Equilibrium Binding Constants Research Articles

DMNP as an Effective Cage for Ln3+: A Spectroscopic and Thermodynamic Study.

Dimethoxynitrophenyl-EDTA (DMNP) is a popular calcium cage that is frequently used to investigate the role of Ca2+ in signaling processes in vivo. Lanthanides have been used in Ca2+ biomimetics due to similarities in coordination properties of Ln3+ and Ca2+ that may enable fluorescence and NMR studies of functional and structural properties of Ca2+ binding proteins. In this study, we show that Tb3+, Eu3+, and Nd3+ bind strongly to DMNP in a 1:1 ratio. Isothermal titration calorimetric measurements of Ca2+ displacement by Ln3+ in DMNP provide the equilibrium binding constants for Ln3+DMNP complexation with association constants, K11 = (1.2 ± 0.7) × 1012 M-1 for Eu3+, (2.5 ± 1.7) × 1012 M-1 for Nd3+, and (2.8 ± 0.8) × 1012 M-1 for Tb3+. The kinetics and thermodynamics of Ca2+, Mg2+, and Tb3+ release from DMNP were characterized using photothermal beam deflection (PBD). Ligand release from the DMNP cage was rapid and occurred within 10 μs upon cage photofragmentation and was associated with similar reaction volume and enthalpy changes that can be attributed to the photoreleased ion solvation. In the case of Ca2+DMNP photodissociation at subsaturating Ca2+ concentrations, we observed a slower phase with a lifetime of 300 μs that we attribute to Ca2+ rebinding to unphotolyzed DMNP. These results demonstrate that DMNP can serve as an effective photolabile cage for oxophilic Ln3+ that has similar coordination properties to Ca2+ and Mg2+.

Study on the molecular interactions between givosiran and theophylline by surface-enhanced Raman spectroscopy, biolayer interferometry, and visible/fluorescence spectroscopy

Givosiran is a small-interfering ribonucleic acid (siRNA) drug used to treat acute hepatic porphyria. As an exogenous double-stranded RNA, givosiran has a specific spatial conformation. When they coexist, givosiran and small-molecule drugs may interact through non-covalent bonds, which may affect their efficacies. This study aimed to evaluate the molecular interaction between givosiran and three structurally-related small-molecule drugs. We combined surface-enhanced Raman spectroscopy, biolayer interferometry, and visible/fluorescence spectroscopy with a methylene blue probe to study the molecular interaction between givosiran and theophylline, caffeine, and theobromine. Molecular interactions were observed only between theophylline and givosiran, with an affinity constant (KD) of 64.2 μM. The main binding forces were hydrogen bonds and van der Waals forces, and the equilibrium binding constant (KA) was 1.855 × 104 L·mol−1. This study focused on the 5 W (Whether, Where, Why, hoW about the tendency, hoW strong) issues of the molecular interaction between givosiran and small-molecule chemical drugs. Our findings contradict the previous perception that the probability of drug–drug interactions (DDI) between nucleic acid and small-molecular drugs is low. Our study illustrates that a combination of direct and indirect analytical methods will pave a new way for future siRNA-related unknown interactions studies.

Amine functionalized supported ionic liquid membranes (SILMs) for CO2/N2 separation

Supported ionic liquid membranes (SILMs), containing aprotic N-heterocyclic anion ionic liquids (AHA ILs) in an inorganic inert support, exhibit CO2/N2 permselectivity values as high as 640 at 35.0 °C and 0.03 bar CO2, which represents conditions similar to post-combustion carbon capture (PCCC) from a natural gas power plant. A Fickian model fit to the experimental data estimates CO2 permeability at direct air capture (DAC) conditions of 10,400 barrer and a CO2/N2 permselectivity of 4000 for the best performing IL, triethyl(octyl)phosphonium 4-bromopyrazolide ([P2228][4-BrPyra]). The most important criterion for high selectivity is a large equilibrium constant for binding between the IL and CO2, which results in high CO2 solubility. ILs with smaller molar volumes and with no fluoroalkyl chains enhance N2 rejection. Low viscosity and high IL molar density also enhance CO2/N2 permselectivity and CO2 permeance.

Sensitivity and Selectivity Detection Studies of Fe (III) Using Cu(I) Complex of Schiff Base Material

The liver, bones, kidneys, teeth, and central nervous system sustain serious damage as a result of heavy metal ions entering the human food chain. In order to improve public health, new techniques must be developed for the rapid, easy, simple, reliable, low-cost, and reliable identification of toxic metal ions.Naked eye detection of hazardous metal ions with Cu (I) fluorescent properties of Cu(I) Complex of with 2, 2′-bipyridine and trans cinnamic acid were investigated. The structure of the fluorescent Cu (I) complex was characterized by conductivity measurement, elemental analysis, UV-Visible and FT-IR. The Cu (I) complex was soluble in dimethylsulfoxide, distilled water, methanol and insoluble in tetrahydrofuran. In the applications, firstly the color of the Cu(I) complex was compared with/without metal ions, and then the measurements were made in the UV-Vis spectrophotometer to exhibit selective and sensitive to Fe3+ ions in DMSO (dimethylsulfoxide) / H2O (water) (v/v, 1:1). Cu(I) complex exhibited absorbance band at 323 nm in dimethylsulfoxide. The absorbance intensity was decreased by Fe (III) and behaves as a turn-off sensor. The sensor showed high selectively and sensitivity toward Fe (III) over the other cations in dimethlsulfoxide solution. The equilibrium binding constant of Cu (I) complex with Fe (III) was 1.9x10 4 M -1 as calculated using stern –volmer equation. The limit of detection was also determined and calculated as 0.219 μM. Based on facts obtained from this study, the author suggests the Copper (I) complex response to Fe (III) rapidly and a large number of consecutive ions showed almost no obvious absorbance change during detection. Copper (I) complex could act as cost effective, selective, specific and sensor for detection of Fe (III) ions over other metal ions.

Assessment of polystyrene nano plastics effect on human salivary α-amylase structural alteration: Insights from an in vitro and in silico study

The study found that the enzyme activity of human salivary α-amylase (α-AHS) was competitively inhibited by nanoplastic polystyrene (PS-NPs), with a half-inhibitory concentration (IC50) of 92 μg/mL, while the maximum reaction rate (Vmax) remained unchanged at 909 μg/mL•min. An increase in the concentration of PS-NPs led to a quenching of α-AHS fluorescence with a slight red shift, indicating a static mechanism. The binding constant (Ka) and quenching constant (Kq) were calculated to be 2.92 × 1011 M−1 and 1.078 × 1019 M−1• S−1 respectively, with a hill coefficient (n) close to one and an apparent binding equilibrium constant (KA) of 1.54 × 1011 M−1. Molecular docking results suggested that the interaction between α-AHS and PS-NPs involved π-anion interactions between the active site Asp197, Asp300 residues, and van der Waals force interactions affecting the Tyr, Trp, and other residues. Fourier transform infrared (FT-IR) and circular dichroism (CD) analyses revealed conformational changes in α-AHS, including a loss of secondary structure α-helix and β-sheet. The study concludes that the interaction between α-AHS and PS-NPs leads to structural and functional changes in α-AHS, potentially impacting human health. This research provides a foundation for further toxicological analysis of MPs/NPs in the human digestive system.

Multimerizations, Aggregation, and Transfer Reactions of Small Numbers of Molecules.

Chemical equilibria of multimerizations in systems with small numbers of particles exhibit a behavior seemingly at odds with that observed macroscopically. In this paper, we apply the recently proposed expression of equilibrium constant for binding, which includes cross-correlations in reactants' concentrations, to write an equilibrium constant for the formation of clusters larger than two (e.g., trimer, tetramer, and pentamer) as series of two-body reactions. Results obtained by molecular dynamics simulations demonstrate that the value of this expression is constant for all concentrations and system sizes, as well as at an onset of a phase transition to an aggregated state, where densities in the system change discontinuously. In contrast, the value of the commonly utilized expression of equilibrium constant, which ignores correlations, is not constant and its variations can reach few orders of magnitude. Considering different paths for the same multimer formation, with elementary reactions of any order, yields different expressions for the equilibrium constant, yet, with exactly the same value. This is also true for routes with essentially zero probability to occur. Existence of different expressions for the same equilibrium constant imposes equalities between averages of correlated, along with uncorrelated, concentrations of participating species. Moreover, a relation between an average particle number and relative fluctuations derived for two-body reactions is found to be obeyed here as well despite couplings to additional equilibrium reactions in the system. Analyses of transfer reactions, where association and dissociation events take place on both sides of the chemical equation, further indicate the necessity to include cross-correlations in the expression of the equilibrium constant. However, in this case, the magnitudes of discrepancies of the uncorrelated expression are smaller, likely because of partial cancellation of correlations, which exist on both the reactant and product sides.

A comprehensive biophysical and theoretical study on the binding of dexlansoprazole with human serum albumin

Pharmacokinetics, pharmacodynamics, and therapeutic activity of drugs are defined by the affinities between the drug and biological macromolecules through molecular recognition. The present work is on the investigation of the interaction of new generation competent proton pump inhibitor dexlansoprazole (DLP) with human serum albumin (HSA). Fluorescence and UV–visible spectroscopy studies were adopted for quantification of the interaction in terms of the quenching constant (KSV), bimolecular quenching constant (Kq), equilibrium-binding constant (Kb), the number of binding sites (n), and the thermodynamic parameters. A hypochromic effect on the addition of HSA to DLP from the absorption studies shows their interaction. The quenching and binding constants from fluorescence studies were 104 and 105 M−1 order, respectively. A higher quenching constant with increasing temperature reflects a collisional-type quenching mechanism. A higher binding constant at a higher temperature shows an increase in the rate of binding, and the negative value of the free energy change shows the spontaneity of the binding. The sign and magnitude of the change in entropy (ΔS0 = 737.12 ± 2 J mol−1 K−1) and enthalpy (ΔH0 = 193.58 ± 1.7 kJ mol−1) shows that binding is an entropy-driven process with hydrophobic force as the major driving force. Site marker competitive displacement studies using 1-Anilino-8-naphthalene sulfonate (ANS), warfarin, and ibuprofen confirmed the biding of DLP in subdomain IIA of HSA. Binding-induced structural changes were evident from the changes in the circular dichroism (CD) signatures. Molecular docking of DLP in HSA showed the position of DLP in the warfarin binding site of HSA and corroborated the experimental findings.

Biophysical studies of peptoid interactions with phospholipids.

Using second harmonic generation and fluorescence anisotropy methods, we monitored adsorption of water-soluble peptoids to phospholipid bilayers at varying pH conditions and temperatures. We compared equilibrium binding constants and relative surface coverages of different peptoids sequences as we varied the cholesterol content, head group charge and physical phase of the lipids. We also collected pressure-area isotherms of lipid monolayers in the presence and absence of peptoids to gain insight into the physical principles that govern how peptoid sequence and membrane composition alter peptoid-lipid interactions.

Building a quantitative and predictive model of 5'SS selection by human U1 snRNP using RNA-map.

Alternative splicing of pre-mRNA greatly expands the protein coding potential of our genome and accounts for extensive cell and tissue-specific variation in gene expression. Spliceosome assembly depends on the recognition of the 5′ splice site (5′SS) at the exon-intron boundary by the U1 small nuclear ribonucleoprotein (U1 snRNP). Critically, mutations in either U1 or the 5′SS can lead to human disease, including cancer. Despite decades of investigation, accurately predicting 5′SS selection and alternative splicing outcomes is difficult, suggesting new approaches are needed. Here, we present our progress in developing a quantitative and predictive model of 5′SS selection by human U1 snRNP grounded in biochemical understanding. We used the RNA on a massively parallel array (RNA-MaP) platform to measure equilibrium binding constants of reconstituted human U1 snRNP to >60,000 unique RNAs. Our designed RNA library features all possible GU and GC variants in addition to thousands of identified mutations, allowing us to build a comprehensive and predictive model of 5′SS/U1 interactions at the transcriptome scale. Overall, our results will provide a first principles understanding of 5′SS selection and will serve as a foundation for future work to systematically determine the effects of other splicing factors and splice site usage in vivo.

Theoretical prediction of nanomolar and sequence-selective binding of synthetic supramolecular cucurbit[7]uril to N-terminal Leu-containing tripeptides.

Molecular recognition towards peptides and proteins with high affinity by synthetic supramolecular hosts is important but challenging. In this work, we investigate the molecular recognition of the synthetic cucurbit[7]uril (CB[7]) to 17 designed N-terminal Leu-containing tripeptides in aqueous medium by molecular dynamics (MD) simulation and screen out tripeptides with high binding affinity. It is found that, compared to LGG, only the third residue is Arg (R), the binding affinity of CB[7] to LGR reaches nanomolar level with binding equilibrium constant (Ka) of 1.1 × 109 M-1. The CB[7] recognition to the N-terminal Leu-containing tripeptides is highly sequence dependent; whether changing the sequence order (from LGR to LRG) or increasing the sequence length (from LGR to LGGR), Ka decreases by about three orders of magnitude. Interestingly, substituting N-terminal Leu for its isomer Ile, the binding of CB[7] to tripeptides weakens significantly with Ka decreasing by 3-8 orders of magnitude. Thus CB[7] can effectively distinguish N-terminal Leu-containing tripeptides from N-terminal Ile-containing tripeptides. Importantly, we predict that when R is as C-terminus, regardless of N-terminal residue being of aromatic type or Leu, the binding strength is always close to the nanomolar level. Therefore, R can be introduced to rationally design novel peptides with high binding affinity to CB[7] in practical applications.

Native holdup (nHU) to measure binding affinities from cell extracts

Characterizing macromolecular interactions is essential for understanding cellular processes, yet most methods currently used to detect protein interactions from cells are qualitative. Here, we introduce the native holdup (nHU) approach to estimate equilibrium binding constants of protein interactions directly from cell extracts. Compared to other pull-down-based assays, nHU requires less sample preparation and can be coupled to any analytical methods as readouts, such as Western blotting or mass spectrometry. We use nHU to explore interactions of SNX27, a cargo adaptor of the retromer complex and find good agreement between in vitro affinities and those measured directly from cell extracts using nHU. We discuss the strengths and limitations of nHU and provide simple protocols that can be implemented in most laboratories.

Structural Basis and Mechanism of Cyclophilin A‐Tau Interaction in Alzheimer’s Disease and Neuroinflammation

AbstractBackgroundTau protein that comprises neurofibrillary tangles in Alzheimer’s Disease (AD) interact with the abundant immunophilin protein Cyclophilin A (CypA) due to its peptidyl‐prolyl cis‐trans isomerase activity.MethodUsing surface plasmon resonance (SPR), nuclear magnetic resonance (NMR) and cell culture, structural details of CypA/tau interaction and its functional consequences are characterized.ResultUsing SPR, we observed a binding equilibrium constant (KD) of full‐length tau at ∼10 µM and identified the tau binding pocket on CypA using NMR. CypA and tau uptake by microglia was measured by immunofluorescence microscopy and flow cytometry. CypA‐tau interactions within microglia cell lines affect neuroinflammation by altering proinflammatory and anti‐inflammatory cytokine secretion responses.ConclusionCypA binds to tau with high affinity and specificity, which has functional significance in tau conformation, interaction, aggregation, and in neuroinflammation.

In Vitro Characterization of a Nuclear Receptor-like Domain of the Xylanase Regulator 1 from Trichoderma reesei.

Engineering transcription factors is an interesting research target gaining increasing attention, such as in the case of industrially used organisms. With respect to sustainability, biomass-degrading saprophytic fungi, such as Trichoderma reesei, are promising industrial work horses because they exhibit a high secretory capacity of native and heterologously expressed enzymes and compounds. A single-point mutation in the main transactivator of xylanase and cellulase expressions in T. reesei Xyr1 led to a strongly deregulated and enhanced xylanase expression. Circular dichroism spectroscopy revealed a change in secondary structure caused by this mutation. According to electrophoretic mobility shift assays and determination of the equilibrium-binding constants, the DNA-binding affinity of the mutated Xyr1 was considerably reduced compared to the wild-type Xyr1. Both techniques were also used to investigate the allosteric response to carbohydrates (D-glucose-6-phosphate, D-xylose, and sophorose) signalling the repression or induction of Xyr1 target genes. The mutated Xyr1 no longer exhibited a conformational change in response to these carbohydrates, indicating that the observed deregulation is not a simple matter of a change in DNA-binding of the transactivator. Altogether, we postulate that the part of Xyr1 where the mutation is located functions as a nuclear receptor-like domain that mediates carbohydrate signals and modulates the Xyr1 transactivating activity.

Supramolecular association studied by Fluorescence correlation spectroscopy.

A comprehensive description of a supramolecular system involves a full understanding of its thermodynamic and dynamic properties, as well as detailed knowledge of its structure. Fluorescence Correlation Spectroscopy (FCS) constitutes a powerful technique to acquire this information. Fluorescence correlation curves show a characteristic diffusion term that is related to the binding equilibrium constant or other thermodynamic properties of the supramolecular system. The association and dissociation rate constants of the binding process can be determined in FCS when the relaxation time of the binding is faster than the observation time-a regime called fast-exchange dynamics - in opposition to the slow-exchange regime. In all cases, structural information can be inferred from the diffusional properties of the supramolecular complexes. A short overview of the use of FCS for the study of supramolecular systems is given with examples which belong to the fast and slow regime.

Step toward Probing the Nonannular Belt of Membrane Proteins.

Integral membrane proteins are embedded in the biological membrane, where they carry out numerous biological processes. Although lipids present in the membrane are crucial for membrane protein function, it remains difficult to characterize many lipid binding events to membrane proteins, such as those that form the annular belt. Here, we use native mass spectrometry along with the charge-reducing properties of trimethylamine N-oxide (TMAO) to characterize a large number of lipid binding events to the bacterial ammonia channel (AmtB). In the absence of TMAO, significant peak overlap between neighboring charge states is observed, resulting in erroneous abundances for different molecular species. With the addition of TMAO, the weighted average charge state (Zavg) was decreased. In addition, the increased spacing between nearby charge states enabled a higher number of lipid binding species to be observed while minimizing mass spectral peak overlap. These conditions helped us to determine the equilibrium binding constants (Kd) for up to 16 lipid binding events. The binding constants for the first few lipid binding events display the highest affinity, whereas the binding constants for higher lipid binding events converge to a similar value. These findings suggest a transition from nonannular to annular lipid binding to AmtB.

Prediction of protein–ligand binding affinity from sequencing data with interpretable machine learning

Protein–ligand interactions are increasingly profiled at high throughput using affinity selection and massively parallel sequencing. However, these assays do not provide the biophysical parameters that most rigorously quantify molecular interactions. Here we describe a flexible machine learning method, called ProBound, that accurately defines sequence recognition in terms of equilibrium binding constants or kinetic rates. This is achieved using a multi-layered maximum-likelihood framework that models both the molecular interactions and the data generation process. We show that ProBound quantifies transcription factor (TF) behavior with models that predict binding affinity over a range exceeding that of previous resources; captures the impact of DNA modifications and conformational flexibility of multi-TF complexes; and infers specificity directly from in vivo data such as ChIP-seq without peak calling. When coupled with an assay called KD-seq, it determines the absolute affinity of protein–ligand interactions. We also apply ProBound to profile the kinetics of kinase–substrate interactions. ProBound opens new avenues for decoding biological networks and rationally engineering protein–ligand interactions.

On Computing Equilibrium Binding Constants for Protein-Protein Association in Membranes.

Protein association in lipid membranes is fundamental to membrane protein function and of great biomedical relevance. All-atom and coarse-grained models have been extensively used to understand the protein-protein interactions in the membrane and to compute equilibrium association constants. However, slow translational and rotational diffusion of protein in membrane presents challenges to the effective sampling of conformations defining the ensembles of free and bound states contributing to the association equilibrium and the free energy of dimerization. We revisit the homodimerization equilibrium of the TM region of glycophorin A. Conformational sampling is performed using umbrella sampling along previously proposed one-dimensional collective variables and compared with sampling over a two-dimensional collective variable space using the MARTINI v2.2 force field. We demonstrate that the one-dimensional collective variables suffer from restricted sampling of the native homodimer conformations leading to a biased free energy landscape. Conversely, simulations along the two-dimensional collective variable effectively characterize the thermodynamically relevant native and non-native interactions contributing to the association equilibrium. These results demonstrate the challenges associated with accurately characterizing binding equilibria when multiple poses contribute to the bound state ensemble.

Design mitochondria-specific fluorescent turn-on probes targeting G-quadruplexes for live cell imaging and mitophagy monitoring study

A series of new mitochondrial-selective fluorescent probe was designed and synthesized based on the integration of two planar molecular scaffolds of benzo-indole/indole with a p-substituted styrene moiety. The small-sized probes are flexible and rotatable via an ethylene bridge. The restriction of free rotation upon interaction with targeting biomolecules of G-quadruplexes in mitochondria generates strong emission in visible range (575–615 nm). Cell imaging study showed that the ligands are targeting mitochondria but not nucleus. Competition experiments showed that the ligand BYM is highly selective towards mitochondrial G4-DNA structures against other non-G4 nucleic acid structures including single-/double-stranded DNA and hairpin. The equilibrium binding constant (Keq) of BYM interacting with mitochondrial G4-DNA (mt6363, Keq = 10.8 × 106 M−1) is almost 1000-fold higher than that of mitochondrial double-stranded DNA (Keq = 0.01 × 106 M−1). The probe also showed high sensitivity (LOD = 1.52 nM) and good linear relationship (R2 = 0.9983) with the mitochondrial G4-DNA. The delivery of BYM to mitochondria is not mitochondrial membrane potential dependent but mainly through the permeability transition pore on the mitochondrial inner membrane. In addition, BYM exhibits low cytotoxicity against a number of human cancer and noncancerous cells, indicating that BYM could be a good ligand for bioorthogonal study in live cells by targeting the mitochondrial G4-DNA structures. In the present study, BYM was demonstrated in monitoring the dynamic process of mitochondrial autophagy. The small-sized fluorescent probe possessing high photostability, selectivity and sensitivity targeting mitochondrial G-quadruplexes may able to provide a useful chemical tool for real-time study of mitochondrial functions in live cells.

Caffeine Enhances the Toxicity of Platinum-Based Drugs at the Molecular Level Even Outside of the Intracellular Environment: A Single-Molecule Force Spectroscopy Study.

It is well reported in the literature that caffeine, the most consumed alkaloid around the world, enhances the anticancer effects of the drug cisplatin by inhibiting DNA repair by the cellular machinery. Here, we perform single-molecule force spectroscopy assays with optical tweezers to show that caffeine enhances the toxicity not only of cisplatin but also of various different platinum-based drugs already at the molecular level, using samples containing only double-stranded (ds)DNA, platinum drugs, and the alkaloid in a simple phosphate buffer, that is, completely out of the complex environment found inside real living cells. In fact, our results show that caffeine acts as an allosteric catalyst which increases the effective equilibrium binding constant between DNA and the platinum drugs, also interfering in the cooperativity of the binding reactions. To the best of our knowledge, this is the first time that such a property of caffeine was demonstrated and characterized from a pure physicochemical perspective, outside the cellular environment. Thus, the present work provides new insights into the use of this alkaloid for current chemotherapeutic applications.

Cupric Ions Selectively Modulate TRAAK-Phosphatidylserine Interactions.

TRAAK and TREK2 are two-pore domain K+ (K2P) channels and are modulated by diverse factors including temperature, membrane stretching, and lipids, such as phosphatidic acid. In addition, copper and zinc, both of which are essential for life, are known to regulate TREK2 and a number of other ion channels. However, the role of ions in the association of lipids with integral membrane proteins is poorly understood. Here, we discover cupric ions selectively modulate the binding of phosphatidylserine (PS) to TRAAK but not TREK2. Other divalent cations (Ca2+, Mg2+, and Zn2+) bind both channels but have no impact on binding PS and other lipids. Additionally, TRAAK binds more avidly to Cu2+ and Zn2+ than TREK2. In the presence of Cu2+, TRAAK binds similarly to PS with different acyl chains, indicating a crucial role of the serine headgroup in coordinating Cu2+. High-resolution native mass spectrometry (MS) enables the determination of equilibrium binding constants for distinct Cu2+-bound stoichiometries and uncovered the highest coupling factor corresponds to a 1:1 PS-to-Cu2+ ratio. Interestingly, the next three highest coupling factors had a ∼1.5:1 PS-to-Cu2+ ratio. Our findings bring forth the role of cupric ions as an essential cofactor in selective TRAAK-PS interactions.