Determination of protein-protein interactions at the single-molecule level using optical tweezers.

Biomolecular interactions are at the base of all physical processes within living organisms; the study of these interactions has led to the development of a plethora of different methods. Among these, single-molecule (in singulo) experiments have become relevant in recent years because these studies can give insight into mechanisms and interactions that are hidden for ensemble-based (in multiplo) methods. The focus of this review is on optical tweezer (OT) experiments, which can be used to apply and measure mechanical forces in molecular systems. OTs are based on optical trapping, where a laser is used to exert a force on a dielectric bead; and optically trap the bead at a controllable position in all three dimensions. Different experimental approaches have been developed to study protein­protein interactions using OTs, such as: (1) refolding and unfolding in trans interaction where one protein is tethered between the beads and the other protein is in the solution; (2) constant force in cis interaction where each protein is bound to a bead, and the tension is suddenly increased. The interaction may break after some time, giving information about the lifetime of the binding at that tension. And (3) force ramp in cis interaction where each protein is attached to a bead and a ramp force is applied until the interaction breaks. With these experiments, parameters such as kinetic constants (koff, kon), affinity values (KD), energy to the transition state ΔG≠, distance to the transition state Δx≠ can be obtained. These parameters characterize the energy landscape of the interaction. Some parameters such as distance to the transition state can only be obtained from force spectroscopy experiments such as those described here.

Comparison of mobility shift affinity capillary electrophoresis and capillary electrophoresis frontal analysis for binding constant determination between human serum albumin and small drugs.

In this study, two capillary electrophoresis-based ligand binding assays, namely, mobility shift affinity capillary electrophoresis (ms-ACE) and capillary electrophoresis-frontal analysis (CE-FA), were applied to determine binding parameters of human serum albumin toward small drugs under similar experimental conditions. The substances S-amlodipine (S-AML), lidocaine (LDC), l-tryptophan (l-TRP), carbamazepine (CBZ), ibuprofen (IBU), and R-verapamil (R-VPM) were used as the main binding partners. The scope of this comparative study was to estimate and compare both the assays in terms of their primary measure's precision and the reproducibility of the derived binding parameters. The effective mobility could be measured with pooled CV values between 0.55% and 7.6%. The precision of the r values was found in the range between 1.5% and 10%. Both assays were not universally applicable. The CE-FA assay could successfully be applied to measure the drugs IBU, CBZ, and LDC, and the interaction toward CBZ, S-AML, l-TRP, and R-VPM could be determined using ms-ACE. The average variabilities of the estimated binding constants were 64% and 67% for CE-FA and ms-ACE, respectively.

Effect of N- and C-Terminal Amino Acids on the Interfacial Binding Properties of Phospholipase D from Vibrio parahaemolyticus.

The effects of N-terminal (1⁻34 amino acids) and C-terminal (434⁻487 amino acids) amino acid sequences on the interfacial binding properties of Phospholipase D from Vibrio parahaemolyticus (VpPLD) were characterized by using monomolecular film technology. Online tools allowed the prediction of the secondary structure of the target N- and C-terminal VpPLD sequences. Various truncated forms of VpPLD with different N- or C-terminal deletions were designed, based on their secondary structure, and their membrane binding properties were examined. The analysis of the maximum insertion pressure (MIP) and synergy factor "a" indicated that the loop structure (1⁻25 amino acids) in the N-terminal segment of VpPLD had a positive effect on the binding of VpPLD to phospholipid monolayers, especially to 1,2-dimyristoyl-sn-glycero-3-phosphoserine and 1,2-dimyristoyl-sn-glycero-3-phosphocholine. The deletion affecting the N-terminus loop structure caused a significant decrease of the MIP and synergy factor a of the protein for these phospholipid monolayers. Conversely, the deletion of the helix structure (26⁻34 amino acids) basically had no influence on the binding of VpPLD to phospholipid monolayers. The deletion of the C-terminal amino acids 434⁻487 did not significantly change the binding selectivity of VpPLD for the various phospholipid monolayer tested here. However, a significant increase of the MIP value for all the phospholipid monolayers strongly indicated that the three-strand segment (434⁻469 amino acids) had a great negative effect on the interfacial binding to these phospholipid monolayers. The deletion of this peptide caused a significantly greater insertion of the protein into the phospholipid monolayers examined. The present study provides detailed information on the effect of the N- and C-terminal segments of VpPLD on the interfacial binding properties of the enzyme and improves our understanding of the interactions between this enzyme and cell membranes.

Investigation of α-Synuclein Amyloid Fibrils Using the Fluorescent Probe Thioflavin T.

In this work, α-synuclein amyloid fibrils-the formation of which is a biomarker of Parkinson's disease-were investigated using the fluorescent probe thioflavin T (ThT). The experimental conditions of protein fibrillogenesis were chosen so that a sufficient number of continuous measurements could be performed to characterize and analyze all stages of this process. The reproducibility of fibrillogenesis and the structure of the obtained aggregates (which is a critical point for further investigation) were proven using a wide range of physical-chemical methods. For the determination of ThT-α-synuclein amyloid fibril binding parameters, the sample and reference solutions were prepared using equilibrium microdialysis. By utilizing absorption spectroscopy of these solutions, the ThT-fibrils binding mode with a binding constant of about 104 M-1 and stoichiometry of ThT per protein molecule of about 1:8 was observed. Fluorescence spectroscopy of the same solutions with the subsequent correction of the recorded fluorescence intensity on the primary inner filter effect allowed us to determine another mode of ThT binding to fibrils, with a binding constant of about 106 M-1 and stoichiometry of about 1:2500. Analysis of the photophysical characteristics of the dye molecules bound to the sites of different binding modes allowed us to assume the possible localization of these sites. The obtained differences in the ThT binding parameters to the amyloid fibrils formed from α-synuclein and other amyloidogenic proteins, as well as in the photophysical characteristics of the bound dye, confirmed the hypothesis of amyloid fibril polymorphism.

Structural Features of Amyloid Fibrils Formed from the Full-Length and Truncated Forms of Beta-2-Microglobulin Probed by Fluorescent Dye Thioflavin T.

The persistence of high concentrations of beta-2-microglobulin (ß2M) in the blood of patients with acute renal failure leads to the development of the dialysis-related amyloidosis. This disease manifests in the deposition of amyloid fibrils formed from the various forms of ß2M in the tissues and biological fluids of patients. In this paper, the amyloid fibrils formed from the full-length ß2M (ß2m) and its variants that lack the 6 and 10 N-terminal amino acids of the protein polypeptide chain (ΔN6ß2m and ΔN10ß2m, respectively) were probed by using the fluorescent dye thioflavin T (ThT). For this aim, the tested solutions were prepared via the equilibrium microdialysis approach. Spectroscopic analysis of the obtained samples allowed us to detect one binding mode (type) of ThT interaction with all the studied variants of ß2M amyloid fibrils with affinity ~104 M-1. This interaction can be explained by the dye molecules incorporation into the grooves that were formed by the amino acids side chains of amyloid protofibrils along the long axis of the fibrils. The decrease in the affinity and stoichiometry of the dye interaction with ß2M fibrils, as well as in the fluorescence quantum yield and lifetime of the bound dye upon the shortening of the protein amino acid sequence were shown. The observed differences in the ThT-ß2M fibrils binding parameters and characteristics of the bound dye allowed to prove not only the difference of the ΔN10ß2m fibrils from other ß2M fibrils (that can be detected visually, for example, by transmission electron microscopy (TEM), but also the differences between ß2m and ΔN6ß2m fibrils (that can not be unequivocally confirmed by other approaches). These results prove an essential role of N-terminal amino acids of the protein in the formation of the ß2M amyloid fibrils. Information about amyloidogenic protein sequences can be claimed in the development of ways to inhibit ß2M fibrillogenesis for the treatment of dialysis-related amyloidosis.

Zinc Oxide Nanoparticle-Poly I:C RNA Complexes: Implication as Therapeutics against Experimental Melanoma.

There is current interest in harnessing the combined anticancer and immunological effect of nanoparticles (NPs) and RNA. Here, we evaluate the bioactivity of poly I:C (pIC) RNA, bound to anticancer zinc oxide NP (ZnO-NP) against melanoma. Direct RNA association to unfunctionalized ZnO-NP is shown by observing change in size, zeta potential, and absorption/fluorescence spectra upon complexation. RNA corona was visualized by transmission electron microscopy (TEM) for the first time. Binding constant (Kb = 1.6-2.8 g-1 L) was determined by modified Stern-Volmer, absorption, and biological surface activity index analysis. The pIC-ZnO-NP complex increased cell death for both human (A375) and mouse (B16F10) cell lines and suppressed tumor cell growth in BALB/C-B16F10 mouse melanoma model. Ex vivo tumor analysis indicated significant molecular activity such as changes in the level of phosphoproteins JNK, Akt, and inflammation markers IL-6 and IFN-γ. High throughput proteomics analysis revealed zinc oxide and poly I:C-specific and combinational patterns that suggested possible utility as an anticancer and immunotherapeutic strategy against melanoma.

Studying small molecule-aptamer interactions using MicroScale Thermophoresis (MST).

Aptamers are potent and versatile binding molecules recognizing various classes of target molecules. Even challenging targets such as small molecules can be identified and bound by aptamers. Studying the interaction between aptamers and drugs, antibiotics or metabolites in detail is however difficult due to the lack of sophisticated analysis methods. Basic binding parameters of these small molecule-aptamer interactions such as binding affinity, stoichiometry and thermodynamics are elaborately to access using the state of the art technologies. The innovative MicroScale Thermophoresis (MST) is a novel, rapid and precise method to characterize these small molecule-aptamer interactions in solution at microliter scale. The technology is based on the movement of molecules through temperature gradients, a physical effect referred to as thermophoresis. The thermophoretic movement of a molecule depends - besides on its size - on charge and hydration shell. Upon the interaction of a small molecule and an aptamer, at least one of these parameters is altered, leading to a change in the movement behavior, which can be used to quantify molecular interactions independent of the size of the target molecule. The MST offers free choice of buffers, even measurements in complex bioliquids are possible. The dynamic affinity range covers the pM to mM range and is therefore perfectly suited to analyze small molecule-aptamer interactions. This section describes a protocol how quantitative binding parameters for aptamer-small molecule interactions can be obtained by MST. This is demonstrated by mapping down the binding site of the well-known ATP aptamer DH25.42 to a specific region at the adenine of the ATP molecule.

Good use of fruit wastes: eco-friendly synthesis of silver nanoparticles, characterization, BSA protein binding studies.

A simple and eco-friendly methodology for the green synthesis of silver nanoparticles (AgNPs) using a mango seed extract was evaluated. The AgNPs were characterized by ultraviolet-visible spectrophotometry, Fourier transform infrared spectroscopy, transmission electron microscopy, energy dispersive X-ray spectroscopy, and X-ray diffraction. The interaction between the green synthesized AgNPs and bovine serum albumin (BSA) in an aqueous solution at physiological pH was examined by fluorescence spectroscopy. The results confirmed that the AgNPs quenched the fluorophore of BSA by forming a ground state complex in aqueous solution. This fluorescence quenching data were also used to determine the binding sites and binding constants at different temperatures. The calculated thermodynamic parameters (ΔG°, ΔH° and ΔS°) suggest that the binding process occurs spontaneously through the involvement of electrostatic interactions. The synchronous fluorescence spectra showed a blue shift, indicating increasing hydrophobicity. Copyright © 2015 John Wiley & Sons, Ltd.

Drug Distribution to Retinal Pigment Epithelium: Studies on Melanin Binding, Cellular Kinetics, and Single Photon Emission Computed Tomography/Computed Tomography Imaging.

Melanin binding is known to affect the distribution and elimination of ocular drugs. The purpose of this study was to evaluate if the extent of drug uptake to primary retinal pigment epithelial (RPE) cells could be estimated based on in vitro binding studies with isolated melanin and evaluate the suitability of single photon emission computed tomography/computed tomography (SPECT/CT) in studying pigment binding in vivo with pigmented and albino rats. Binding of five compounds, basic molecules timolol, chloroquine, and nadolol and acidic molecules methotrexate and 5(6)-carboxy-2',7'-dichlorofluorescein (CDCF), was studied using isolated melanin from porcine choroid-RPE at pH 5.0 and 7.4. The uptake to primary porcine RPE cells was studied with timolol, chloroquine, methotrexate, and CDCF. The cell study setting was modeled using parameters from the in vitro binding study. In vivo kinetics of 3-[I-123]-iodochloroquine was studied by the SPECT/CT method in albino and pigmented rats. All basic compounds bound to melanin at both pH values, whereas the acidic compounds bound more at pH 5.0 than at pH 7.4. The basic compounds (chloroquine, timolol) showed significant cellular uptake, unlike the acidic compounds (methotrexate, CDCF). On the basis of the modeling, melanin binding was a major factor governing the overall drug distribution to the RPE cells. Likewise, melanin binding explained distribution of 3-[I-123]-iodochloroquine in the pigmented RPE, whereas drug accumulation was not seen in the albino rat. This study demonstrates the suitability of noninvasive SPECT/CT imaging in monitoring ocular melanin binding in vivo. These studies are a useful step toward understanding the pharmacokinetic impact of melanin binding.

Evaluation and optimization of the metal-binding properties of a complex ligand for immobilized metal affinity chromatography.

The simultaneous determination of two binding parameters for metal ions on an immobilized metal affinity chromatography column was performed by frontal chromatography. In this study, the binding parameters of Cu(2+) to l-glutamic acid were measured, the metal ion-binding characteristics of the complex ligand were evaluated. The linear correlation coefficients were all greater than 99%, and the relative standard deviations of two binding parameters were 0.58 and 0.059%, respectively. The experiments proved that the frontal chromatography method was accurate, reproducible, and could be used to determine the metal-binding parameters of the affinity column. The effects of buffer pH, type, and concentration on binding parameters were explored by uniform design experiment. Regression, matching and residual analyses of the models were performed. Meanwhile, the optimum-binding conditions of Cu(2+) on the l-glutamic acid-silica column were obtained. Under these binding conditions, observations and regression values of two parameters were similar, and the observation values were the best. The results demonstrated that high intensity metal affinity column could be effectively prepared by measuring and evaluating binding parameters using frontal chromatography combined with a uniform design experiment. The present work provided a new mode for evaluating and preparing immobilized metal affinity column with good metal-binding behaviors.

Binding of flavonoids to yeast aldehyde dehydrogenase: a molecular mechanism and computational approach.

The interaction of three flavonoids, apigenin, fisetin and quercetin with yeast aldehyde dehydrogenase, ALDH was studied by spectroscopic and molecular docking methods. A combination of both static and dynamic processes interaction mechanism for the binding of flavonoids with ALDH was found. The interaction takes place with moderate binding and the interaction was driven by hydrophobic contacts. The microenvironments of the fluorescent amino acids changed upon flavonoids binding. The distances between ALDH and flavonoids determined by Förster Resonant Energy Transfer (FRET) confirmed the results obtained by fluorescence. The structure of ALDH against thermal denaturation was stabilized by apigenin and destabilized by fisetin and quercetin. Molecular docking simulation showed that all flavonoids bind to the same site of ALDH and confirmed the moderate binding straight found in fluorescence.Communicated by Ramaswamy H. Sarma.

Interaction of MnTOEtPyP4 porphyrin with DNA.

The binding of water-soluble meso-tetra-(4N-oxyethylpyridyl) porphyrin (H2TOEtPyP4) and its manganese (III) derivative (MnTOEtPyP4) with calf thymus DNA have been quantitatively studied using UV/Vis spectrophotometry, Circular Dichroism (CD), thermal melting curves and viscometry. The results show, that porphyrins interact with DNA via one binding mode at low relative concentrations (r) and two binding modes at high values of r. The binding constant (Kb) and stoichiometry (n) were determined from binding isotherms for both porphyrin-DNA complexes. The thermal melting analysis indicates that the double-helical structure of DNA molecules is stabilizing in presence of studied porphyrins. At certain concentrations of porphyrin, two-stage melting curves were observed, which indicates the existence of two different binding modes. Obtained results show that MnTOEtPyP4 associates with DNA duplex via outside binding mode.Communicated by Ramaswamy H. Sarma.

Microscale thermophoresis as a powerful growing analytical technique for the investigation of biomolecular interaction and the determination of binding parameters.

Thein vitropanel of technologies to address biomolecular interactions are in play, however microscale thermophoresis is continuously increasing in use to represent a key player in this arena. This review highlights the usefulness of microscale thermophoresis in the determination of molecular and biomolecular affinity interactions. This work reviews the literature from January 2016 to January 2022 about microscale thermophoresis. It gives a summarized overview about both the state-of the art and the development in the field of microscale thermophoresis. The principle of microscale thermophoresis is also described supported with self-created illustrations. Moreover, some recent advances are mentioned that showing application of the technique in investigating biomolecular interactions in different fields. Finally, advantages as well as drawbacks of the technique in comparison with other competing techniques are summarized.

Interaction of doxorubicin delivered by superparamagnetic iron oxide nanoparticles with DNA.

We studied the interaction of superparamagnetic iron oxide nanoparticles (SPIONs), covered by trisodium citrate, with doxorubicin (DOX) and DNA using the spectrophotometric method. We calculated the binding parameters in the binary (DOX-SPION and SPION-DNA) and the ternary (DOX-SPION-DNA) systems. Our studies showed that the nanoparticles do not interact with DNA. We also observed that one nanoparticle loads rather a large number of DOX molecules with a quite high binding constant value (kDOX-SPION = 1.2 × 104 M-1). The DNA addition to the DOX-SPION system induces DOX release from the SPION surface and the formation of DOX-DNA complexes. The presence of nanoparticles has almost no effect on the constant of doxorubicin binding to DNA (kDOX-DNA ≈ 3 × 104 M-1). At high DNA concentrations, almost all DOX molecules bind to DNA. Accordingly, the use of SPIONs as DOX carriers does not require an increased drug dose to achieve a therapeutic effect. Thus, SPIONs are perspective nanocarriers for DOX delivery.

Analytical methods for obtaining binding parameters of drug-protein interactions: A review.

The study of drug-protein interactions can reveal the corresponding binding mechanisms, providing valuable information for the early phase drug development and development of new drugs. This article reviews the methods used for obtaining the binding parameters of drug-protein systems. The methods include equilibrium dialysis, high-performance affinity chromatography, capillary electrophoresis, spectroscopy, calorimetry, competition and displacement, mass spectrometry, fluorescence resonance energy transfer, and thermal stability shift analysis. Relevant parameters include the association constant, number of binding sites, thermodynamic properties, binding force types, binding site types, binding distances, changes in protein conformation, and changes in protein stability. In addition, the review also summarizes the principles, advantages, and limitations of each method in detail. The comparison of parameter information can not only guide method selection but also provide valuable reference information for in-depth exploration of drug-protein interaction mechanisms.

pH-dependent complex formation of Zn-meso-tetra(4-N-hydroxyethylpyridyl) porphyrin with cancer DNA.

The complex formation between the synthetic water-soluble Zn-meso-tetra(4-N-hydroxyethylpyridyl) porphyrin (ZnTOEPyP4) and cancer DNA in comparison to healthy DNA was investigated using the UV/VIS spectrophotometry method in phosphate-buffered saline at different pHs. The increasing of DNA/porphyrin ratio leads to hypochromicity and red shift in the Soret band, which indicate the complexation of the ZnTOEPyP4 with DNA. The results show that the binding constant (Kb) and the exclusion parameter (n) of ZnTOEPyP4 with DNA strongly depend upon the pH. The Kbof ZnTOEPyP4 with cancer DNA is higher than with normal DNA.Communicated by Ramaswamy H. Sarma.

Studies to explore the UVA photosensitizing action of 9-phenylacridine in cells by interaction with DNA.

Acridine and its derivatives are well known for their DNA binding properties. In this report, we present our findings on evaluating different binding parameters of the interaction of 9-phenylacridine (ACPH) with DNA. Absorption spectroscopic studies including standard and reverse titration, the effects of ionic strength and temperature on titration, and Job plot analysis were done to calculate the binding constant and determine the different thermodynamic parameters and stoichiometry of the binding. Spectrofluorimetry and circular dichroism (CD) spectral titration were also utilized to confirm these findings. The results indicated that ACPH binds to DNA reversibly through non-electrostatic interactions by hydrogen bonding and van der Waals interactions. The binding constant and the number of binding sites were of the order 103 M-1 and ≈2, respectively with a binding stoichiometry of 1:4. The binding of ACPH with DNA was spontaneous, exothermic and enthalpy-driven. The extent of uptake of ACPH in B16 melanoma cells was estimated. As this compound absorbs in the UVA region, the effect of treatment with ACPH prior to UVA exposure was assessed to evaluate its phototoxicity in these cells. Our results indicated that the binding to DNA enhanced damage to sensitize cells to killing through apoptosis. Our findings indicated its potential to act as a photosensitizer.

Dengue fusion peptide in Langmuir monolayers: A binding parameter study.

Membrane fusion is known to be the primary mechanism of entry of flaviviruses into host cells. Several studies reported the investigation of the membrane fusion mechanism mediated by the fusion peptide, a component of the membrane protein surrounding the flaviviruses. In this study, we investigated the interaction of Dengue fusion peptide (FLAg) with Langmuir monolayers to uncover the role of membrane charges and organization in its membrane binding. Binding parameters of FLAg were obtained by measuring its adsorption onto Langmuir monolayers of different types of individual lipids, as well as their mixtures. Specific peptide binding was observed in the presence of charged lipid monolayers at different pHs, revealing that the lipid composition of the membrane modulates peptide interaction, and the preference of the peptide for negatively charged lipids.

Studies of eosin Y - DNA interaction using a competitive binding assay.

The interaction between xanthene dye eosin Y and double stranded DNA has been studied by spectrophotometry. The conventional titration study does not show the interaction in the eosin Y - DNA system. Therefore, the competitive binding assay was carried out. The DNA-targeted ligands proflavine and methylene blue were used as competitors. Multivariate curve resolution - alternative least squares method (MCR-ALS) was applied to analyze the spectrophotometric titration data. The experimental binding isotherms were fitted by Scatchard and McGee equations. The binding constant of eosin Y with DNA was found to be 1.7·104 M-1. It is shown that the competitive binding assay requires consideration of heteroassociation for the correct determination of ligand-DNA binding parameters.

New aryl Schiff bases of thiadiazole derivative of ibuprofen as DNA binders and potential anticancer drug candidates.

The work presented in this paper describes the synthesis of two new aryl Schiff bases [(E)-N-(4-(benzyloxy)-3-methoxybenzylidene)-5-(1-(4-isobutylphenyl)ethyl)-1,3,4-thiadiazol-2-amine] (ASB-1) and [(E)-N-(4-(benzyloxy)benzylidene)-5-(1-(4-isobutylphenyl)ethyl)-1,3,4-thiadiazol-2-amine] (ASB-2). These compounds were characterized by different analytical techniques and then studied for DNA binding. Binding studies were carried out at neutral pH (7.0) and at 37 °C by theoretical and experimental methods including DFT, molecular docking, spectroscopy (UV-visible, fluorescence), cyclic voltammetry (CV) and viscometry. Further investigations of these compounds were done on hepatocellular carcinoma; Huh-7 cancer cell line. Binding constant, free energy change and binding site size, i.e. Kb, ΔG and n were evaluated which indicated that both ASB-1 and ASB-2 bind significantly and spontaneously with the DNA. However, data revealed relatively greater binding of ASB-1 with DNA. Spectral and voltammetric results were found supportive of each other. Binding site sizes and viscosity measurements verified the mixed binding mode of interactions as observed in molecular docking analysis, i.e. intercalation with groove binding. DNA binding studies were very well correlated with the in-vitro studies performed on Huh-7 cell line as well as normal HEK-293 cell lines. The compound ASB-1 not only showed greater binding affinity toward DNA but also showed greater anticancer potency with least IC50 value as compared to ASB-2.