Equilibrium studies of a fluorescent tacrolimus binding to surfactant protein A

Tacrolimus (FK506) is a hydrophobic immunosuppressive agent used in kidney, liver, and lung transplantation. The objective of this study was to characterize the binding of FK506 to surfactant protein A (SP-A), an abundant lipoprotein found in the alveolar fluid that functions as part of the innate immune system in the lung. We have synthesized a novel derivative of FK506 in which a dansyl moiety was covalently bound via cadaverine to the C22 position of the FK506 molecule (DNS-FK). Using the fluorescence and anisotropy properties of DNS-FK, we demonstrated that tacrolimus avidly binds to SP-A with an apparent equilibrium association constant (K(app)) of 10(7)M(-1) and a Gibbs binding free energy of -40 kJ mol(-1)K(-1). Derivatization of FK506 at the C22 position did not block FK506 binding to the cytosolic immunophilin FK506-binding protein (FK-BP) or human serum albumin (HSA), both used as controls of tacrolimus-binding proteins. K(app) for FK-BP/DNS-FK and HSA/DNS-FK complexes were 1.5 x 10(7) and 10(7)M(-1), respectively. The high sensitivity of this analytical technique makes it suitable for binding analysis of FK506 to proteins.     

Synthesis and microtubule binding of fluorescent paclitaxel derivatives

The preparation of two new fluorescent derivatives of paclitaxel in which the fluorophore is bonded to paclitaxel at the C-10 position is reported. Both analogues, 10-deacetyl-10-(m-aminobenzoyl)paclitaxel (1, BTax) and 10-deacetyl-10-[7-(diethylamino) coumarin-3-carbonyl]paclitaxel (2, CTax) retain good activity as promoters of in vitro tubulin assembly. Microtubule binding enhances the emission intensity of both probes.     

Equilibrium dialysis studies of polyamine binding to DNA

Binding constants and binding site sizes for the interactions of the polyamines spermine (+4), spermidine (+3), and putrecine (+2) with helical DNA have been determined as a function of ionic conditions and temperature by equilibrium dialysis using ¹⁴C-labeled polyamines. In addition, competition equilibrium dialysis has been used to determine binding parameters for the divalent cations putrescine and Mg²⁺ from the competitive effect of these ions on the binding of spermine or spermidine. In all cases, the logarithm of the binding constant (log K_obs) varies linearly with the logarithm of the monovalent salt concentration; the slopes d log K_obs/d log[NaCl] are proportional to the valence of the ligand, and values of the extrapolated binding constants at 1M NaCl obtained from the intercepts are small (of order 1–10M^?1). In those cases examined, K_obs is insensitive to temperature; the free energy of binding is predominantly entropic. Consequently, polymines as DNA-binding ligands behave analogously to the oligolysìnes investigated previously [cf. Record, Lohman & de Haseth (1976) J. Mol. Biol. 107 , 145–158; Lohman, de Haseth & Record (1980) Biochemistry 19 , 3522–3530]. The interactions of these oligocations with DNA are predominantly electrostatic and are driven by the release of thermodynamically bound electrolyte ions from the vicinity of the DNA. The extent to which these oligocations are localized at individual phosphate binding sites or delocalized on the DNA molecule is currently not known.     

Kinetic studies of recA protein binding to a fluorescent single-stranded polynucleotide

Fluorescence spectroscopy was used to investigate the binding of Escherichia coli recA protein to a single-stranded polynucleotide. Poly(deoxy-1,N6-ethenoadenylic acid) was prepared by reaction of chloroacetaldehyde with poly(deoxyadenylic acid). The fluorescence of poly(deoxy-1,N6-ethenoadenylic acid) was enhanced upon recA protein binding. The kinetics of the binding process were studied as a function of several parameters: ionic concentration (KCl and MgCl2), pH, nature of the nucleoside triphosphate [adenosine 5'-triphosphate or adenosine 5'-O-(gamma-thiotriphosphate)], protein and polynucleotide concentrations, polynucleotide chain length, and order of sequential additions. The observed kinetic curves exhibited a lag phase followed by a slow binding process characteristic of a nucleation-elongation mechanism with an additional slow step governing the rate of the association process. The lag phase reflecting the nucleation step was not observed when the protein was first bound to the polynucleotide before addition of adenosine 5'-triphosphate. Adenosine 5'-triphosphate induced a dissociation of the recA protein, which was immediately followed by binding of the recA-adenosine 5'-triphosphate-Mg2+ ternary complex. The origin of this "mnemonic effect" and of the different kinetic steps is discussed with respect to protein conformational changes and aggregation phenomena.     

Modulation of the binding characteristics of a fluorescent nucleotide derivative to the sarcoplasmic reticulum adenosinetriphosphatase

Trinitrophenyladenosine monophosphate (TNP-AMP) binding to the phosphorylated Ca2+-ATPase of sarcoplasmic reticulum results in manyfold higher fluorescence intensity and longer lifetimes of the nucleotide analogue, as compared to TNP-AMP binding to the nonphosphorylated enzyme. This is observed when the phosphoenzyme intermediate is formed either from ATP or from inorganic phosphate (Pi). An important question is whether the TNP-AMP fluorescence properties can also reflect the kinetically defined interconversions of different phosphoenzyme species during catalysis. We have approached this question by manipulating the phosphorylation conditions in a manner which is known to result in accumulation of different species of the phosphoenzyme, i.e., by variations in pH, substrates, and K+ and Ca2+ concentrations. Decreasing pH or increasing [K+] caused large decreases in fluorescence intensity at a given concentration of TNP-AMP under conditions of phosphorylation with either ATP or Pi. In contrast, low to high intravesicular Ca2+ concentrations had no effect on fluorescence during steady-state turnover. TNP-AMP titrations of the phosphorylated enzyme stabilized in different states revealed that H+ and K+ caused a shift in TNP-AMP binding affinity to the site responsible for high fluorescence enhancement, while maintaining approximately the same maximal fluorescence yield at saturation. The fluorescence lifetimes of TNP-AMP bound to phosphoenzyme did not change with variations in pH, [K+], and substrates. We conclude that the environment of that part of the TNP-AMP binding site which binds the trinitrophenyl moiety undergoes a change upon enzyme phosphorylation resulting in enhanced fluorescence yield; this change is invariant between different phosphoenzyme species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Decoration of microtubules by a colchicine derivative

Pure deacetylcolchicine was conjugated to a heme-nonapeptide having peroxidase-like activity. This colchicine derivative binds to soluble tubulin and is able to depolymerize microtubules in vitro. Cytochemical investigations show that substoichiometric concentrations of the derivative bind to microtubules. The microtubules show different labelling patterns. The decoration of labile and sensitive microtubules by the colchicine derivative indicates a direct action of the drug on these structures.     

Equilibrium dialysis studies of the binding of radioprotector compounds to DNA

Radioprotection by WR-2721, S-2-(3-aminopropylamino)ethyl phosphorothioate, is thought to involve its corresponding thiol (WR-1065) or symmetrical disulfide (WR-33278). It has been suggested that these metabolites concentrate close to the DNA target molecule; to test this hypothesis we have measured their in vitro binding to DNA. The binding of WR-33278 (0.05-0.4 mM) to calf thymus DNA (6 mM, with respect to DNA phosphate) was determined at 50, 100, and 150 mM KCl in 1 mM Tris, pH 7, by equilibrium dialysis. The binding of WR-1065 (0.5-8mM) was determined at 25, 50, and 100 mM KCl, under similar conditions, but with 2 mM EDTA and 3 mM dithiothreitol (DTT) added to the dialysis buffer to prevent thiol oxidation. Drug levels were quantitated by HPLC after fluorescent labeling with monobromobimane; disulfide samples were reduced with DTT prior to analysis. Dissociation constants (Kd = [Free Drug] [DNA site]/ [bound drug] ) under these conditions were found to vary with ionic strength, being in the range of 0.02 +/- 0.01 to 0.18 +/- 0.06 mM for WR-33278 and 0.43 +/- 0.24 to 3.5 +2/- 1.5 mM for WR-1065. WR-2721, glutathione, cysteine, and DTT showed no detectable binding to DNA in 25 mM KCl. However, cysteamine and cystamine did bind to DNA, with unbound drug to bound drug ratios of 8 +/- 2 and 0.6 +/- 0.1, respectively, at total drug concentrations of 1 mM. Cystamine and WR-1065 bound to DNA with comparable affinity under similar conditions. These results indicate that binding of WR-33278 and WR-1065 by DNA phosphate are probably significant in the mechanism of radioprotection by WR-2721.     

Equilibrium studies on neutral red-DNA binding.

Spectrophotometric binding studies were undertaken on the interaction of neutral red with native and heat-denatured, sonicated, calf thymus DNA in a 0.2M ionic strength buffer containing Tris–sodium acetate–potassium chloride at 25°C. The pK_A of neutral red was found to be 6.81. At pH 5 the binding of protonated neutral red was complicated even at low concentration ratios of dye to DNA. In the pH range 7.5–8.5 the tight binding process could be studied and it was found that both protonated and free base species of neutral red significantly bind with DNA having association constants (in terms of polynucleotide phosphate) of 5.99 × 10³ M^?1 and 0.136 × 10³ M^?1, respectively, for native DNA and 7.48 × 10³ M^?1 and 0.938 × 10³ M^?1, respectively, for denatured DNA. The pK_A value of the neutral red–DNA complexes were 8.46 for native DNA and 7.72 for denatured DNA. These results are discussed in terms of possible binding mechanisms.     

Macromolecular accessibility of fluorescent taxoids bound at a paclitaxel binding site in the microtubule surface

The macromolecular accessibility of the paclitaxel binding site in microtubules has been investigated using a fluorescent taxoid and antibodies against fluorescein, which cannot diffuse into the microtubule lumen. The formation of a specific ternary complex of microtubules, Hexaflutax (7-O-{N-[6-(fluorescein-4'-carboxamido)-n-hexanoyl]-l-alanyl}paclitaxel) and 4-4-20 IgG (a monoclonal antibody against fluorescein) has been observed by means of sedimentation and electron microscopy methods. The kinetics of binding of the antibody to microtubule-bound Hexaflutax has been measured. The quenching of the observed fluorescence is fast (k+ 2.26 +/- 0.25 x 10(6) m(-1) s(-1) at 37 degrees C), indicating that the fluorescein groups of Hexaflutax are exposed to the outer solvent. The velocity of the reaction is linearly dependent on the antibody concentration, indicating that a bimolecular reaction is being observed. Another fluorescent taxoid (Flutax-2) bound to microtubules has also been shown to be rapidly accessible to polyclonal antibodies directed against fluorescein. A reduced rate of Hexaflutax quenching by the antibody is observed in microtubule-associated proteins containing microtubules or in native cellular cytoskeletons. It can be concluded that the fluorescent taxoids bind to an outer site on the microtubules that is shared with paclitaxel. Paclitaxel would be internalized in a further step of binding to reach the known luminal site, this step being blocked in the case of the fluorescent taxoids. Because the fluorescent ligands are able to induce microtubule assembly, binding to the outer site should be enough to induce assembly by a preferential binding mechanism.     

Fluorescence studies of the binding of a yeast tRNAPhe derivative to Escherichia coli ribosomes.

The equilibrium binding of a highly fluorescent derivative of yeast tRNA^Phe to Escherichia coli 70 S ribosomes was studied fluorimetrically at 7 °C in 25 mm-magnesium. Under these conditions 70 S ribosomes bind two deacylated tRNAs stoichiometrically. An analysis of the binding data using a model in which occupancy of the weaker site requires prior occupancy of the stronger site leads to apparent association constants of (1.00 ± 0.05) × 10⁹m^?1 and (3.4 ± 0.2) × 10⁷m^?1. The use of an independent site model does not change these values appreciably. The observed binding constants do not depend upon the presence or absence of the messenger RNA, poly(U). However, spectroscopic evidence strongly suggests that the anticodons of both bound tRNAs are in contact with the message. This evidence further suggests that in the presence of poly(U) the environment of the hypermodified base adjacent to the anticodon is substantially different in the two sites. This may reflect a difference in the conformation of the anticodon loops or an interaction between the hypermodified base of the weak site tRNA and the anticodon loop of the strong site tRNA.     

Insights into the binding of paclitaxel to human serum albumin: multispectroscopic studies

The interaction of paclitaxel with human serum albumin (HSA) was studied using fluorescence, resonance light scattering, ultraviolet‐visible, circular dichroism and Fourier transform infrared spectroscopy at pH 7.4. Fluorescence data revealed that the fluorescence quenching of HSA by paclitaxel was a static quenching procedure. Time‐resolved fluorescence data also confirmed the quenching mode, which present a constant decay time of about 5 ns. The binding sites were approximately 1 and the binding constant suggested a weak association (324/M at 298 K), which is helpful for the release of the drug to targeted organs. The thermodynamic parameters, ΔG^○, ΔH° and ΔS° were calculated as – 1.06 × 10⁴ J/mol, 361 J/mol per K and 9.7 × 10⁴ J/mol respectively at 298 K, suggesting that binding was spontaneous and was driven mainly by hydrophobic interactions. The binding distance between HSA and paclitaxel was determined to be 2.23 nm based on the Förster theory. Analysis of circular dichroism, ultraviolet‐visible, three‐dimensional fluorescence, Fourier transform infrared and resonance light scattering spectra demonstrated that HSA conformation was slightly altered in the presence of paclitaxel and dimension of the individual HSA molecules were larger after interacting with paclitaxel. These results were confirmed by a molecular docking study. Copyright © 2013 John Wiley & Sons, Ltd.     

Electrostatically biased binding of kinesin to microtubules

The minimum motor domain of kinesin-1 is a single head. Recent evidence suggests that such minimal motor domains generate force by a biased binding mechanism, in which they preferentially select binding sites on the microtubule that lie ahead in the progress direction of the motor. A specific molecular mechanism for biased binding has, however, so far been lacking. Here we use atomistic Brownian dynamics simulations combined with experimental mutagenesis to show that incoming kinesin heads undergo electrostatically guided diffusion-to-capture by microtubules, and that this produces directionally biased binding. Kinesin-1 heads are initially rotated by the electrostatic field so that their tubulin-binding sites face inwards, and then steered towards a plus-endwards binding site. In tethered kinesin dimers, this bias is amplified. A 3-residue sequence (RAK) in kinesin helix alpha-6 is predicted to be important for electrostatic guidance. Real-world mutagenesis of this sequence powerfully influences kinesin-driven microtubule sliding, with one mutant producing a 5-fold acceleration over wild type. We conclude that electrostatic interactions play an important role in the kinesin stepping mechanism, by biasing the diffusional association of kinesin with microtubules.     

Equilibrium binding studies of a tryptophan-shifted mutant of phosphofructokinase from Bacillus stearothermophilus

A tryptophan-shifted mutant of phosphofructokinase (PFK) from Bacillus stearothermophilus has been constructed. This mutant, which is functionally similar to wild-type, provides the opportunity to examine the allosteric properties of PFK under equilibrium conditions. The unique fluorescence properties of the tryptophan-shifted mutant enzyme, W179F/F230W, have been utilized to deduce the thermodynamics of ligand binding and the allosteric perturbations in the absence of catalytic turnover. Specifically, phospho(enol)pyruvate (PEP) and MgADP binding to the mutant PFK can be directly observed using tryptophan fluorescence, and dissociation constants for these ligands have been measured to be equal to 2.71 +/- 0.04 and 90.4 +/- 3.5 microM, respectively. In addition, the homotropic couplings for the allosteric ligands have been assessed for the first time. PEP binds cooperatively with a Hill number of 2.9 +/- 0.3, while MgADP binding is not cooperative. The equilibrium couplings between these ligands and the substrate fructose 6-phosphate (Fru-6-P) have also been determined and follow the same trends with temperature observed under steady-state kinetic assay conditions using wild-type PFK, indicating that the presence of bound MgATP has little influence on the allosteric interactions. Like wild-type PFK, the coupling free energies for the mutant result from largely compensating enthalpy and entropy components at 25 degrees C. Furthermore, the sign of each coupling free energy, which signifies the nature of the allosteric effect, is opposite that of the enthalpy contribution and is therefore due to the larger absolute value of the associated entropy change. This characteristic stands in direct contrast to the thermodynamic basis of the allosteric response in the homologous PFK from E. coli in which the sign of the coupling free energy is established by the sign of the coupling enthalpy.     

Synthesis and photophysical studies on a new fluorescent phenothiazine‐based derivative

A new typical phenothiazine compound functionalized with thienyl‐indandione derivative (PTZTID) was synthesized and characterized using spectral analysis (ultraviolet–visible (UV–vis) light, infrared (IR), ¹H nuclear magnetic resonance (NMR) and ¹³C NMR tools). The UV–vis absorption spectra of the PTZTID solution in 1,4‐dioxane showed two absorption bands attributed to localized aromatic π–π* transitions of conjugated aromatic moieties and intramolecular charge transfer with the characteristics of a π–π* transition. The fluorescence spectra exhibited a maximum emission wavelength at 580 nm. The effect of concentration on photophysical properties took the form of a minor hypsochromic shift, which was attributed to some extent to the occurrence of H‐type aggregation of the PTZTID derivative. Binary solvent effects on the spectroscopic behaviour of PTZTID were measured at different H₂O/1,4‐dioxane ratios. Similarly, when increasing the water content, a hypsochromic shift was observed that resulted from H‐type aggregation. Furthermore, geometry and electronic configurations of PTZTID were studied at density functional theory /B3LYP level and indicated that the compound had a nonplanar (butterfly structure).     

Visual arrestin binding to microtubules involves a distinct conformational change

Recently we found that visual arrestin binds microtubules and that this interaction plays an important role in arrestin localization in photoreceptor cells. Here we use site-directed mutagenesis and spin labeling to explore the molecular mechanism of this novel regulatory interaction. The microtubule binding site maps to the concave sides of the two arrestin domains, overlapping with the rhodopsin binding site, which makes arrestin interactions with rhodopsin and microtubules mutually exclusive. Arrestin interaction with microtubules is enhanced by several "activating mutations" and involves multiple positive charges and hydrophobic elements. The comparable affinity of visual arrestin for microtubules and unpolymerized tubulin (K(D) > 40 mum and >65 mum, respectively) suggests that the arrestin binding site is largely localized on the individual alphabeta-dimer. The changes in the spin-spin interaction of a double-labeled arrestin indicate that the conformation of microtubule-bound arrestin differs from that of free arrestin in solution. In sharp contrast to rhodopsin, where tight binding requires an extended interdomain hinge, arrestin binding to microtubules is enhanced by deletions in this region, suggesting that in the process of microtubule binding the domains may move in the opposite direction. Thus, microtubule and rhodopsin binding induce different conformational changes in arrestin, suggesting that arrestin assumes three distinct conformations in the cell, likely with different functional properties.     

Cytoplasmic dynein mediates adenovirus binding to microtubules

During infection, adenovirus (Ad) capsids undergo microtubule-dependent retrograde transport as part of a program of vectorial transport of the viral genome to the nucleus. The microtubule-associated molecular motor, cytoplasmic dynein, has been implicated in the retrograde movement of Ad. We hypothesized that cytoplasmic dynein constituted the primary mode of association of Ad with microtubules. To evaluate this hypothesis, an Ad-microtubule binding assay was established in which microtubules were polymerized with taxol, combined with Ad in the presence or absence of microtubule-associated proteins (MAPs), and centrifuged through a glycerol cushion. The addition of purified bovine brain MAPs increased the fraction of Ad in the microtubule pellet from 17.3% +/- 3.5% to 80.7% +/- 3.8% (P < 0.01). In the absence of tubulin polymerization or in the presence of high salt, no Ad was found in the pellet. Ad binding to microtubules was not enhanced by bovine brain MAPs enriched for tau protein or by the addition of bovine serum albumin. Enhanced Ad-microtubule binding was also observed by using a fraction of MAPs purified from lung A549 epithelial cell lysate which contained cytoplasmic dynein. Ad-microtubule interaction was sensitive to the addition of ATP, a hallmark of cytoplasmic dynein-dependent microtubule interactions. Immunodepletion of cytoplasmic dynein from the A549 cell lysate abolished the MAP-enhanced Ad-microtubule binding. The interaction of Ad with both dynein and dynactin complexes was demonstrated by coimmunoprecipitation. Partially uncoated capsids isolated from cells 40 min after infection also exhibited microtubule binding. In summary, the primary mode of Ad attachment to microtubules occurs though cytoplasmic dynein-mediated binding.