Binding Site Polarity Research Articles

Michler’s hydrol blue elucidates structural differences in prion strains

Yeast prions provide self-templating protein-based mechanisms of inheritance whose conformational changes lead to the acquisition of diverse new phenotypes. The best studied of these is the prion domain (NM) of Sup35, which forms an amyloid that can adopt several distinct conformations (strains) that confer distinct phenotypes when introduced into cells that do not carry the prion. Classic dyes, such as thioflavin T and Congo red, exhibit large increases in fluorescence when bound to amyloids, but these dyes are not sensitive to local structural differences that distinguish amyloid strains. Here we describe the use of Michler's hydrol blue (MHB) to investigate fibrils formed by the weak and strong prion fibrils of Sup35NM and find that MHB differentiates between these two polymorphs. Quantum mechanical time-dependent density functional theory (TDDFT) calculations indicate that the fluorescence properties of amyloid-bound MHB can be correlated to the change of binding site polarity and that a tyrosine to phenylalanine substitution at a binding site could be detected. Through the use of site-specific mutants, we demonstrate that MHB is a site-specific environmentally sensitive probe that can provide structural details about amyloid fibrils and their polymorphs.

Polarity of the ATP binding site of the Na+,K+-ATPase, gastric H+,K+-ATPase and sarcoplasmic reticulum Ca2+-ATPase

A fluorescence ratiometric method utilizing the probe eosin Y is presented for estimating the ATP binding site polarity of P-type ATPases in different conformational states. The method has been calibrated by measurements in a series of alcohols and tested using complexation of eosin Y with methyl-β-cyclodextrin. The results obtained with the Na+,K+-, H+,K+- and sarcoplasmic reticulum Ca2+-ATPases indicate that the ATP binding site, to which eosin is known to bind, is significantly more polar in the case of the Na+,K+- and H+,K+-ATPases compared to the Ca2+-ATPase. This result was found to be consistent with docking calculations of eosin with the E2 conformational state of the Na+,K+-ATPase and the Ca2+-ATPase. Fluorescence experiments showed that eosin binds significantly more strongly to the E1 conformation of the Na+,K+-ATPase than the E2 conformation, but in the case of the Ca2+-ATPase both fluorescence experiments and docking calculations showed no significant difference in binding affinity between the two conformations. This result could be due to the fact that, in contrast to the Na+,K+- and H+,K+-ATPases, the E2-E1 transition of the Ca2+-ATPase does not involve the movement of a lysine-rich N-terminal tail which may affect the overall enzyme conformation. Consistent with this hypothesis, the eosin affinity of the E1 conformation of the Na+,K+-ATPase was significantly reduced after N-terminal truncation. It is suggested that changes in conformational entropy of the N-terminal tail of the Na+, K+- and the H+,K+-ATPases during the E2-E1 transition could affect the thermodynamic stability of the E1 conformation and hence its ATP binding affinity.

Phenolic Bis-styrylbenzo[ c]-1,2,5-thiadiazoles as Probes for Fluorescence Microscopy Mapping of Aβ Plaque Heterogeneity.

A fluorescent bis-styryl-benzothiadiazole (BTD) with carboxylic acid functional groups (X-34/Congo red analogue) showed lower binding affinity toward Aβ1-42 and Aβ1-40 fibrils than its neutral analogue. Hence, variable patterns of neutral OH-substituted bis-styryl-BTDs were generated. All bis-styryl-BTDs showed higher binding affinity to Aβ1-42 fibrils than to Aβ1-40 fibrils. The para-OH on the phenyl rings was beneficial for binding affinity while a meta-OH decreased the affinity. Differential staining of transgenic mouse Aβ amyloid plaque cores compared to peripheral coronas using neutral compared to anionic bis-styryl ligands indicate differential recognition of amyloid polymorphs. Hyperspectral imaging of transgenic mouse Aβ plaque stained with uncharged para-hydroxyl substituted bis-styryl-BTD implicated differences in binding site polarity of polymorphic amyloid plaque. Most properties of the corresponding bis-styryl-BTD were retained with a rigid alkyne linker rendering a probe insensitive to cis-trans isomerization. These new BTD-based ligands are promising probes for spectral imaging of different Aβ fibril polymorphs.

Dipolar 3-methoxychromones as bright and highly solvatochromic fluorescent dyes

Herein, three environment-sensitive (solvatochromic) fluorescent dyes presenting a strong electron acceptor 3-methoxychromone unit and varied electron donor 2-aryl were developed. All three dyes showed remarkable polarity-dependent shifts of the emission maximum, which increase with extension of the dye conjugation. For the 3-methoxychromone bearing a 7-(diethylamino)-9,9-dimethylfluoren-2-yl donor group the difference between the excited and the ground state dipole moments, estimated from the Lippert-Mataga expression, reached 20 D, which is among the largest reported for neutral dipolar fluorophores. Moreover, the new dyes are characterized by significant two-photon absorption cross-section (up to 450 GM) and large fluorescence quantum yields. The strong decrease in the fluorescence quantum yields of the dyes in polar protic solvents was observed together with the increase in the non-radiative deactivation rates, which can originate from twisted intramolecular charge transfer and intermolecular proton transfer phenomena. In comparison to the parent 3-hydroxychromone derivatives, the new dyes presented significantly improved photostability, which confirms that photodegradation of 3-hydroxychromones occurs from a product of the excited-state intramolecular proton transfer (phototautomer). Finally, an application of the new dyes for probing local binding site polarity of serum albumin was shown. This new class of fluorescent dyes may serve as attractive building blocks for future molecular sensors utilizing environment-sensitive fluorophores.

Development of a quinoxaline-based fluorescent probe for quantitative estimation of protein binding site polarity

The compound 2-[(1E)-2-(1H-pyrrol-2-yl)ethenyl]-quinoxaline (PQX) is a promising fluorescent chromophore for the estimation of protein binding site polarity, due to its full-color solvatochromic fluorescence. A linear relationship was obtained between the peak emission wavenumber and E(T)(N) (normalized solvent polarity). The BSA binding site polarity was estimated from the solvatochromic plot.

Spectroscopic characterization of diverse amyloid fibrils in vitro by the fluorescent dye Nile red

The fluorescence of Nile red (9-diethylamino-5H-benzophenoxazine-5-one) is quenched in aqueous solutions but shows augmented fluorescence in hydrophobic environments. Nile red fluorescence was blue shifted and strongly augmented in the presence of various amyloid fibrils assayed under acidic as well as neutral pH conditions. Fibrils grown from lysozyme and insulin (at pH 1.6 and 65 °C), transthyretin (TTR) fibrils grown from the acid unfolded monomer (pH 2.0, 21 °C) or from the dissociated tetramer starting from native protein under less acidic conditions (pH 4.4, 37 °C) were detected. Nile red was also successfully employed in detecting Aβ1-42 and human prion protein (PrP90-231) amyloid fibrils grown at neutral pH. Nile red was amyloid fibril specific and did not fluoresce appreciably in the presence of the monomeric precursor proteins. Stoke's shifts of the wavelength maximum of Nile red bound to various fibrils were different (ranging from 615 nm to 638 nm) indicating sensitivity to the tertiary structure in its respective binding sites of different amyloid proteins. A polarity assay using ethanol-water mixtures and pure octanol ranging from dielectric constants between 10 and 70 showed a linear correlation of Nile red Stoke's shift and allowed assignment of amyloid fibril binding site polarity. Fluorescence resonance energy transfer between Thioflavin T (ThT) and Nile red was proven to be efficient and co-staining was employed to discriminate between conformational isoforms of Aβ1-42 amyloid fibrils grown under agitated and quiescent conditions. This paper demonstrates the complementary use of this fluorometric method for conformational typing of amyloid structures.

Ligand Recognition by RAR and RXR Receptors: Binding and Selectivity

Fundamental biological functions, most notably embriogenesis, cell growth, cell differentiation, and cell apoptosis, are in part regulated by a complex genomic network that starts with the binding (and activation) of retinoids to their cognate receptors, members of the superfamily of nuclear receptors. We have studied ligand recognition of retinoic receptors (RXRalpha and RARgamma) using a molecular-mechanics-based docking method. The protocol used in this work is able to rank the affinity of pairs of ligands for a single retinoid receptor, the highest values corresponding to those that adapt better to the shape of the binding site and generate the optimal set of electrostatic and apolar interactions with the receptor. Moreover, our studies shed light onto some of the energetic contributions to retinoid receptor ligand selectivity. In this regard we show that there is a difference in polarity between the binding site regions that anchor the carboxylate in RAR and RXR, which translates itself into large differences in the energy of interaction of both receptors with the same ligand. We observe that the latter energy change is canceled off by the solvation energy penalty upon binding. This energy compensation is borne out as well by experiments that address the effect of site-directed mutagenesis on ligand binding to RARgamma. The hypothesis that the difference in binding site polarity might be exploited to build RXR-selective ligands is tested with some compounds having a thiazolidinedione anchoring group.

Novel Two-Band Ratiometric Fluorescence Probes with Different Location and Orientation in Phospholipid Membranes

3-hydroxyflavone (3-HF) derivatives are very attractive fluorescence sensors due to their ability to respond to small changes in their microenvironment via a dramatic alteration of the relative intensities of their two well-separated emission bands. We developed fluorescence probes with locations at different depths and orientations of 3-HF moiety in the phospholipid bilayer, which determine their fluorescence behavior. While the spectral shifts of the probes correlate with their binding site polarity, the intensity ratio is a complex parameter that is also sensitive to the local hydration. We demonstrate that even the deeply located probes sense this hydration effect, which can be modulated by the charge of the lipid heads and is anisotropic with respect to the bilayer plane. Thus the two-band ratiometric fluorescence probes can provide multiparametric information on the properties of lipid membranes at different depths.

Probing AOT Reverse Micelles with Two-Color Fluorescence Dyes Based on 3-Hydroxychromone

Reverse micelles formed by AOT (Aerosol OT, sodium bis(2-ethylhexyl) sulfosuccinate) in hexane at different mole fractions of water (W0) were studied with novel two-band ratiometric fluorescence probes based on 3-hydroxychromone. In the range of W0 from 0 to 30, the intensity ratio at emission band maxima changes up to 18 times. The profile of the fluorescence response is different at low and high W0. This suggests that at the initial step of hydration the increase of binding site polarity of the probes is accompanied by the loss of their specific interaction with ionic headgroups at the micellar interface, while at higher W0 they respond by redistribution to apolar phase.

Binding site polarity and ligand affinity of homologous fatty acid-binding proteins from animals with different body temperatures

Binding affinity and binding-pocket polarity is determined for intracellular fatty acid-binding protein (FABP) from aerobic muscle of Chaenocephalus aceratus, the Antarctic icefish, and from rat heart. FABPs bind fatty acids via weak-bond forces (both ionic and hydrophobic), and these bond forces are temperature sensitive, yet FABPs are present in animals whose body temperatures range over nearly 40 degrees C. To investigate FABP's sensitivity to body temperature, fatty acid binding affinity (Kd) was determined for both rat heart-FABP and icefish heart-FABP at two physiological temperatures (0 degrees C or 37 degrees C). Saturated and unsaturated fatty acids (16:0 and 16:1), delivered in model membranes (liposomes) whose composition is typical of either Antarctic fish (16:0/22:6 phosphatidylcholine) or mammals (bovine-heart phosphatidylcholine) were examined. Incubation at 0 degree C or 37 degrees C dose not significantly affect Kd for rat heart FABP, regardless of liposome composition or fatty acid ligand (Kd = 0.686 +/- 0.127 - 1.129 +/- 0.356 microM at 0 degree C, 0.775 +/- 0.307 - 1.605 +/- 0.427 microM at 37 degrees C). Incubation temperature significantly affects icefish FABP's affinity for 16:1 (0.626 +/- 0.093 microM at 37 degrees C vs. 1.896 +/- 0.343 microM at 0 degree C for fatty acid presented in Antarctic fish liposomes; 0.331 +/- 0.101 microM at 37 degrees C vs. 0.949 +/- 0.121 microM at 0 degree C for bovine heart liposomes) but not 16:0. Kd is not significantly different between FABPs under any set of conditions (with one exception: Kd is significantly lower in rat FABP vs. icefish FABP for 16:0 at 0 degree C for fatty acids delivered in bovine heart liposomes). Although Kd values are largely equivalent between the two FABPs, relative contributions from ionic vs. hydrophobic weak-bond forces are different between the two animals. Rat heart FABP has a binding pocket that is significantly more nonpolar than that of icefish FABP (as measured by quantum yield of the bound fluorescent fatty-acid analogue (PA-DPH); Q = 0.067 +/- 0.008 vs. 0.034 +/- 0.005 at 0 degree C, 0.030 +/- 0.003 vs. 0.019 +/- 0.002 at 37 degrees C). This suggests that rat-heart FABP realizes a micromolar Kd with a greater reliance upon hydrophobic interactions than does icefish FABP.

Fluorescence emission from adsorbed bovine serum albumin and albumin-bound 1-anilinonaphthalene-8-sulfonate studied by TIRF

Total internal reflection fluorescence (TIRF) spectroscopy was used to analyse the conformational changes of bovine serum albumin (BSA) upon adsorption to silica surfaces. The intrinsic fluorescence emitted by BSA tryptophans and the fluorescence of BSA-bound ligand 1-anilinonaphthalene-8-sulfonate (ANS) were analysed prior to and after the removal of nonadsorbed protein. It was found that the irreversibly adsorbed BSA emits a blue-shifted intrinsic fluorescence (11 nm) and that the surface-adsorbed BSA-bound ANS fluorescence emission is red-shifted (12 nm). It was concluded that the conformational change in adsorbed BSA involves the whole BSA molecule: tryptophans become exposed to a less polar environment while the binding site polarity is increased. The conformation of irreversibly adsorbed BSA is not similar to the unfolded conformation of heat-denatured BSA. The red-edge excitation of adsorbed BSA-bound ANS indicates a slower spectral relaxation in the adsorbed protein as compared with the protein in solution. The fluorescence intensity contributions from irreversibly adsorbed species (BSA with and without bound ANS) suggest some changes in either ANS-BSA binding affinity and/or ANS fluorescence enhancement.

Spin probes for binding site polarity

Spin probes for binding site polarity

Quantitative estimation of protein binding site polarity. Fluorescence of N-arylaminonaphthalenesulfonates.

Quantitative estimation of protein binding site polarity. Fluorescence of N-arylaminonaphthalenesulfonates.