Isothermal Titration Microcalorimetry Research Articles

Human FABP1 T94A variant enhances cholesterol uptake

Although expression of the human liver fatty acid binding protein (FABP1) T94A variant alters serum lipoprotein cholesterol levels in human subjects, nothing is known whereby the variant elicits these effects. This issue was addressed by in vitro cholesterol binding assays using purified recombinant wild-type (WT) FABP1 T94T and T94A variant proteins and in cultured primary human hepatocytes expressing the FABP1 T94T (genotyped as TT) or T94A (genotyped as CC) proteins. The human FABP1 T94A variant protein had 3-fold higher cholesterol-binding affinity than the WT FABP1 T94T as shown by NBD-cholesterol fluorescence binding assays and by cholesterol isothermal titration microcalorimetry (ITC) binding assays. CC variant hepatocytes also exhibited 30% higher total FABP1 protein. HDL- and LDL-mediated NBD-cholesterol uptake was faster in CC variant than TT WT human hepatocytes. VLDL-mediated uptake of NBD-cholesterol did not differ between CC and TT human hepatocytes. The increased HDL- and LDL-mediated NBD-cholesterol uptake was not associated with any significant change in mRNA levels of SCARB1, LDLR, CETP, and LCAT encoding the key proteins in lipoprotein cholesterol uptake. Thus, the increased HDL- and LDL-mediated NBD-cholesterol uptake by CC hepatocytes may be associated with higher affinity of T94A protein for cholesterol and/or increased total T94A protein level.

Effects of water content and chain length of n-alkane on the interaction enthalpy between the droplets in water/sodium bis(2-ethylhexyl)-sulfosuccinate/n-alkane microemulsions.

The concentration-dependent enthalpies of mixing for water/sodium bis(2-ethylhexyl)-sulfosuccinate (AOT)/n-alkane microemulsions with different water contents ω0 and chain lengths n of n-alkane were determined by isothermal titration microcalorimetry (ITC) and flow-mixing microcalorimetry at 298.15 K and used to calculate the interaction enthalpies (-ΔH(C)) between the droplets. It was found that -ΔH(C) increased with ω0, and changed from negative to positive at about ω0 = 10. The investigation of the dependence of -ΔH(C) on n revealed that the values of -ΔH(C) were negative and had a minimum for ω0 = 5; while they were positive and had a maximum for ω0 = 15. These phenomena were discussed based on the competition of the overlapping contribution of the surfactant tails between two neighbouring droplets and the penetration contribution of the solvent molecules into the surfactant tails. These results indicated the important role of entropy in the stability of the microemulsion systems.

The effect of sarafloxacin on Cu/ZnSOD structure and activity

The effect of sarafloxacin to Cu/ZnSOD was evaluated via investigating the change in Cu/ZnSOD structure and the structure basis activity upon sarafloxacin binding. Multi-spectroscopic methods, isothermal titration microcalorimetry (ITC) and molecular docking method were adopted in this study. Sarafloxacin binds to Cu/ZnSOD mainly through hydrophobic and hydrogen bond forces and tends to be saturated as the molar ratio of sarafloxacin to Cu/ZnSOD reaches 4. The binding changed the microenvironment around Tyr and the secondary structure of Cu/ZnSOD but did not affect the activity of Cu/ZnSOD. Molecular docking study revealed that sarafloxacin binds into a hydrophobic area with possibility to form hydrogen bonds with Tyr 108, Asp 25, Pro 100 and Ser 103 of Cu/ZnSOD. The binding area locates on the surface of β-barrel close to the second Greek key loop (GK2) and V-loop but far away from the active site and active site channel of Cu/ZnSOD. These promoted the understanding of the experiment phenomenons. The binding of sarafloxacin does not affect the activity of Cu/ZnSOD should attribute to the binding not to change the microenvironment of Cu/ZnSOD active site and active site channel.

Constructing Gemini‐Like Surfactants with Single‐Chain Surfactant and Dicarboxylic Acid Sodium Salts

AbstractConstruction of gemini‐like surfactants using the cationic single‐chain surfactant cetyltrimethylammonium bromide C16H33N(CH3)3Br2 (CTAB) and the anionic dicarboxylic acid sodium salt NaOOC(CH2)n‐2COONa (CnNa2, n = 4, 6, 8, 10, 12) by way of non‐covalent interactions has been investigated by surface tension measurements, hydrogen‐1 nuclear magnetic resonance (1H NMR) spectroscopy and isothermal titration microcalorimetry (ITC). The critical micelle concentrations (cmc) of the CTAB/CnNa2 mixtures are obviously lower than that of CTAB and strongly depend on the mixing ratio. Moreover, the cmc values of the CTAB/CnNa2 mixtures decrease gradually with an increasing methylene chain length of CnNa2, indicating hydrophobic interaction between the hydrocarbon chains of CTAB and CnNa2 facilitates micellization of the mixtures. In particular, the ITC curves and 1H NMR spectra indicate that the binding ratio of CTAB to CnNa2, except C4Na2, is around 2:1, i.e., (CTAB)2CnNa2. Additionally, CTAB/CnNa2 mixtures are soluble in a whole molar ratio and concentration ranges have been studied, even at the electrical neutralization point. Therefore, these results reveal that highly soluble gemini‐like surfactants are conveniently constructed with oppositely‐charged cationic single‐chain surfactants and dicarboxylic acid sodiums. In an attempt at improving the performance of surfactants this work provides guidance for choosing additives that form gemini‐like surfactants via an uncomplicated synthesis.

Disaggregation ability of different chelating molecules on copper ion-triggered amyloid fibers.

Dysfunctional interaction of amyloid-β (Aβ) with excess metal ions is proved to be related to the etiology of Alzheimer's disease (AD). Using metal-binding compounds to reverse metal-triggered Aβ aggregation has become one of the potential therapies for AD. In this study, the ability of a carboxylic acid gemini surfactant (SDUC), a widely used metal chelator (EDTA), and an antifungal drug clioquinol (CQ) in reversing the Cu(2+)-triggered Aβ(1-40) fibers have been systematically studied by using turbidity essay, BCA essay, atomic force microscopy, transmission electron microscopy, and isothermal titration microcalorimetry. The results show that the binding affinity of Cu(2+) with CQ, SDUC, and EDTA is in the order of CQ > EDTA > SDUC, while the disaggregation ability to Cu(2+)-triggered Aβ(1-40) fibers is in the order of CQ > SDUC > EDTA. Therefore, the disaggregation ability of chelators to the Aβ(1-40) fibers does not only depend on the binding affinity of the chelators with Cu(2+). Strong self-assembly ability of SDUC and π-π interaction of the conjugate group of CQ also contributes toward the disaggregation of the Cu(2+)-triggered Aβ(1-40) fibers and result in the formation of mixed small aggregates.

Micellization of anionic gemini surfactants and their interaction with polyacrylamide

A series of anionic gemini surfactants have been synthesized. The surface properties and micellization process of as-prepared sulfonate gemini surfactants (SGS) and carboxylate gemini surfactant (CGS) have been studied by surface tension measurement and isothermal titration microcalorimetry. Meanwhile, the interaction of these five surfactants with polyacrylamide (PAM) was investigated using surface tension, steady-state fluorescence measurement, and isothermal titration microcalorimetry. The results show that the critical micelle concentrations (CMCs) of above-mentioned surfactants are more than 1 order of magnitude lower than those of corresponding single chain surfactants. Moreover, the enthalpy of micelle formation (ΔH mic) for the investigated gemini surfactants is negative. In the surfactant–PAM systems, the thermodynamic parameters of binding have also been determined. The conclusion may be drawn that the binding strength of SGS onto PAM is stronger than that of CGS, resulting from more compact structure of SGS aggregates. With increasing surfactant hydrophobicity, the values of ΔH agg become more exothermic and a ΔS agg decrease was observed. Therefore, the interaction between SGS and PAM is enthalpy-driven.

Surfactant selection principle for reducing critical micelle concentration in mixtures of oppositely charged gemini surfactants.

Cationic quaternary ammonium gemini surfactants C(n)H(2n+1)(CH3)2N(+)CH2CHCHCH2(CH3)2N(+)C(n)H(2n+1)2Br(-) (C(n)C4C(n), n = 12, 8, 6) with alkyl spacers, C(n)H(2n+1)(CH3)2N(+)CH2CHOHCHOHCH2(CH3)2N(+)C(n)H(2n+1)2Br(-) (C(n)C4(OH)2C(n), n = 12, 8, 6, 4) with two hydroxyl groups in alkyl spacers, and cationic ammonium single-chain surfactants C(n)H(2n+1)(CH3)2N(+)Br(-) (C(n)TAB, n = 12, 8, 6) have been chosen to fabricate oppositely charged surfactant mixtures with anionic sulfonate gemini surfactant C12H25N(CH2CH2CH2SO3(-))CH2CH2CH2(CH3)2N(CH2CH2CH2SO3(-))C12H252Na (C12C3C12(SO3)2). Surface tension, electrical conductivity, and isothermal titration microcalorimetry (ITC) were used to study their surface properties, aggregation behaviors, and intermolecular interactions. The mixtures of C12C3C12(SO3)2/C(n)C4(OH)2C(n) (n = 12, 8) and C12C3C12(SO3)2/C12C4C12 show anomalous larger critical micelle concentration (CMC) than C12C3C12(SO3)2, while the mixtures of C12C3C12(SO3)2/C(n)C4(OH)2C(n) (n = 6, 4), C12C3C12(SO3)2/C(n)C4(OH)2C(n) (n = 6, 4), and C12C3C12(SO3)2/C(n)TAB (n = 12, 8, 6) exhibit much lower CMC than C12C3C12(SO3)2. The results indicate that strong hydrophobic interactions between the alkyl chains assisted by strong electrostatic attractions between the headgroups and hydrogen bonds between the spacers lead to the formation of less surface active premicellar aggregates in bulk solution, resulting in the increase of CMC. If these interactions are weakened or inhibited, less surface active premicellar aggregates are no longer formed in the mixtures, and thus the CMC values are reduced. The work reveals that the combination of two surfactants with great self-assembling ability separately may have strong intermolecular binding interactions; however, their mixtures do not always generate superior synergism properties. Only moderate intermolecular interaction can generate the strongest synergism in CMC reduction.

Electrochemical detection of the 2-isobutyl-3-methoxypyrazine model odorant based on odorant-binding proteins: The proof of concept

We developed an electrochemical assay for the detection of odorant molecules based on a rat odorant-binding protein (rOBP3). We demonstrated that rOBP3 cavity binds 2-methyl-1,4-naphtoquinone (MNQ), an electrochemical probe, as depicted from the decrease of its electrochemical signal, and deduced the dissociation constant, KdMNQ=0.5(±0.2)μM. The amount of MNQ displaced from rOBP3 by 2-isobutyl-3-methoxypyrazine (IBMP), a model odorant molecule, was measured using square-wave voltammetry. The release of MNQ by competition led to an increase of the electrochemical response. In addition, this method allowed determination of the dissociation constant of rOBP3 for IBMP, KdIBMP=0.5(±0.1)μM. A negative control was performed with a non-binding species, caffeic acid (CA). The determined binding affinity values were confirmed using a fluorescent competitive binding assay and isothermal titration microcalorimetry. This electrochemical assay opens the way for designing robust, reliable and inexpensive odorant biosensors.

A thermochemical study of thiol collector surface reactions on galena

The mechanism whereby thiol collectors adsorb onto a mineral surface has been widely studied. Most of these studies examine the mineral surface at equilibrium state and, often, well above monolayer coverage. However, the formation of species at the mineral surface is a complex process that is both kinetically and thermodynamically controlled. This study uses isothermal titration microcalorimetry to measure the heat evolved during the reaction of a variety of different thiol collectors and collector mixtures with a galena mineral surface.It was observed that, for ethyl xanthate (SEX) and isobutyl xanthate (SIBX), there are sequential reactions occurring at the mineral surface at sub-monolayer coverages. A proposal is put forward for the reactions based on literature and the microcalorimetric, dissolution and pH data from this investigation. DTC (isobutyl dithiocarbamate) had very similar heats of reaction to the xanthates. The major difference was that there was no secondary reaction noticed in the case of DTC. This led to the postulation that the reaction mechanism was different in the case of DTC, with DTC reacting directly with the galena surface. DTP (diethyl dithiophosphate) showed no reaction with the galena surface at all and this was confirmed with adsorption isotherm studies. Enthalpies of reaction of mixtures of SEX with DTP were an additive effect of the enthalpies of each of the pure collectors, meaning that addition of DTP lowered the enthalpy of the SEX reaction proportionally to the amount of DTP added. There was no evidence of a synergistic reaction occurring between the xanthate and the DTP as suggested by previous flotation experiments. This suggests that there is an alternative reaction mechanism occurring to explain improved flotation performance when using SEX-DTP mixtures with galena.

Self-assembled structures of anionic hydrophobically modified polyacrylamide with star-shaped trimeric and hexameric quaternary ammonium surfactants.

The self-assembly of a 1% hydrophobically modified and 30% hydrolyzed polyacrylamide (C12PAM) with cationic star-shaped oligomeric surfactants has been investigated by isothermal titration microcalorimetry, turbidimetry, ζ potential, scanning electron microscopy, and (1)H NMR techniques. The oligomeric surfactants are composed of quaternary dodecyldimethylammonium ions with three or six hydrophobic chains connected by a polyamine spacer at the headgroup level, abbreviated as DTAD and PAHB, respectively. DTAD/C12PAM and PAHB/C12PAM mixed systems undergo the same aggregate transitions with increases in surfactant concentration from soluble networklike aggregates to precipitated denser and more cross-linked structures and then to soluble spherical aggregates. The networklike aggregates are generated at very low surfactant concentration. However, at the corresponding surfactant concentration without C12PAM, DTAD cannot form aggregates and PAHB forms only networklike aggregates with a very loose structure. The strong electrostatic and hydrophobic interaction of DTAD and PAHB with C12PAM and the hydrophobic interaction between the alkyl chains of DTAD and PAHB themselves evidently promote the formation of networklike aggregates. As the surfactant concentration increases, cationic surfactants become excessive. The molecular configuration is changed by the stronger hydrophobic association among the DTAD and PAHB molecules and the enhanced electrostatic repulsion between the mixed aggregates. Thus, the networklike aggregates transfer to spherical aggregates.

Complexation of β-cyclodextrin with a gemini surfactant studied by isothermal titration microcalorimetry and surface tensiometry.

We report on the inclusion complex formation of β-cyclodextrin (βCD) with a cocogem surfactant (counterion-coupled gemini surfactant; (bis(4-(2-alkyl)benzenesulfonate)-Jeffamine salt, abbreviated as ABSJ), studied by isothermal titration calorimetry (ITC) and surface tension (SFT) measurements. We measured the critical micelle concentration (cmc) of ABSJ in water by the two experimental techniques in the temperature range 283-343 K, and determined the thermodynamic parameters of the complex formation directly by ITC and indirectly by the SFT. The stoichiometry (N), the binding constant (K), and the enthalpy of complexation were determined, and the Gibbs free energy and the entropy term were calculated from the experimental data. A novel method is presented for the determination of N and K by using surface tensiometry.

Ag I ‐Directed Triple‐Stranded Helicates with meta ‐Ethynylpyridine Ligands

A series of triple-stranded complexes, [1a3Ag]BF4, [1b3Ag]BF4, [2a3Ag2](BF4)2, [2b3Ag2](BF4)2, and [33Ag3](BF4)3, have been obtained through the self-assembly of AgI ions with p-phenylene ethynylpyridine ligands. X-ray crystallographic studies unambiguously demonstrated a AgI-directed monodentate triple-stranded helical structure in the solid state for 1b and 2b. A combination of NMR spectroscopy, HRMS (ESI), and isothermal titration microcalorimetry studies further confirmed that strands 1, 2, and 3 are also able to form triple-stranded helical structures in the presence of AgI ions in solution.

Unique DC-SIGN clustering activity of a small glycomimetic: A lesson for ligand design.

DC-SIGN is a dendritic cell-specific C-type lectin receptor that recognizes highly glycosylated ligands expressed on the surface of various pathogens. This receptor plays an important role in the early stages of many viral infections, including HIV, which makes it an interesting therapeutic target. Glycomimetic compounds are good drug candidates for DC-SIGN inhibition due to their high solubility, resistance to glycosidases, and nontoxicity. We studied the structural properties of the interaction of the tetrameric DC-SIGN extracellular domain (ECD), with two glycomimetic antagonists, a pseudomannobioside (1) and a linear pseudomannotrioside (2). Though the inhibitory potency of 2, as measured by SPR competition experiments, was 1 order of magnitude higher than that of 1, crystal structures of the complexes within the DC-SIGN carbohydrate recognition domain showed the same binding mode for both compounds. Moreover, when conjugated to multivalent scaffolds, the inhibitory potencies of these compounds became uniform. Combining isothermal titration microcalorimetry, analytical ultracentrifugation, and dynamic light scattering techniques to study DC-SIGN ECD interaction with these glycomimetics revealed that 2 is able, without any multivalent presentation, to cluster DC-SIGN tetramers leading to an artificially overestimated inhibitory potency. The use of multivalent scaffolds presenting 1 or 2 in HIV trans-infection inhibition assay confirms the loss of potency of 2 upon conjugation and the equal efficacy of chemically simpler compound 1. This study documents a unique case where, among two active compounds chemically derived, the compound with the lower apparent activity is the optimal lead for further drug development.

Thermodynamics of complexes formation by ITC in methanol/water=9/1 (v/v) solution: A case study

Abstract Most enzymes that participate in the biochemistry of nucleic acids require divalent metal ion cofactors to promote activity. Development of potent inhibitors, acting against those viral enzymes operating via a cooperative two-metal ion mechanism, such as HIV integrase (IN) and RNase H, hepatitis C virus polymerase and influenza endonuclease, requires optimizing the binding affinity to the target, which is dictated by the binding free energy composed of both enthalpic and entropic contributions. They can be obtained by using isothermal titration microcalorimetry. We have defined an experimental procedure for obtaining reliable thermodynamic data in methanol/water = 9/1 0.1 M KCl as solvent, used to overcome solubility problems. In this way we have measured the heats of formation of the complexes formed by N-(4-fluorobenzyl)-5-hydroxy-2-isopropyl-1-methyl-6-oxo-1,6-dihydroxypyrimidine-4-carboxylate (HL, a model of Raltegravir, the antiretroviral drug produced by Merck & Co.), and a series of divalent metal ions of biological interest (Mg(II), Mn(II), Co(II) and Zn(II)), whose speciation was previously determined by potentiometry.

Phosphorus-containing cyclodextrin polymers: metal cations and hydroxyapatite affinities

The aim of this study was to highlight the properties of novel phosphorus-containing β-cyclodextrin polymers (CDP) and mainly their dual complexing abilities toward hydrophobic guests and (bio)minerals. Affinity of CDP toward divalent metal cations, calcium, magnesium and zinc, has been investigated by isothermal titration microcalorimetry (ITC). The complexation constants K were around 1.1–6.2 × 104 L mol−1 and were in the order Ca2+ < Mg2+ < Zn2+. The K regular increase with the CDP molecular weights could be attributed to a cooperative binding of the cations by the monophosphate groups carried by the CDPs. Regarding their CD and phosphorus functionalities, the dual complexing abilities toward amphiphilic guests and divalent cations can occur independently as evidenced from ITC experiments performed in presence of both species. Finally, strong interactions between CDPs and hydroxyapatite have been highlighted by X-ray photoelectron spectrometry with adsorbed amount in the range of 2 mg/m2. CDPs are thus promising materials endowed with dual functionalities which could serve in biomaterials to simultaneously entrap bioactive molecules and capture (bio)minerals.

Synthesis of a new dextran-PEG-β-cyclodextrin host polymer using “Click” chemistry

The objective of this work was to synthesize a new type of dextran-based host polymer in which β-cyclodextrin (βCD) moieties are linked to the dextran backbone via flexible hydrophilic poly (ethylene glycol) (PEG) spacer. Short PEG-acrylate (Mn~375) was coupled with 5-hexynoic acid and grafted to the thiolated dextran using thiol-ene “click” chemistry. In the following step 6-monodeoxy-6-monoazido-βCD was “clicked” to the obtained alkyne terminated Dext-g-PEG copolymer (Dext-g-PEG-Hex-5-ynoate) using a copper (I)-catalyzed azide–alkyne cycloaddition. The reaction conditions and catalytic system were optimized. The synthesized Dext-g-PEG-βCD polymers have been characterized by 1H-NMR spectroscopy and isothermal titration microcalorimetry and they show good binding properties comparing with conventional cross-linked pβCD.

Interactions of cationic trimeric, gemini and monomeric surfactants with trianionic curcumin in aqueous solution

Interactions of trianionic curcumin (Cur(3-)) with a series of cationic surfactants, monomeric surfactant dodecyl trimethylammonium bromide (DTAB), dimeric surfactant hexamethylene-1,6-bis(dodecyldimethylammonium bromide) (12-6-12) and trimeric surfactant tri(dodecyldimethylammonioacetoxy)diethyltriamine trichloride (DTAD), have been investigated in aqueous solution of pH 13.0. Surface tension and spectral measurements indicate that the cationic surfactants display a similar surfactant concentration dependent interaction process with Cur(3-), involving three interaction stages. At first the three cationic surfactants electrostatically bind on Cur(3-) to form the surfactant-Cur(3-) complex. Then the bound and unbound cationic surfactants with Cur(3-) aggregate into surfactant-Cur(3-) mixed micelles through hydrophobic interactions above the critical micelle concentration of the surfactants (CMCC) in the presence of Cur(3-). Finally excess unbound surfactants self-assemble into micelles like those without Cur(3-). For all the three surfactants, the addition of Cur(3-) only decreases the critical micelle concentration of 12-6-12 but does not affect the critical micelle concentration of DTAB and DTAD. As the oligomeric degree of surfactants increases, the intermolecular interaction of the cationic surfactants with Cur(3-) increases and the surfactant amount needed for Cur(3-) encapsulation decreases. Compared with 12-6-12, either the weaker interaction of DTAB with Cur(3-) or stronger interaction of DTAD with Cur(3-) limits the stability or solubility of Cur(3-) in surfactant micelles. Therefore, gemini surfactant 12-6-12 is the best choice to effectively suppress Cur(3-) degradation at very low concentrations. Isothermal titration microcalorimetry, surface tension and (1)H NMR results reveal that 12-6-12 and Cur(3-) form a (12-6-12)2-Cur(3-) complex and start to form micelles at extremely decreased concentrations, where either 12-6-12 or Cur(3-) works as a bridge linkage and the resultant structure exhibits the characteristics of oligomeric surfactants.

Effects of Branched-Chain Alcohols on Surface Activity and Micellization of Gemini Surfactants

Gemini surfactants are a kind of novel and efficient surfactants and alcohols are one of the most widely used additives to surfactant products in industries and daily life. Understanding the effects of alcohols on surface activities and micellization of gemini surfactants will promote the applications of gemini surfactants. Effects of linear-chain alcohol (1-pentanol) and branched-chain alcohols (2-hexanol and 3-heptanol) with the same main chain as 1-pentanol on the surface activity and micellization of cationic ammonium gemini surfactants C12CSC12Br2 (S=2, 4, 6, 8, 10, 12) in aqueous solution have been investigated by surface tension, electrical conductivity, isothermal titration microcalorimetry (ITC), cryogenic transmission electron microscopy (Cryo-TEM), and NMR techniques. The branched-chain alcohols have not shown obvious effects on the critical micelle concentration of surfactants (CMC), the size and the morphology of micelles due to loose microstructure and weak interactions between the alcohols and the gemini surfactant molecules. However, the addition of branched-chain alcohols greatly decreases the surface tension of surfactants and the surface tension decreases with the increase of the branching factor of the alcohols. In addition, the surfactant concentration required to reduce the surface tension of the solvent by 20 mN/m (C20) and the surface tension values of the surfactants at CMC (γCMC) decrease obviously with the increment of the branching factor of the alcohols at a fixed alcohol concentration due to the increase of the hydrophobic chain density at the air/solution interface. The results suggest that the branched-chain alcohols influence the self-assembly of surfactants more obviously at the air/solution interface than in micelles. Moreover, there is a second critical concentration in the surface tension curves of C12C10C12Br2 and C12C12C12Br2 with a longer spacer, indicating that the hydrophobic chains of these gemini surfactants are not packed tightly even above CMC due to the flexibility of their spacers and low CMC, and are packed more tightly with the increase of the surfactant concentration. The addition of alcohols decreases the second critical concentration remarkably with the increase of their branching factor, which means that the branched alcohols have a more obvious effect on the surface activity of gemini surfactants with a longer spacer. This work helps us to understand the effects of branched-chain alcohols on the self-assembly of gemini surfactants at air/solution interface and in bulk solution, and may provide some guidance on how to choose alcohols to adjust surface activities and micellization of gemini surfactants.

Fructose 1-phosphate is the one and only physiological effector of the Cra (FruR) regulator of Pseudomonas putida

Fructose-1-phosphate (F1P) is the preferred effector of the catabolite repressor/activator (Cra) protein of the soil bacterium Pseudomonas putida but its ability to bind other metabolic intermediates in vivo is unclear. The Cra protein of this microorganism (CraPP) was submitted to mobility shift assays with target DNA sequences (the PfruB promoter) and candidate effectors fructose-1,6-bisphosphate (FBP), glucose 6-phosphate (G6P), and fructose-6-phosphate (F6P). 1mM F1P was sufficient to release most of the Cra protein from its operators but more than 10mM of FBP or G6P was required to free the same complex. However, isothermal titration microcalorimetry failed to expose any specific interaction between CraPP and FBP or G6P. To solve this paradox, transcriptional activity of a PfruB-lacZ fusion was measured in wild-type and ΔfruB cells growing on substrates that change the intracellular concentrations of F1P and FBP. The data indicated that PfruB activity was stimulated by fructose but not by glucose or succinate. This suggested that CraPP represses expression in vivo of the cognate fruBKA operon in a fashion dependent just on F1P, ruling out any other physiological effector. Molecular docking and dynamic simulations of the Cra-agonist interaction indicated that both metabolites can bind the repressor, but the breach in the relative affinity of CraPP for F1P vs FBP is three orders of magnitude larger than the equivalent distance in the Escherichia coli protein. This assigns the Cra protein of P. putida the sole role of transducing the presence of fructose in the medium into a variety of direct and indirect physiological responses.

Multi‐Colored Fibers by Self‐Assembly of DNA, Histone Proteins, and Cationic Conjugated Polymers

AbstractThe development of biomolecular fiber materials with imaging ability has become more and more useful for biological applications. In this work, cationic conjugated polymers (CCPs) were used to construct inherent fluorescent microfibers with natural biological macromolecules (DNA and histone proteins) through the interfacial polyelectrolyte complexation (IPC) procedure. Isothermal titration microcalorimetry results show that the driving forces for fiber formation are electrostatic and hydrophobic interactions, as well as the release of counterions and bound water molecules. Color‐encoded IPC fibers were also obtained based on the co‐assembly of DNA, histone proteins, and blue‐, green‐, or red‐ (RGB‐) emissive CCPs by tuning the fluorescence resonance energy‐transfer among the CCPs at a single excitation wavelength. The fibers could encapsulate GFP‐coded Escherichia coli BL21, and the expression of GFP proteins was successfully regulated by the external environment of the fibers. These multi‐colored fibers show a great potential in biomedical applications, such as biosensor, delivery, and release of biological molecules and tissue engineering.