Nicole Glasser

Soybean phosphatidylcholine vesicles containing plant sterols : a fluorescence anisotropy study

The typical plant sterols (sitosterol, stigmasterol and campesterol) were compared with respect to their ability to regulate membrane fluidity of soybean phosphatidylcholine (PC) vesicles. Fluidity changes were monitored by the steady-state fluorescence anisotropy with 1,6-diphenyl-1,3,5-hexatriene as a probe and assigned to a measure of the acyl chain orientational order. Sitosterol and campesterol appear to be the most suitable sterols in ordering the acyl chains of soybean lecithin bilayers, even more efficient than cholesterol, the standard of reference for sterol effects on membranes, suggesting that they play a significant role in the regulation of plant membrane properties. Stigmasterol is shown to be much less active. Cycloartenol, a biosynthetic precursor of plant sterols, increases the acyl chain order with the same efficiency as cholesterol. We also investigated the effects of two unusual sterols, 24-methylpollinastanol and 14 alpha,24-dimethylcholest-8-en-3 beta-ol, which were shown to accumulate in plants treated with fungicides belonging to two important classes, N-substituted morpholines and triazoles, respectively. These two sterols exhibit a behavior very similar to that of stigmasterol. The results are discussed in terms of sterol effects on the molecular packing of soybean PC bilayers.

8-vinyl-deoxyadenosine, an alternative fluorescent nucleoside analog to 2′-deoxyribosyl-2-aminopurine with improved properties

We report here the synthesis and the spectroscopic characterization of 8-vinyl-deoxyadenosine (8vdA), a new fluorescent analog of deoxyadenosine. 8vdA was found to absorb and emit in the same wavelength range as 2′-deoxyribosyl-2-aminopurine (2AP), the most frequently used fluorescent nucleoside analog. Though the quantum yield of 8vdA is similar to that of 2AP, its molar absorption coefficient is about twice, enabling a more sensitive detection. Moreover, the fluorescence of 8vdA was found to be sensitive to temperature and solvent but not to pH (around neutrality) or coupling to phosphate groups. Though 8vdA is base sensitive and susceptible to depurination, the corresponding phosphoramidite was successfully prepared and incorporated in oligonucleotides of the type d(CGT TTT XNX TTT TGC) where N = 8vdA and X = A, T or C. The 8vdA-labeled oligonucleotides gave more stable duplexes than the corresponding 2AP-labeled sequences when X = A or T, indicating that 8vdA is less perturbing than 2AP and probably adopts an anti conformation to preserve the Watson–Crick H-bonding. In addition, the quantum yield of 8vdA is significantly higher than 2AP in all tested oligonucleotides in both their single strand and duplex states. The steady-state and time-resolved fluorescence parameters of 8vdA and 2AP were found to depend similarly on the nature of their flanking residues and on base pairing, suggesting that their photophysics are governed by similar mechanisms. Taken together, our data suggest that 8vdA is a non perturbing nucleoside analog that may be used with improved sensitivity for the same applications as 2AP.

Purification, characterization and ion binding properties of human brain S100b protein

Human brain S100b (beta beta) protein was purified using zinc-dependent affinity chromatography on phenyl-Sepharose. The calcium- and zinc-binding properties of the protein were studied by flow dialysis technique and the protein conformation both in the metal-free form and in the presence of Ca2+ or Zn2+ was investigated with ultraviolet spectroscopy, sulfhydryl reactivity and interaction with a hydrophobic fluorescence probe 6-(p-toluidino)naphthalene-2-sulfonic acid (TNS). Flow dialysis measurements of Ca2+ binding to human brain S100b (beta beta) protein revealed six Ca2+-binding sites which we assumed to represent three for each beta monomer, characterized by the macroscopic association constants K1 = 0.44 X 10(5) M-1; K2 = 0.1 X 10(5) M-1 and K3 = 0.08 X 10(5) M-1. In the presence of 120 mM KCl, the affinity of the protein for calcium is drastically reduced. Zinc-binding studies on human S100b protein showed that the protein bound two zinc ions per beta monomer, with macroscopic constants K1 = 4.47 X 10(7) M-1 and K2 = 0.1 X 10(7) M-1. Most of the Zn2+-induced conformational changes occurred after the binding of two zinc ions per mole of S100b protein. These results differ significantly from those for bovine protein and cast doubt on the conservation of the S100 structure during evolution. When calcium binding was studied in the presence of zinc, we noted an increase in the affinity of the protein for calcium, K1 = 4.4 X 10(5) M-1; K2 = 0.57 X 10(5) M-1; K3 = 0.023 X 10(5) M-1. These results indicated that the Ca2+- and Zn2+-binding sites on S100b protein are different and suggest that Zn2+ may regulate Ca2+ binding by increasing the affinity of the protein for calcium.

Pirenzepine Promotes the Dimerization of Muscarinic M1 Receptors through a Three-step Binding Process

Ligand binding to G protein-coupled receptors is a complex process that involves sequential receptor conformational changes, ligand translocation, and possibly ligand-induced receptor oligomerization. Binding events at muscarinic acetylcholine receptors are usually interpreted from radioligand binding studies in terms of two-step ligand-induced receptor isomerization. We report here, using a combination of fluorescence approaches, on the molecular mechanisms for Bodipy-pirenzepine binding to enhanced green fluorescent protein (EGFP)-fused muscarinic M1 receptors in living cells. Real time monitoring, under steady-state conditions, of the strong fluorescence energy transfer signal elicited by this interaction permitted a fine kinetic description of the binding process. Time-resolved fluorescence measurements allowed us to identify discrete EGFP lifetime species and to follow their redistribution upon ligand binding. Fluorescence correlation spectroscopy, with EGFP brightness analysis, showed that EGFP-fused muscarinic M1 receptors predominate as monomers in the absence of ligand and dimerize upon pirenzepine binding. Finally, all these experimental data could be quantitatively reconciled into a three-step mechanism, with four identified receptor conformational states. Fast ligand binding to a peripheral receptor site initiates a sequence of conformational changes that allows the ligand to access to inner regions of the protein and drives ligand-receptor complexes toward a high affinity dimeric state.

Distinct motifs of neuropeptide Y receptors differentially regulate trafficking and desensitization.

Activated human neuropeptide Y Y1 receptors rapidly desensitize and internalize through clathrin‐coated pits and recycle from early and recycling endosomes, unlike Y2 receptors that neither internalize nor desensitize. To identify motifs implicated in Y1 receptor desensitization and trafficking, mutants with varying C‐terminal truncations or a substituted Y2 C‐terminus were constructed. Point mutations of key putative residues were made in a C‐terminal conserved motif [φ‐H‐(S/T)‐(E/D)‐V‐(S/T)‐X‐T] that we have identified and in the second intracellular i2 loop. Receptors were analyzed by functional assays, spectrofluorimetric measurements on living cells, flow cytometry, confocal imaging and bioluminescence resonance energy transfer assays for β‐arrestin activation and adaptor protein (AP‐2) complex recruitment. Inhibitory GTP‐binding protein‐dependent signaling of Y1 receptors to adenylyl cyclase and desensitization was unaffected by C‐terminal truncations or mutations, while C‐terminal deletion mutants of 42 and 61 amino acids no longer internalized. Substitutions of Thr357, Asp358, Ser360 and Thr362 by Ala in the C‐terminus abolished both internalization and β‐arrestin activation but not desensitization. A Pro145 substitution by His in an i2 consensus motif reported to mediate phosphorylation‐independent recruitment of β‐arrestins affected neither desensitization, internalization or recycling kinetics of activated Y1 receptors nor β‐arrestin activation. Interestingly, combining Pro145 substitution by His and C‐terminal substitutions significantly attenuates Y1 desensitization. In the Y2 receptor, replacement of His155 with Pro at this position in the i2 loop motif promotes agonist‐mediated desensitization, β‐arrestin activation, internalization and recycling. Overall, our results indicate that β‐arrestin‐mediated desensitization and internalization of Y1 and Y2 receptors are differentially regulated by the C‐terminal motif and the i2 loop consensus motif.

Bimane- and acrylodan-labeled S100 proteins. Role of cysteines-85.alpha. and -84.beta. in the conformation and calcium binding properties of S100aa and S100b (.beta..beta.) proteins

Bovine brain S100 alpha alpha, S100a (alpha beta), and S100b (beta beta) protein dimers were labeled with the sulfydryl-specific fluorescent probes monobromo(trimethylammonio)bimane (bimane) and 6-acryloyl-2-(dimethylamino)naphthalene (acrylodan) at cysteines-85 alpha and -84 beta. The conformation and fluorescence properties of the S100 proteins derived were studied by means of anion-exchange chromatography on a Mono Q column using a fast protein chromatography system and fluorescence intensity, maximum emission wavelength, and polarization measurements. Spectroscopic studies on the intrinsic absorption and fluorescence properties of S100 alpha alpha and S100b proteins chemically modified on cysteines-85 alpha and -84 beta with iodoacetamide completed this study. Several arguments suggest that the alkylated S100 proteins undergo conformational changes that are mainly characterized by the destabilization of the quaternary protein structure, which provokes a slow dimer-monomer equilibrium at high protein concentrations and induces total subunit dissociation at low ones. Calcium binding studies on bimane-S100 alpha alpha and -S100b proteins showed that alkylated proteins had a much higher calcium binding affinity than native protein and that the antagonistic effect of KCl on calcium binding was much less pronounced. These results confirmed our previous observations that the affinities of calcium binding sites II alpha and II beta in S100 proteins are highly dependent on protein conformation [Baudier, J., & Gerard, D. (1986) J. Biol. Chem. 261, 8204-8212].

Contribution of a tyrosine-based motif to cellular trafficking of wild-type and truncated NPY Y1 receptors

The human NPY Y(1) receptor undergoes fast agonist-induced internalization via clathrin-coated pits then recycles back to the cell membrane. In an attempt to identify the molecular determinants involved in this process, we studied several C-terminal truncation mutants tagged with EFGP. In the absence of agonist, Y(1) receptors lacking the last 32 C-terminal amino acids (Y(1)Δ32) are constitutively internalized, unlike full-length Y(1) receptors. At steady state, internalized Y(1)Δ32 receptors co-localize with transferrin, a marker of early and recycling endosomes. Inhibition of constitutive internalization of Y(1)Δ32 receptors by hypertonic sucrose or by co-expression of Rab5aS34N, a dominant negative form of the small GTPase Rab5a or depletion of all three isoforms of Rab5 indicates the involvement of clathrin-coated pits. In contrast, a truncated receptor lacking the last 42 C-terminal amino acids (Y(1)Δ42) does not constitutively internalize, consistent with the possibility that there is a molecular determinant responsible for constitutive internalization located in the last 10 amino acids of Y(1)Δ32 receptors. We show that the agonist-independent internalization of Y(1)Δ32 receptors involves a tyrosine-based motif YXXΦ. The potential role of this motif in the behaviour of full-length Y(1) receptors has also been explored. Our results indicate that a C-terminal tyrosine-based motif is critical for the constitutive internalization of truncated Y(1)Δ32 receptors. We suggest that this motif is masked in full-length Y(1) receptors which do not constitutively internalize in the absence of agonist.