Binding Stoichiometry Research Articles

FERM domains recruit ample PI(4,5)P2s to form extensive protein-membrane attachments

The four-point-one ezrin-radixin-moesin homology (FERM) protein domain is a multifunctional protein-lipid binding site, constituting an integral part of numerous membrane-associated proteins. Its interaction with the lipid phosphatidylinositol-4,5-bisphosphate (PIP2), located at the inner leaflet of eukaryotic plasma membranes, is important for localization, anchorage, and activation of FERM-containing proteins. FERM-PIP2 complexes structurally determined so far exclusively feature a 1:1 binding stoichiometry of protein and lipid, with a few basic FERM residues neutralizing the −4 charge of the bound PIP2. Whether this picture from static crystal structures also applies to the dynamic interaction of FERM domains on PIP2 membranes is unknown. We here quantified the stoichiometry of FERM-PIP2 binding in a lipid bilayer using atomistic molecular dynamics simulations and experiments on solid supported membranes for the FERM domains of focal adhesion kinase and ezrin. In contrast to the structural data, we find much higher average stoichiometries of FERM-PIP2 binding, amounting to 1:3 or 1:4 ratios, respectively. In simulations, the full set of basic residues at the membrane interface, 7 and 15 residues for focal adhesion kinase and ezrin, respectively, engages in PIP2 interactions. In addition, Na ions enter the FERM-membrane binding interface, compensating negative PIP2 charges in case of high charge surpluses from bound PIP2. We propose the multivalent binding of FERM domains to PIP2 in lipid bilayers to significantly enhance the stability of FERM-membrane binding and to render the FERM-membrane linkage highly adjustable.

Investigating Native Metal Ion Binding Sites in Mammalian Histidine-Rich Glycoprotein.

Mammalian histidine-rich glycoprotein (HRG) is a highly versatile and abundant blood plasma glycoprotein with a diverse range of ligands that is involved in regulating many essential biological processes, including coagulation, cell adhesion, and angiogenesis. Despite its biomedical importance, structural information on the multi-domain protein is sparse, not least due to intrinsically disordered regions that elude high-resolution structural characterization. Binding of divalent metal ions, particularly ZnII, to multiple sites within the HRG protein is of critical functional importance and exerts a regulatory role. However, characterization of the ZnII binding sites of HRG is a challenge; their number and composition as well as their affinities and stoichiometries of binding are currently not fully understood. In this study, we explored modern electron paramagnetic resonance (EPR) spectroscopy methods supported by protein secondary and tertiary structure prediction to assemble a holistic picture of native HRG and its interaction with metal ions. To the best of our knowledge, this is the first time that this suite of EPR techniques has been applied to count and characterize endogenous metal ion binding sites in a native mammalian protein of unknown structure.

β-Cyclodextrin and Folic Acid Host-Guest Interaction Binding Parameters Determined by Taylor Dispersion Analysis and Affinity Capillary Electrophoresis.

The thermodynamic properties of molecular recognition in host-guest inclusion complexes can be studied by Taylor dispersion analysis (TDA). Host-guest inclusion complexes have modest size, and it is possible to get convergent results fast, achieving greater certainty for the obtained thermodynamic properties. Cyclodextrins (CDs) and their derivatives can be used as drug carriers that can boost stability, solubility, and bioavailability of physiologically active substances. A simple and effective approach for assessing the binding properties of CD complexesthat are critical in the early stages of drug and formulation development is needed to fully understand the process of CD and guest molecules' complex formation. In this work, TDA was successfully used to rapidly determine interaction parameters, including binding constant and stoichiometry, between β-CD and folic acid (FA) along with the diffusivities of the free FA and its complex with β-CD. Additionally, the FA diffusion coefficient obtained by TDA was compared to the results previously obtained by NMR. Affinity capillary electrophoresis (ACE) was also used to compare the binding constants obtained by different methods. The results showed that the binding constants obtained by ACE were somewhat lower than those obtained by the two TDA procedures. This article is protected by copyright. All rights reserved.

PEGylation Effects on the Interaction of Sphingomyelin Nanoemulsions with Serum Albumin: A Thermodynamic Investigation

AbstractThe recent focus in the development of novel nanosystems for biomedical applications lays firmly on their interactions with biomolecules. Thermodynamic parameters driving the interaction between nanoparticles and proteins provide insights into complex processes at bio/nanointerface. The present work aims to investigate the binding mechanisms and the dominant contributions that determine the adsorption processes during the interactions of a model protein, that is, bovine serum albumin, with a new type of drug delivery systems, Vitamin E/sphingomyelin nanoemulsions, plain and coated with polyethylene glycol, and d‐ɑ‐tocopheryl polyethylene glycol succinate. The binding parameters (binding constant, binding stoichiometry, enthalpy, Gibbs energy, and entropy changes of binding) are evaluated by the isothermal titration calorimetry with a MicroCaliTC200 equipment. The effect of nanoemulsions on the protein stability is examined by measuring the thermodynamic parameters for the protein's unfolding (heat capacity; enthalpy, entropy, and free energy changes) with a NanoDSC (TA Instrument) apparatus. The thermodynamic profile shows for all compositions an entropy‐driven interaction dominated by hydrophobic forces due to the rearrangements/displacement of the surrounding water molecules, while maintaining the native conformation of the protein. All the information acquired by thermodynamic approach may significantly enhance the knowledge with special focus on PEGylated nanoemulsions used for biomedical applications.

Efficient synthesis of α/β-isomeric oxadiazolyl and triazolopyridyl BODIPYs for sensing of Hg2+ ions and pH sensors

A simple sequential one-pot protocol has been developed to prepare oxadiazolyl and triazolopyridyl-BODIPY derivatives 4–6 by employing iodine (III)-promoted oxidative cyclization of BODIPY hydrazide-hydrazones which are readily accessible from the reaction of formyl BODIPYs with arylhydrazides. Photophysical studies of the prepared compounds 4–6 showed a significant red shift in absorption and emission maxima when compared to that of the parent BODIPY. Presence of triazolopyridyl substituent in 6a has affected its binding ability with metal ions and has rendered unique photophysical response by 6a selectively in presence of Hg2+ ions. Compound 6a showed high affinity towards Hg2+ (Ka = 1.8 × 104 M−1) with 2.1 μM limit of detection. The binding stoichiometry between 6a and Hg2+ was determined to be 2:1 by Job's plot analysis. Under acidic conditions, compounds 4c, 5c, 6a and 6b bearing dimethylamino- and triazolopyridyl moieties at α/β-positions of BODIPY unit exhibit blue shifted absorption (10–15 nm) and emission spectra (7–34 nm). Photophysical response of the compounds 4c, 5c, 6a and 6b on changing pH was validated by DFT calculations.

A Selective Excited-State Intramolecular-Proton-Transfer (ESIPT) Sensor for Copper(II) Based on Chelation-Enhanced Quenching and "Off-On" Detection of Amino Acids.

We report the synthesis of 2-(4,5-diphenyl-1H-imidazole-2-yl)phenol (TPI-9) as an interesting fluorescent molecule displaying Excited-State Intramolecular-Proton-Transfer (ESIPT) with stokes shift of 120 nm. Phenolic compounds with the ability to form intramolecular hydrogen bonds and subsequent proton transfer are known as ESIPT fluorophores. Proton accepting ability can increase significantly by tailoring electron-donating groups. With the assistance of an environment-friendly organocatalyst, 10-camphor sulfonic acid (10-CSA), TPI-9 was synthesized to introduce substituents with electron-donating abilities to develop an efficient ESIPT mechanism. Factors influencing the emission, such as solvent, pH, and metal ions, are investigated. Quenching of fluorescence by Cu2+ through chelation enhancement quenching effect with a high selectivity allowed the establishment of a Cu2+ sensor with an LoD of 0.57 ppm and a ratiometric estimation with an LoD of 0.73 ppm. Metal binding (2 : 1) stoichiometry and quenching constant (0.0072 mol-1 s-1 ) are calculated from Job's and Stern-Volmer plots. Density functional theory (DFT) calculations are in accordance with the experimental results. Competitive replacement of TPI-9 by amino acids restores ESIPT, consequently, the fluorescence. Thus, an "off-on" fluorescence sensor for amino acid estimation is developed under 1 minute incubation. A linear relationship between amino acid concentration and fluorescence intensity is in 0-20 μg/mL range, and the LoD is less than 2.2 μg/mL.

A highly selective “on–off” fluorescent sensor for detection of Fe3+ ion in protein and aqueous media: Synthesis, structural characterization, and computational studies

We describe herein the design and synthesis of a smart PGA chemosensor characterized by various techniques viz., FT-IR, UV–visible, 1H NMR, 13C NMR, Mass spectroscopy, and single crystal X-ray diffraction studies. The X-ray crystal analysis revealed that the PGA chemosensor crystallized in the orthorhombic crystal system with P212121 space group. The photophysical properties of the synthesized PGA chemosensor were examined by employing absorption and fluorescence spectral analysis. PGA revealed turn-on sensing responses towards the selective recognition of Fe3+ over other metal ions via CN isomerization processes. PGA also shows reversible effects upon the addition of EDTA. The 2:1 binding stoichiometry was calculated using Job’s plot and the value of detection limit was found to be 0.265 × 10-5 M, which is far lower than the permitted limit for drinking water according to WHO guidelines. The sensing mode of PGA towards Fe3+ was explored by employing FT-IR and 1H NMR studies. Density Functional Theory (DFT) studies, were employed which closely agreed with the experimental results. The PGA chemosensor has been successfully used to detect Fe3+ in various water samples and BSA medium. Furthermore, confocal imaging studies were examined, which supports the sensing ability of PGA for detection of Fe3+ in aqueous and protein medium.

A simple TICT/ICT based molecular probe exhibiting ratiometric fluorescence Turn-On response in selective detection of Cu2+

The work reported herein describes photophysical behavior of a new fluorescent probe APDA. The probe exhibited sensitivity for Cu2+ ion in 80% aqueous acetonitrile medium. Probe shows dual emission due to TICT and ICT states and upon interaction with different metal ions Cu2+ induces hydrolysis of imine bond to rejuvenate aldehyde derivative with ratiometric “turn–On” fluorescence response, through ICT state while emission due to TICT state get diminished. Similarly, the synthesized model compound ADA under similar experimental condition supported hydrolysis of imine bond in the presence of copper. Jobs plot analysis, based on emission titration data suggested a 1:1 binding stoichiometry between probe APDA and Cu2+ ion. The limit of detection (LOD) was found to be 4.4 × 10-8 M (44 nM). The probe has been applied on test paper strip to detect Cu2+ ion with naked-eye sensitive response. Also, the cell imaging studies revealed the potential applicability of probe to detect Cu2+ ion in live cells. The mechanism of sensing was confirmed by 1H, 13C NMR, FTIR and mass spectrometry data analysis.

A colorimetric and turn-on fluorescent sensor for rapid and selective detection of Fe3+ ion based on azo compound of 4-((4-(dimethylamino) phenyl)diazenyl)-N-(pyridin-2-yl)benzamide

A new colorimetric and turn-on fluorescent sensor based on an azo compound of 4-((4-(dimethylamino) phenyl) diazenyl)-N-(pyridin-2-yl)benzamide dye (L) was designed, synthesized and investigated. The structure of L was characterized by FT-IR, 1HNMR, 13CNMR and elemental analysis. The binding structures and sensing mechanisms of L toward iron were investigated using the various spectroscopic studies and theoretical calculations. Sensor exhibited highly selective and sensitive detection toward Fe3+ in acetonitrile aqueous solution. Compared to other metal cations, in case of iron, the color change from yellow to red was visible by the naked eye and a selective enhancement of fluorescence as well. The Job's method confirmed the formation of complex between L and Fe3+ with 1:1 binding stoichiometry. Furthermore, the detection limit (1.3 nM) for Fe3+ was much lower than that recommended by EPA (Environmental Protection Agency) for drinking water (5.37 µM). The theoretical calculations confirmed the coordination between nitrogen atom of pyridine and the oxygen atom of the carbonyl (C=O) group of L with Fe3+.

Study on the Interaction Mechanism of Methoxy Polyethylene Glycol Maleimide with Sweet Potato β-Amylase.

In this study, sweet potato β-amylase (SPA) was modified by methoxy polyethylene glycol maleimide (molecular weight 5000, Mal-mPEG5000) to obtain the Mal-mPEG5000-SPA modified β-amylase and the interaction mechanism between SPA and Mal-mPEG5000 was investigated. the changes in the functional groups of different amide bands and modifications in the secondary structure of enzyme protein were analyzed using infrared spectroscopy and circular dichroism spectroscopy. The addition of Mal-mPEG5000 transformed the random curl in the SPA secondary structure into a helix structure, forming a folded structure. The Mal-mPEG5000 improved the thermal stability of SPA and protected the structure of the protein from breaking by the surrounding. The thermodynamic analysis further implied that the intermolecular forces between SPA and Mal-mPEG5000 were hydrophobic interactions and hydrogen bonds due to the positive values of ΔHθ and ΔSθ. Furthermore, the calorie titration data showed that the binding stoichiometry for the complexation of Mal-mPEG5000 to SPA was 1.26, and the binding constant was 1.256 × 107 mol/L. The binding reaction resulted from negative enthalpy, indicating that the interaction of SPA and Mal-mPEG5000 was induced by the van der Waals force and hydrogen bonding. The UV results showed the formation of non-luminescent material during the interaction, the Fluorescence results confirmed that the mechanism between SPA and Mal-mPEG5000 was static quenching. According to the fluorescence quenching measurement, the binding constant (KA) values were 4.65 × 104 L·mol-1 (298K), 5.56 × 104 L·mol-1 (308K), and 6.91 × 104 L·mol-1 (318K), respectively.

A smart chemosensor with different response mechanisms to multi-analytes: Chromogenic and fluorogenic recognition of Cu2+, Fe3+, and Zn2+

Single-component sensors that can detect multiple analytes have drawn great research attentions. Herein, a smart chemosensor TPBS that can recognize multiple targets was developed by connecting triphenylamine with benzothiazole via an imine link. The N atoms with lone pair electrons on the flexible imine link served as potential coordination sites to allow for variations in charge transfer and molecular configurations. In CH3CN, TPBS simultaneously achieved the recognition of Cu2+ through a chromogenic response (colorless to red) via ligand-metal charge transfer, and the recognition of Fe3+ through a “turn-on” fluorogenic response (non-emission to blue) via the chelation-enhanced fluorescence effect. In aqueous solution, TPBS recognized Zn2+ via a “turn-on” fluorogenic response (non-emission to green) based on the aggregation-induced emission behavior. The detection limits were as low as 0.45 μM, 3.24 μM, and 3.22 μM for Cu2+, Fe3+, and Zn2+, respectively. A 1:1 binding stoichiometry and exact complex modes between TPBS and the target metal ions were deduced using Job's plot and FT-IR analyses. Both experiments and theoretical calculations supported the deduced coordination mechanisms. The ability of TPBS to recognize Cu2+, Fe3+, and Zn2+ was then tested in real-life water samples and through on-site detection of Zn2+ using test strips.

A spectroscopic and quantum chemical calculation method for the characterisation of metal ions complexed with propyl gallate and procyanidins

The deprotonation mechanism for the phenolic hydroxyl and the complexing of metal ions with a commonly used food additive, propyl gallate (PG) were studied theoretically and experimentally. The interaction of procyanidins [PC, epicatechin16 (4 → 8) catechin], and its basic monomeric unit catechin (CA) with metal ions was studied by the fluorescence quenching spectra. The results showed that the 9-OH quinoid PG was formed at higher pH (10.9) by the oxidization of phenolic hydroxyl. The binding affinities (Ka) and stoichiometry of these metal ions with PG were determined. The Al3+ in PG-Al complex [Al(PG)(H2O)2Cl2]- was coordinated at the 8,9-OH doubly deprotonated catechol site with double chloride ions (Cl-) and double water molecules (H2O). The fluorescence quenching titration with Sn2+, Zn2+, Cu2+, Al3+ and Fe3+ revealed that the stoichiometries of metal-bound PC were 1:1, 2:3, 2:3, 2:3 and 1:1, respectively. The presence of bovine serum albumin (BSA) could enhance the complexing strength of PC with metal ions.

Spectroscopic and TD-DFT studies on sequential fluorescent detection of Cu(II) and HS- ions in an aqueous solution

A new highly selective fluorescent probe AK1 was designed, synthesized and characterized, by conventional techniques, including single crystal XRD analysis, for sequential sensing of Cu(II) and HS- ions in an aqueous solution. The probe showed significant fluorescence quenching in the presence of Cu(II), while other metal ions produced only minor changes in the fluorescence of AK1. Job’s plot and mass and EPR spectral analyses indicated a 2:1 binding stoichiometry between AK1 and Cu(II) ions. The limit of detection (LOD) of AK1 for Cu(II) was determined to be 7.7 µM, which is far lower than the USEPA limit (∼20 µM) for drinking water. Subsequently, the in situ generated AK1-Cu(II) complex acts as a highly selective and sensitive probe for HS- ion, with an enhancement in fluorescence, even in the presence of different anions. Notably, the probe could detect and quantify Cu(II) and HS- ions in real water samples. Moreover, the optical properties of the probe in the absence and presence of Cu(II) and HS- ions were explained by density functional theory (DFT) and time-dependent density functional theory (TD-DFT) calculations.

Green synthesis of naphthyl derivative as an optical sensor for the detection of l-carnitine in food samples.

An economical and green approach to the synthesis of naphthyl derivative for detection of l-carnitine (3-hydroxy-4-N-trimethyl-aminobutyrate) is practically important. We developed a naphthyl derivative as a probe showing 'turn-on' response towards l-carnitine selectively at pH 7.2 through ICT mechanism with a good limit of detection (LOD) of 0.126 μM. Using Job's plot for determining the binding stoichiometry, it was found that probe could form a more stable complex (1:1) with carnitine. The binding constant (K) between probe and carnitine was calculated as 8 × 107 M-1 using the Benesi-Hildebrand plot. The binding interaction of the probe with l-carnitine was confirmed by nuclear magnetic resonance titrations, Fourier-transform infrared spectroscopy, photo physical studies and density functional theory calculations. Meanwhile, the probe can be used to quantitatively detect carnitine in food samples.

ESIPT-based dual-emissive perimidine derivative as a rapid and sensitive sensor for Cu2+ and Al3+: Construction of memory device, 2-to-1 encoder and 1-to-2 decoder

An ESIPT based fluorescent perimidine derivative oPSDAN was developed and characterized by 1H NMR, 13C NMR and mass spectroscopy. The study of the photo-physical properties of the sensor unveiled its selectivity and sensitivity towards Cu2+ and Al3+ ions. The sensing of ions was accompanied by colorimetric change (for Cu2+) as well as emission turn-off response. The binding stoichiometries of sensor oPSDAN with Cu2+ ion and Al3+ ions were determined to be 2:1 and 1:1, respectively. The binding constants and detection limits for Cu2+ and Al3+ were calculated from the UV–vis and fluorescence titration profiles as, 7.1 × 104 M−1, 1.9 × 104 M−1 and 9.89 nM, 1.5 × 10−8 M, respectively. The mechanism was established by 1H NMR as well as mass titrations and was supported by DFT and TD-DFT calculations. The UV–vis and fluorescence spectral results were further utilized for construction of memory device, encoder and decoder. Sensor-oPSDAN was also tested for determining Cu2+ ions in drinking water.

Metal‐free hydroarylation derived ferrocene‐dihydrocoumarin as ultrasensitive dual‐channel probe selectively for picric acid

4‐Ferrocenyl‐6,7‐dimethoxy‐3,4‐dihydrocoumarin (3) was synthesized by intermolecular hydroarylation of phenol from β‐ferrocene acrylic acid, and structure has been thoroughly characterized and confirmed by analysing X‐ray single‐crystal data. Compound 3 serves as highly selective fluorescent probe towards explosive picric acid (PA) through quenching of fluorescence in aqueous media. Probe 3 ensures high selectivity for PA in the background presence of various nitroaromatics, anions, cations and some phenols. The LoD for PA has been estimated to be 25.0 ppb using fluorescent probe 3. Binding stoichiometry and binding site of 3 towards PA have been supported by Job's plot, ESI‐mass, performing NMR titration and eventually by developing a model compound 4‐ferrocenyl‐3,4‐dihydrocoumarin 4, which strongly suggested 6,7‐dimethoxy groups as best probable binding site for PA. Overall, present approach deals with development of a ferrocenyl‐dihydrocoumarin conjugate for highly selective and ultrasensitive ‘turn‐off’ fluorescent probe for PA, which has been well‐supported through rational spectral analyses as well as through synthesizing a model compound.

Biophysical and molecular modelling analysis of the binding of β-resorcylic acid with bovine serum albumin

The bovine serum albumin (BSA) is a model carrier protein that transports and deposits the various endogenous and exogenous ligands in the body. β-resorcylic acid (BR) is a phyto-phenolic molecule mainly used in the food industry because of its antibacterial, flavouring and preservative nature. The interactions of small ligand molecules with proteins are of great importance for researchers from the pharmacology perspective. This article explains the mechanism of binding of BR with BSA with the help of multi-spectroscopic approaches and molecular modelling. The UV absorption and fluorescence spectroscopy affirmed the formation of BSA-BR complex. The thermodynamic studies confirmed the nature of the reaction to be spontaneous and exothermic, and the stabilisation of the complex by van der Waals interactions and hydrogen bonding. The energy transfer was found to occur with a high probability. BR induced the microenvironmental and conformational alterations BSA as revealed by synchronous spectra, 3D fluorescence maps, circular dichroism (CD), resonance-enhanced Rayleigh scattering (RRS), red edge excitation shift ( REES) and Fourier transform infrared (FT-IR). The β-cyclodextrin (β-cd), metals and vitamins largely influenced the BR-BSA binding strength. Competitive displacement assays further elucidated the BR binds to BSA at subdomain IIIA (site II). Molecular docking and simulations computationally validated the results of the wet lab experiments. • The BSA-BR complex was affirmed with 1:1 binding stoichiometry and quenching in static mode. • The binding reaction was spontaneous and exothermic, involving van der Waals forces and hydrogen bonding. • The binding strength was found to change in the presence of metal ions, vitamins and β-cd. • BR binds with BSA near subdomain IIIA (site II). • BR induced minor changes in the conformation and microenvironment in BSA.

A novel fluorescent sensor based on an enzyme-free system for highly selective and sensitive detection of glyphosate and malathion in real samples

Malathion (Mal) and glyphosate (Glyp) are widely used organophosphorus pesticides to prevent or control insects and weed in agriculture. Unfortunately, the extensive and frequent commercial use of these compounds on farmland has caused their presence as residues in crops and livestock. Therefore, the design of new sensors with enhanced analyte selectivity and sensitivity is critical in this field. Here, we developed an optical sensor for the detection of malathion and glyphosate. The sensor, N,N-dimethyl-4-(3-(pyrazin-2-ylimino)prop-1-en-1-yl)aniline (CNP), was synthesized in a one-step reaction by condensation of 4-(dimethylamino)cinnamaldehyde with 2-aminopyrazine. CNP gives a rapid color change, absorbance and fluorescence response to Glyp and Mal in the presence of the metal ions and pesticides tested in the working system. The binding stoichiometry between the sensor and the pesticides (Glyp and Mal) was investigated by some spectrophotometric methods and the sensing mechanisms were eludicated by NMR titration experiments, suggesting that CNP responds to Glyp via H-bonding and to Mal via the coordination. The sensor has a high selectivity and rapid response to the pesticides (Glyp and Mal) with low detection limits. The sensor was also successfully used to monitor the pesticides in real samples; Mal in apple and tap water samples, and Glyp in potato, tap water and soil samples.

A highly sensitive coumarin-based fluorescent probe for visual detection of Cu2+ in aqueous solution and its bioimaging in living cells

In this work, a highly sensitive fluorescent probe methyl (E)-2-((7-(diethylamino)-2-oxo-2H-chromen-3-yl)methylene)hydrazine-1-carboxylate (SJL) was synthesized from 7-(diethylamino)-2-oxo-2H-chromene-3-carbaldehyde and methyl hydrazinecarboxylate and characterized by various spectroscopic techniques. Highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) energies of SJL and SJL-Cu2+ complexes were estimated by DFT calculations, thus elucidating the conformation of the complexes. The probe SJL formed a stable complex and displayed a remarkable fluorescence quenching response to Cu2+ with a quenching efficiency of approximately 82.3% in aqueous solution. The binding stoichiometry of SJL-Cu2+ was determined to be 1: 1 based on the Job's plot method, and it exhibited a good linear relationship at ultra-low concentrations with a detection limit of 0.6 μM. The optimum pH range for SJL was 5–9. SJL could be made into test strips for the characterization of Cu2+. In particular, the probe has been used to visualize copper ions in Hela cells and glassfish larvae.

Probing single-molecule DNA-protein interactions using ABEL-FRET and ABEL-PIFE.

Single-molecule FRET and PIFE (protein induced fluorescence enhancement) are powerful techniques to probe molecular conformation and binding kinetics of DNA-protein interactions. We combine these techniques with Anti-Brownian Electrokinetic (ABEL) trapping to establish new modalities called ABEL-FRET and ABEL-PIFE. We demonstrate that ABEL-FRET achieves ultrahigh, shot-noise limited resolution of smFRET efficiency in solution without surface tethering. ABEL-PIFE enables simultaneous mapping of binding stoichiometry and sequence context of binding location. We highlight these new capabilities using examples of DNA processing enzymes.