazone bond renders the cage stable in water, where it can take advantage of the hydrophobic effect for host-guest recognition. In addition to the internal binding site, namely, the inner cavity, the octahedral cage possesses four "windows", each of which represents an external binding site allowing peripheral complexation. These internal and external binding sites endow the cage with the capability to bind a broad range of guests whose sizes could either be smaller than or exceed the volume of the cage's inner cavity. Upon accommodation of a chiral guest, one of the two cage enantiomers becomes more favored than the other, producing circular-dichroism (CD) signals. The CD signal intensity of the cage is observed to be proportional to the ee value of the chiral guest, allowing a quantitative determination of the latter.

A Self‐Assembled Cage for Wide‐Scope Chiral Recognition in Water

AbstractHerein, we report the self‐assembly of an anionic homochiral octahedral cage by condensing six Ga3+ cations and four trisacylhydrazone ligands. The robust nature of the hydrazone bond renders the cage stable in water, where it can take advantage of the hydrophobic effect for host–guest recognition. In addition to the internal binding site, namely, the inner cavity, the octahedral cage possesses four “windows”, each of which represents an external binding site allowing peripheral complexation. These internal and external binding sites endow the cage with the capability to bind a broad range of guests whose sizes could either be smaller than or exceed the volume of the cage′s inner cavity. Upon accommodation of a chiral guest, one of the two cage enantiomers becomes more favored than the other, producing circular‐dichroism (CD) signals. The CD signal intensity of the cage is observed to be proportional to the ee value of the chiral guest, allowing a quantitative determination of the latter.