Free Ligand In Solution Research Articles

Computational Analysis of Heat Capacity Effects in Protein-Ligand Binding.

Heat capacity effects in protein-ligand binding as measured by calorimetric experiments have recently attracted considerable attention, particularly in the field of enzyme inhibitor design. A significant negative heat capacity change upon ligand binding implies a marked temperature dependence of the binding enthalpy, which is of high relevance for attempts to optimize protein-ligand interactions. In this work, we address the question of how well such heat capacity changes can be predicted by computer simulations. We examine a series of human thrombin inhibitors that all bind with ΔCp values of about -0.4 kcal/mol/K and calculate heat capacity changes from plain molecular dynamics simulations of the bound and free states of the enzyme and ligand. The results show that accurate ΔCp estimates within a few tenths of a kcal/mol/K of the experimental values can be obtained with this approach. This allows us to address the structural and energetic origin of the negative heat capacity changes for the thrombin inhibitors, and it is found that conformational equilibria of the free ligands in solution make a major contribution to the observed effect.

Understanding the Buoy Effect of Surface-Enriched Pt Complexes in Ionic Liquids: A combined ARXPS and Pendant Drop Study.

Recently, we demonstrated that Pt catalyst complexes dissolved in the ionic liquid (IL) [C4C1Im][PF6] can be deliberately enriched at the IL surface by introducing perfluorinated substituents, which act like buoys dragging the metal complex towards the surface. Herein, we extend our angle-resolved X-ray photoelectron spectroscopy (ARXPS) studies at complex concentrations between 30 and 5%mol down to 1%mol and present complementary surface tension pendant drop (PD) measurements under ultra clean vacuum conditions. This combination allows for connec-ting the microscopic information on the IL/gas interface from ARXPS with the macroscopic property surface tension. The surface enrichment of the Pt complexes is found to be most pronounced at 1%mol. It also displays a strong temperature dependence, which was not observed for 5%mol and above, where the surface is already saturated with the complex. The surface enrichment deduced from ARXPS is also reflected by the pronounced decrease in surface tension with increasing concentration of the catalyst. We furthermore observe by ARXPS and PD a much stronger surface affinity of the buoy-complex as compared to the free ligands in solution. Our results are highly interesting for an optimum design of ionic catalyst solutions contact areas with a surrounding reactant/product phase, such as in SILP catalysis.

Aqueous Speciation of Tetravalent Actinides in the Presence of Chloride and Nitrate Ligands.

Speciation of hexachloride tetravalent uranium, neptunium, and plutonium species in aqueous media has been investigated using density functional theory in the presence of inner sphere ligands such as chloride, nitrate, and solvent molecules. All possible structures with the formula [AnIV(Cl)x(H2O)y(NO3)z]4-x-z (An = U, Np, and Pu; x = 0-6, y = 0-8, and z = 0-6) were considered to explore the speciation chemical space of each actinide. The nature of the mixed-ligand complexes present in solution is controlled by the concentration of free ligands in solution. A low chloride concentration is suitable to drive the speciation away from the highly thermodynamically stable hexachloride species. Furthermore, the formation of dimeric species can proceed through both olation and oxolation mechanisms. Oxolation is preferred for monomers that contain fewer water ligands, while olation becomes favorable for complexes with more water ligands.

Halogen Atoms in the Protein-Ligand System. Structural and Thermodynamic Studies of the Binding of Bromobenzotriazoles by the Catalytic Subunit of Human Protein Kinase CK2.

Binding of a family of brominated benzotriazoles to the catalytic subunit of human protein kinase CK2 (hCK2α) was used as a model system to assess the contribution of halogen bonding to protein–ligand interaction. CK2 is a constitutively active pleiotropic serine/threonine protein kinase that belongs to the CMGC group of eukaryotic protein kinases (EPKs). Due to the addiction of some cancer cells, CK2 is an attractive and well-characterized drug target. Halogenated benzotriazoles act as ATP-competitive inhibitors with unexpectedly good selectivity for CK2 over other EPKs. We have characterized the interaction of bromobenzotriazoles with hCK2α by X-ray crystallography, low-volume differential scanning fluorimetry, and isothermal titration calorimetry. Properties of free ligands in solution were additionally characterized by volumetric and RT-HPLC measurements. Thermodynamic data indicate that the affinity increases with bromo substitution, with greater contributions from 5- and 6-substituents than 4- and 7-substituents. Except for 4,7-disubstituted compounds, the bromobenzotriazoles adopt a canonical pose with the triazole close to lysine 68, which precludes halogen bonding. More highly substituted benzotriazoles adopt many additional noncanonical poses, presumably driven by a large hydrophobic contribution to binding. Some noncanonical ligand orientations allow the formation of halogen bonds with the hinge region. Consistent with a predominantly hydrophobic interaction, the isobaric heat capacity decreases upon ligand binding, the more so the higher the substitution.

Effects of Osmolytes on Ligand Binding to Dihydropteroate Synthase from Bacillus anthracis.

Osmolyte interactions with ligands can affect their affinity for proteins and are dependent upon the cosolute and the functional groups of the ligand. Here, we explored ligand binding to Bacillus anthracis dihydropteroate synthase (BaDHPS) under osmotic stress conditions. Osmolyte effects were specific to the cosolute and ligand, suggesting interaction of the osmolytes with the free ligands in solution. The association rates of pterin pyrophosphate were mostly unaffected by the osmolytes, except for a 2-fold decrease in the presence of 1 M trehalose, while the dissociation rates decreased in most osmolyte solutions. The viscosity and dielectric constant of the solution did not correlate with the effects of the osmolytes. Experimental results were compared with predicted preferential interaction coefficients (Δμ23/RT) between the osmolytes and ligands. The Δμ23/RT were able to predict the experimental data for most of the osmolytes. Trehalose and proline effects did not correlate with the predicted values, indicating that these two osmolytes may affect binding in more complex ways than simple preferential interactions. Additionally, osmolytes weakly interacted with the sulfa drug sulfathiazole, which altered its affinity for BaDHPS, suggesting that these types of weak interactions can also impact drug binding. As osmolytes affect ligands binding to two different folate cycle enzymes (DHFRs and DHPS), we predicted how ligand binding to other folate cycle enzymes will be altered by the presence of osmolytes.

ConfID: an analytical method for conformational characterization of small molecules using molecular dynamics trajectories.

The conformational space of small molecules can be vast and difficult to assess. Molecular dynamics (MD) simulations of free ligands in solution have been applied to predict conformational populations, but their characterization is often based on clustering algorithms or manual efforts. Here, we introduce ConfID, an analytical tool for conformational characterization of small molecules using MD trajectories. The evolution of conformational sampling and population frequencies throughout trajectories is calculated to check for sampling convergence while allowing to map relevant conformational transitions. The tool is designed to track conformational transition events and calculate time-dependent properties for each conformational population detected. Toolkit and documentation are freely available at http://sbcb.inf.ufrgs.br/confid. marcelo.poleto@ufv.br or bigrisci@inf.ufrgs.br. Supplementary data are available at Bioinformatics online.

Raman Tweezers Microspectroscopy of Functionalized 4.2 nm Diameter CdSe Nanocrystals in Water Reveals Changed Ligand Vibrational Modes by a Metal Cation

We demonstrated the possibility of acquiring Raman spectra of colloidal quantum dots (QDs) at low concentration in water with a size as small as 2.5 nm in diameter using Raman tweezers microspectroscopy. We measured the spectra of CdSe QDs capped with thioglycerol and with l-cysteine. This technique was applied to probe the interaction between Co2+ and Cys–CdSe QDs whose fluorescence emission is quenched in the presence of this metal cation. The quenching mechanism was so far hypothetical. The Raman spectra of Cys–CdSe QDs recorded in the absence and in the presence of Co2+ demonstrated the binding of Co2+ cations to the carboxylate groups of the l-cysteine ligand grafted on the surface of the 4.2 nm CdSe QDs. The frequency of modes for the grafted ligand is changed with respect to the free ligand in solution. Considering the vibrational coupling between the excitonic state and the ligand, we inferred that the binding of a metal cation to the grafted ligand modifies this coupling, so that exciton relaxati...

Selective hydrogenation of cinnamaldehyde by unsupported and few layer graphene supported platinum concave nanocubes exposing {110} facets stabilized by a long-chain amine

Free and supported platinum concave nanocubes exposing {110} facets have been prepared by a wet-chemistry route and used as catalysts for the selective hydrogenation of cinnamaldehyde. The nanocubes were directly grown on few layer graphene support in the presence of octadecylamine as a stabilizing agent. Immobilization improves both activity and selectivity towards the desired unsaturated alcohol. The presence of the organic stabilizer guarantees the stability of the nanoparticle morphology and influences the catalyst performances. While the activity is favored by small nanoparticles and low ligand contents, the selectivity to the unsaturated alcohol is favored by high ligand contents, most likely due to steric effects that favor adsorption through the C=O moiety. Apart from stabilizing the nanocubes, the support interacts with the long chain amine ligand, behaving as a ligand reservoir. Thus, an equilibrium is established between the ligand interacting with the nanoparticles, the free ligand in solution, and the ligand interacting with the support. Ligand redistribution during the catalytic reaction assures a good compromise between activity and selectivity even after three recycling tests. While a direct comparison with other catalysts is not straightforward, the FLG-supported concave nanocubes presented outstanding activity, and selectivity to cinnamyl alcohol higher than 80%.

Kinetics of Cationic-Ligand-Exchange Reactions in Au25 Nanoclusters

Herein, we report the first investigation into the kinetics of cationic-ligand-exchange processes through the reaction of Au25(SCH2CH2Ph)18 with a cationic thiol HS(CH2)11N(CH3)3+, which resulted in different populations of the two thiolate ligands (SR0 and SR+), that is, Au25(SR0)18–x(SR+)x. The ligand-exchange process was monitored by electrospray ionization mass spectrometry, and the reaction kinetics is discussed in combination with 1H NMR results; these techniques reveal that the kinetics of the cationic-ligand-exchange process, which is different from a typical neutral-thiol-to-neutral-thiol ligand exchange, is strongly dependent on how the SR+ ligands interact with each other during the ligand-exchange process. There are two main factors that determine the unique reaction kinetics, namely, Coulombic repulsions (i) between the attached SR+ ligands and free ligands in solution, which hinder further ligand exchange, and (ii) among the surface SR+ ligands on the thiolate monolayer, which promote the th...

Investigating the Formation of Giant {Pd72}Prop and {Pd84}Gly Macrocycles Using NMR, HPLC, and Mass Spectrometry.

The formation of giant polyoxometalate (POM) species is relatively underexplored, as their self-assembly process is complex due to the rapid kinetics. Polyoxopalladates (POPds) are a class of POMs based on Pd, the largest of which is the {Pd84}Ac wheel, and its slower kinetics mean the system is more amenable to systematic study. Here, we show that it is possible to follow the assembly of two types of Pd wheels, {Pd84}Gly and the smaller {Pd72}Prop, formed using glycolate and propionate ligands, respectively. We analyzed the formation of {Pd72}Prop and {Pd84}Gly using mass spectrometry (SEC-HPLC-MS and preparative desalting followed by MS). This was accompanied by studies that followed the chemical shift differences between the outer/inner ligands and the free ligand in solution for the {Pd84}Ac, {Pd72}Prop, and {Pd84}Gly species using NMR, which showed it was possible to track the formation of the wheels. Our findings confirm that the macrocycles assemble from smaller building blocks that react together to form the larger species over a period of days. These findings open the way for further structural derivatives and exploration of their host-guest chemistry.

Effects of electrostatic interactions on ligand dissociation kinetics.

We study unbinding of multivalent cationic ligands from oppositely charged polymeric binding sites sparsely grafted on a flat neutral substrate. Our molecular dynamics simulations are suggested by single-molecule studies of protein-DNA interactions. We consider univalent salt concentrations spanning roughly a 1000-fold range, together with various concentrations of excess ligands in solution. To reveal the ionic effects on unbinding kinetics of spontaneous and facilitated dissociation mechanisms, we treat electrostatic interactions both at a Debye-Hückel (DH) (or implicit ions, i.e., use of an electrostatic potential with a prescribed decay length) level and by the more precise approach of considering all ionic species explicitly in the simulations. We find that the DH approach systematically overestimates unbinding rates, relative to the calculations where all ion pairs are present explicitly in solution, although many aspects of the two types of calculation are qualitatively similar. For facilitated dissociation (FD) (acceleration of unbinding by free ligands in solution) explicit-ion simulations lead to unbinding at lower free-ligand concentrations. Our simulations predict a variety of FD regimes as a function of free-ligand and ion concentrations; a particularly interesting regime is at intermediate concentrations of ligands where nonelectrostatic binding strength controls FD. We conclude that explicit-ion electrostatic modeling is an essential component to quantitatively tackle problems in molecular ligand dissociation, including nucleic-acid-binding proteins.

Complexation of Ni(II) by Dimethylglyoxime for Rapid Removal and Monitoring of Ni(II) in Water

The complexation of Ni(II) with dimethylglyoxime (DMG) entrapped within a Nafion membrane and a DMG–sol–gel matrix was studied and compared for different solutions. First and pseudo-second order kinetic models, Elovich, intra-particle, and liquid film diffusion models were applied to evaluate sorption kinetics. Complexation of Ni(II) by DMG entrapped in the polymeric materials followed a pseudo-second order kinetic model; moreover, DMG in Nafion also allowed diffusion-controlled uptake. The pseudo-second order rate constant was significantly higher for the free ligand in solution than for Ni(II) accumulation in the surface-attached DMG-Nafion. The DMG–sol–gel removal ability of Ni(II) was tested using actual mine water. The presence of interferences only insignificantly decreased the removal percentage of Ni(II), thus confirming the high selectivity of DMG towards Ni(II). Also, an electrochemical sensor modified with DMG in Nafion was investigated further for direct electrochemical determination of Ni(II) in untreated mine water. Determination errors and interference effects were low. Thus, this approach represents an effective potential solution for selective Ni(II) removal from mine water as well as a rapid and cheap sensor for on-site monitoring of Ni(II) in mine and environmental waters.

Chiral supramolecular nanoparticles: The study of chiral surface modification of silver nanoparticles by cysteine and its derivatives

Many studies dealing with modified nanoparticles are based on the assumption that organic molecules form single-layer structure on their surface. In this work, we suggest a novel approach to study the important phenomenon of supramolecular chirality at metal nanoparticle surfaces. Self-assembly layers of several ligands, namely l-cysteine, l-penicillamine and α-methyl-l-cysteine, on different silver nanoparticles were studied by means of absorption and electronic circular dichroism spectroscopy in UV–vis range and surface enhanced Raman scattering spectroscopy. The amount of ligands bonded to the surface of nanoparticles was quantified by absorption spectroscopy. Quantification was performed indirectly by measuring concentration of free ligands in solution after incubation with absorption labels 4-toluenesulfonyl chloride or 1-fluoro-2,4-dinitrobenzene. Data were obtained for ligands at concentration levels from 10−6 to 10−3 mol L−1. The results showed the significantly lower adsorbed molecules of methyl derivatives of cysteine than for cysteine themselves. This fact could explain the unexpected behavior of silver nanoparticles that provide ECD bands in the presence of cysteine and not in the presence of its derivatives.

Phototunable Liquid‐Crystalline Phases Made of Nanoparticles

AbstractThe properties of liquid‐crystalline (LC) hybrid systems made of inorganic nanoparticles grafted with photosensitive azo compounds are presented. For materials with a large density of azo ligands at the surface, the LC structure can be reversibly melted by UV light, and the return to the LC state does not require the absorption of visible light. For systems with a lower density of azo ligands, UV light causes shortening of the distance between metal sublayers in the lamellar phase. Interestingly, the azo derivatives attached to the nanoparticle surface show very different kinetics of cis/trans conformational change as compared to the free molecules. The cis form of free ligands in solution is stable for days, whereas the isomerization of molecules attached to the nanoparticle surface to the trans form takes only a few minutes. Apparently, owing to the crowded environment, azo ligands immobilized at a metal surface behave as they would in the condensed state.

Phototunable liquid-crystalline phases made of nanoparticles.

The properties of liquid-crystalline (LC) hybrid systems made of inorganic nanoparticles grafted with photosensitive azo compounds are presented. For materials with a large density of azo ligands at the surface, the LC structure can be reversibly melted by UV light, and the return to the LC state does not require the absorption of visible light. For systems with a lower density of azo ligands, UV light causes shortening of the distance between metal sublayers in the lamellar phase. Interestingly, the azo derivatives attached to the nanoparticle surface show very different kinetics of cis/trans conformational change as compared to the free molecules. The cis form of free ligands in solution is stable for days, whereas the isomerization of molecules attached to the nanoparticle surface to the trans form takes only a few minutes. Apparently, owing to the crowded environment, azo ligands immobilized at a metal surface behave as they would in the condensed state.

Homoleptic and heteroleptic RuII complexes with extended phenanthroline-based ligands

Four RuII complexes of general formula [Ru(PT)3]2+, [Ru(NT)3]2+, [Ru(PT)(bpy)2]2+, and [Ru(NT)(bpy)2]2+, where PT=10,13-bis((triisopropylsilyl)ethynyl)dipyrido[3,2-a:2′,3′-c]phenazine, NT=10,15-bis((triisopropylsilyl)ethynyl)benzo[i]dipyrido[3,2-a:2′,3′-c]phenazine and bpy=2,2′-bipyridine have been synthesized. Their electrochemical and photophysical properties have been studied, with the aid of DFT and TD-DFT theoretical methods. The extended phenanthroline ligand PT exhibits two reduction processes at −0.98 and −1.55 (versus SCE) and no clearly detectable oxidation processes; the more extended analogue NT is substantially easier to reduce by about 300mV. In the four complexes, the two first reduction processes are centered on the extended phenanthroline ligands, and, according to DFT calculations, also oxidations are likely to be located on such moieties. PT and NT exhibit green-yellow fluorescence at 298 and 77K with photoluminescence quantum yields of 3.4% and 62.1%, respectively, at room temperature. At 77K, a strong and long-lived phosphorescence is detected only for PT (λmax=658nm, τ=29ms). In oxygen-free solution at 298K [Ru(PT)3]2+ and [Ru(PT)(bpy)2]2+ exhibit a very weak emission band with λmax at about 700nm, which extends towards the near infrared region and is unambiguously attributed to emission from the lowest triplet level centered on the PT ligand; its emission intensity is strongly enhanced at 77K where lifetimes of 724 and 767ns are measured for [Ru(PT)3]2+ and [Ru(PT)(bpy)2]2+, respectively. The two complexes with the NT ligand show no triplet emission as observed for the PT analogues, but only a faint fluorescence signal attributable to negligible amounts of free ligand in solution. Photophysical data are fully rationalized with DFT methods, which always predict that the lowest triplet excited state is centered on the extended phenanthroline ligand in all of the investigated RuII complexes. The estimated energy of the lowest triplet level of the PT-based compounds is in good agreement with the experimental value determined from the phosphorescence spectra. Moreover, the theoretical model rationalizes the lack of phosphorescence for the NT series, because the lowest triplet is estimated to be at 0.97eV above the ground state (≈1280nm), where non-radiative deactivations prevail.

Measuring ligand–receptor binding kinetics and dynamics using k-space image correlation spectroscopy

Accurate measurements of kinetic rate constants for interacting biomolecules are crucial for understanding the mechanisms underlying intracellular signalling pathways. The magnitude of binding rates plays a very important molecular regulatory role which can lead to very different cellular physiological responses under different conditions. Here, we extend the k-space image correlation spectroscopy (kICS) technique to study the kinetic binding rates of systems wherein: (a) fluorescently labelled, free ligands in solution interact with unlabelled, diffusing receptors in the plasma membrane and (b) systems where labelled, diffusing receptors are allowed to bind/unbind and interconvert between two different diffusing states on the plasma membrane. We develop the necessary mathematical framework for the kICS analysis and demonstrate how to extract the relevant kinetic binding parameters of the underlying molecular system from fluorescence video-microscopy image time-series. Finally, by examining real data for two model experimental systems, we demonstrate how kICS can be a powerful tool to measure molecular transport coefficients and binding kinetics.

Catalytic Mechanism of Cytochrome P450 for 5′-Hydroxylation of Nicotine: Fundamental Reaction Pathways and Stereoselectivity

A series of computational methods were used to study how cytochrome P450 2A6 (CYP2A6) interacts with (S)-(-)-nicotine, demonstrating that the dominant molecular species of (S)-(-)-nicotine in CYP2A6 active site exists in the free base state (with two conformations, SR(t) and SR(c)), despite the fact that the protonated state is dominant for the free ligand in solution. The computational results reveal that the dominant pathway of nicotine metabolism in CYP2A6 is through nicotine free base oxidation. Further, first-principles quantum mechanical/molecular mechanical free energy (QM/MM-FE) calculations were carried out to uncover the detailed reaction pathways for the CYP2A6-catalyzed nicotine 5'-hydroxylation reaction. In the determined CYP2A6-(S)-(-)-nicotine binding structures, the oxygen of Compound I (Cpd I) can abstract a hydrogen from either the trans-5'- or the cis-5'-position of (S)-(-)-nicotine. CYP2A6-catalyzed (S)-(-)-nicotine 5'-hydroxylation consists of two reaction steps, that is, the hydrogen transfer from the 5'-position of (S)-(-)-nicotine to the oxygen of Cpd I (the H-transfer step), followed by the recombination of the (S)-(-)-nicotine moiety with the iron-bound hydroxyl group to generate the 5'-hydroxynicotine product (the O-rebound step). The H-transfer step is rate-determining. The 5'-hydroxylation proceeds mainly with the stereoselective loss of the trans-5'-hydrogen, that is, the 5'-hydrogen trans to the pyridine ring. The calculated overall stereoselectivity of ∼97% favoring the trans-5'-hydroxylation is close to the observed stereoselectivity of 89-94%. This is the first time it has been demonstrated that a CYP substrate exists dominantly in one protonation state (cationic species) in solution, but uses its less-favorable protonation state (neutral free base) to perform the enzymatic reaction.

Coordination properties of axially unfixed chiral dipyridine ligands towards metal and ammonium ions

New chiral dipyridine ligands with an axially unfixed 1,1′-biphenyl bridge were prepared via homocoupling of bromophenyl pyridines. The conformeric ratios of the free ligands in solution and their coordination properties towards metal ions were studied by NMR spectroscopy. X-Ray crystallography of the silver(I) and copper(I) complexes showed 1:1 metal to ligand complexes and S planar chirality. Interestingly, the biphenyl ligands show a 1:2 stepwise binding towards most ammonium ions tested with strong fluorescence enhancement, but a selectively 1:1 binding towards l-ornithine methyl ester hydrochloride with no fluorescence enhancement.

Determination of trace metal speciation parameters by using screen-printed electrodes in stripping chronopotentiometry without deaerating

The objective of this study was to exploit the advantages of stripping chronopotentiometry (SCP) and stripping chronopotentiometry at scanned deposition potential (SSCP) for trace metal speciation analyses by using thin-film mercury screen-printed electrodes (TMF-SPE). At first, the SCP parameters were optimised for TMF-SPE, in order to reach the complete depletion regime. It has been shown that a stripping current higher than or equal to 10 μA allows this regime to be attained without removing oxygen from the solution. Then, these analytical conditions were used for the construction of SSCP curves for Cd-PDCA and Cd-NTA. When the concentration of free ligand in solution was known, the knowledge of the model describing the SSCP curves in absence and presence of a complex and the use of an effective fitting tool enabled estimating the stability constant and the rate constants for complexation. Further studies with complexes of restricted mobility are however necessary to assess the usefulness of this procedure to also estimate the diffusion coefficient of the complexes. Besides, this study showed that this approach was valid even when ligands were not in excess at the electrode during stripping.