Supramolecular luminescent lanthanide dimers for fluoride sequestering and sensing.

Lanthanide complexes (Ln=Eu, Tb, and Yb) that are based on a C2 -symmetric cyclen scaffold were prepared and characterized. The addition of fluoride anions to aqueous solutions of the complexes resulted in the formation of dinuclear supramolecular compounds in which the anion is confined into the cavity that is formed by the two complexes. The supramolecular assembly process was monitored by UV/Vis absorption, luminescence, and NMR spectroscopy and high-resolution mass spectrometry. The X-ray crystal structure of the europium dimer revealed that the architecture of the scaffold is stabilized by synergistic effects of the EuFEu bridging motive, π stacking interactions, and a four-component hydrogen-bonding network, which control the assembly of the two [EuL] entities around the fluoride ion. The strong association in water allowed for the luminescence sensing of fluoride down to a detection limit of 24 nM.

Simultaneous self-assembly of a [2]catenane, a trefoil knot, and a Solomon link from a simple pair of ligands.

A topological triptych: Three molecular links, a [2]catenane, a trefoil knot, and a Solomon link, were obtained in one pot through the self-assembly of two simple ligands in the presence of Zn(II). The approach relied on dynamic covalent chemistry and metal templation.

p-Sulfonato-calix[n]arenes inhibit staphylococcal bicomponent leukotoxins by supramolecular interactions.

PVL (Panton-Valentine leukocidin) and other Staphylococcus aureus ß-stranded pore-forming toxins are important virulence factors involved in various pathologies that are often necrotizing. The present study characterized leukotoxin inhibition by selected SCns (p-sulfonato-calix[n]arenes): SC4, SC6 and SC8. These chemicals have no toxic effects on human erythrocytes or neutrophils, and some are able to inhibit both the activity of and the cell lysis by leukotoxins in a dose-dependent manner. Depending on the type of leukotoxins and SCns, flow cytometry revealed IC50 values of 6-22 µM for Ca2+ activation and of 2-50 µM for cell lysis. SCns were observed to affect membrane binding of class S proteins responsible for cell specificity. Electrospray MS and surface plasmon resonance established supramolecular interactions (1:1 stoichiometry) between SCns and class S proteins in solution, but not class F proteins. The membrane-binding affinity of S proteins was Kd=0.07-6.2 nM. The binding ability was completely abolished by SCns at different concentrations according to the number of benzenes (30-300 µM; SC8>SC6≫SC4). The inhibitory properties of SCns were also observed in vivo in a rabbit model of PVL-induced endophthalmitis. These calixarenes may represent new therapeutic avenues aimed at minimizing inflammatory reactions and necrosis due to certain virulence factors.

Crystal growth of proteins, nucleic acids, and viruses in gels.

Medium-sized single crystals with perfect habits and no defect producing intense and well-resolved diffraction patterns are the dream of every protein crystallographer. Crystals of biological macromolecules possessing these characteristics can be prepared within a medium in which mass transport is restricted to diffusion. Chemical gels (like polysiloxane) and physical gels (such as agarose) provide such an environment and are therefore suitable for the crystallisation of biological macromolecules. Instructions for the preparation of each type of gel are given to urge crystal growers to apply diffusive media for enhancing crystallographic quality of their crystals. Examples of quality enhancement achieved with silica and agarose gels are given. Results obtained with other substances forming gel-like media (such as lipidic phases and cellulose derivatives) are presented. Finally, the use of gels in combination with capillary tubes for counter-diffusion experiments is discussed. Methods and techniques implemented with proteins can also be applied to nucleic acids and nucleoprotein assemblies such as viruses.

Differential action on coregulator interaction defines inverse retinoid agonists and neutral antagonists.

Retinoic acid receptors (RARs) are ligand-dependent transcription factors that control a plethora of physiological processes. RARs exert their functions by regulating gene networks controlling cell growth, differentiation, survival, and death. Uncovering the molecular details by which synthetic ligands direct specificity and functionality of nuclear receptors is key to rational drug development. Here we define the molecular basis for (E)-4-[2-[5,6-Dihydro-5,5-dimethyl-8-(2-phenylethynyl)naphthalen-2-yl]ethen-1-yl]benzoic acid (BMS204,493) acting as the inverse pan-RAR agonist and define 4-[5,6-Dihydro-5,5-dimethyl-8-(quinolin-3-yl)naphthalen-2-carboxamido]benzoic acid (BMS195,614) as the neutral RARalpha-selective antagonist. We reveal the details of the differential coregulator interactions imposed on the receptor by the ligands and show that the anchoring of H12 is fundamentally distinct in the presence of the two ligands, thus accounting for the observed effects on coactivator and corepressor interactions. These ligands will facilitate studies on the role of the constitutive activity of RARs, particularly of the tumor suppressor RARbeta, whose specific functions relative to other RARs have remained elusive.

Identification by high throughput screening of small compounds inhibiting the nucleic acid destabilization activity of the HIV-1 nucleocapsid protein.

Due to its highly conserved zinc fingers and its nucleic acid chaperone properties which are critical for HIV-1 replication, the nucleocapsid protein (NC) constitutes a major target in AIDS therapy. Different families of molecules targeting NC zinc fingers and/or inhibiting the binding of NC with its target nucleic acids have been developed. However, their limited specificity and their cellular toxicity prompted us to develop a screening assay to target molecules able to inhibit NC chaperone properties, and more specifically the initial NC-promoted destabilization of the nucleic acid secondary structure. Since this destabilization is critically dependent on the properly folded fingers, the developed assay is thought to be highly specific. The assay was based on the use of cTAR DNA, a stem-loop sequence complementary to the transactivation response element, doubly labelled at its 5' and 3' ends by a rhodamine 6G fluorophore and a fluorescence quencher, respectively. Addition of NC(12-55), a peptide corresponding to the zinc finger domain of NC, to this doubly-labelled cTAR, led to a partial melting of the cTAR stem, which increases the distance between the two labels and thus, restores the rhodamine 6G fluorescence. Thus, positive hits were detected through the decrease of rhodamine 6G fluorescence. An "in-house" chemical library of 4800 molecules was screened and five compounds with IC(50) values in the micromolar range have been selected. The hits were shown by mass spectrometry and fluorescence anisotropy titration to prevent binding of NC(12-55) to cTAR through direct interaction with the NC folded fingers, but without promoting zinc ejection. These non-zinc ejecting NC binders are a new series of anti-NC molecules that could be used to rationally design molecules with potential anti-viral activities.

Detection and structural features of the betaB2-B3-crystallin heterodimer by radical probe mass spectrometry (RP-MS).

The predilection of the beta-crystallin B2 subunit to interact with the betaB3 subunit rather than self associate is evident by the detection of the betaB2-B3-crystallin heterodimer by native gel electrophoresis and electrospray ionisation time-of-flight (ESI-TOF) mass spectrometry under non denaturing conditions. The complex has been detected for the first time and its molecular mass is measured to be 47,450 +/- 1 Da. Radical probe mass spectrometry (RP-MS) was subsequently applied to investigate the nature of the heterodimer through the limited oxidation of the subunits in the complex. Two peptide segments of the betaB2 subunit and six of the betaB3 subunit were found to oxidise, with far greater oxidation observed within the betaB3 versus the betaB2 subunit. This, and the observation that the oxidation data of betaB2 subunit is inconsistent with the structure of the betaB2 monomer, demonstrates that the protection of betaB2 is conferred by its association with betaB3 subunit within the heterodimer where only the residues of, and towards, its N-terminal domain remain exposed to solvent. The results suggest that the betaB2 subunit adopts a more compacted form than in its monomeric form in order for much of its structure to be enveloped by the betaB3 subunit within the heterodimer.

An optimized MALDI mass spectrometry method for improved detection of lysine/arginine/histidine free peptides.

Transcription factors and their regulators possess "basic amino acid free domains" which modulate transcriptional gene activation. We aimed at optimizing a MALDI mass spectrometry (MS) analytical method for the characterization of such domains after protein enzymatic digestion. A panel of recombinant transcription factors with different basic residue contents was proteolytically digested with the Asp-N endoprotease and resulting peptide mixtures were analyzed by MALDI-MS with alpha-cyano-4-hydroxy-cinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB) as matrix. We found that peptides without lysine, arginine, histidine (Lys/Arg/His free peptides) were efficiently detected in the positive ion mode only when using DHB. These findings proved to be very useful for two different targeted proteomic applications. Indeed, the MALDI-MS/MS identification of the CARM1 proteolytic cleavage site, which happens in a Lys/Arg/His free domain, could only be achieved using the DHB matrix. Moreover, in routine proteomic analyses, the detection efficiency of Lys/Arg/His free C-terminal peptides of two-dimensional gel separated proteins was strongly enhanced when DHB was used instead of CHCA.

Nondenaturing mass spectrometry to study noncovalent protein/protein and protein/ligand complexes: technical aspects and application to the determination of binding stoichiometries.

In the present chapter we detail how mass spectrometry (MS) can be used to characterize noncovalent complexes, especially multimeric proteins and protein/ligand complexes. This original application of MS, also called "supramolecular MS" or "nondenaturing MS," appeared in the early 1990s and has continuously evolved since then. Nondenaturing MS is now fully integrated in structural biology programs and in drug discovery platforms. Indeed, appropriate sample preparation and fine tuning of the instrument make it possible to transfer weak assemblies without disruption from solution into the gas phase of the mass spectrometer. In this chapter we detail experimental conditions (sample preparation, optimization of instrumental parameters, etc.) required for the detection of noncovalent complexes by MS. We then focus on the type of information and accuracy that we get after interpreting electrospray ionization mass spectra obtained under nondenaturing conditions, with emphasis on the determination of the stoichiometry of protein/protein and protein/ligand complexes.

Crystal structure of Thermus thermophilus tRNA m1A58 methyltransferase and biophysical characterization of its interaction with tRNA.

Methyltransferases from the m(1)A(58) tRNA methyltransferase (TrmI) family catalyze the S-adenosyl-l-methionine-dependent N(1)-methylation of tRNA adenosine 58. The crystal structure of Thermus thermophilus TrmI, in complex with S-adenosyl-l-homocysteine, was determined at 1.7 A resolution. This structure is closely related to that of Mycobacterium tuberculosis TrmI, and their comparison enabled us to enlighten two grooves in the TrmI structure that are large enough and electrostatically compatible to accommodate one tRNA per face of TrmI tetramer. We have then conducted a biophysical study based on electrospray ionization mass spectrometry, site-directed mutagenesis, and molecular docking. First, we confirmed the tetrameric oligomerization state of TrmI, and we showed that this protein remains tetrameric upon tRNA binding, with formation of complexes involving one to two molecules of tRNA per TrmI tetramer. Second, three key residues for the methylation reaction were identified: the universally conserved D170 and two conserved aromatic residues Y78 and Y194. We then used molecular docking to position a N(9)-methyladenine in the active site of TrmI. The N(9)-methyladenine snugly fits into the catalytic cleft, where the side chain of D170 acts as a bidentate ligand binding the amino moiety of S-adenosyl-l-methionine and the exocyclic amino group of the adenosine. Y194 interacts with the N(9)-methyladenine ring, whereas Y78 can stabilize the sugar ring. From our results, we propose that the conserved residues that form the catalytic cavity (D170, Y78, and Y194) are essential for fashioning an optimized shape of the catalytic pocket.

Modulation of E-cadherin monomer folding by cooperative binding of calcium ions.

Classical cadherins are transmembrane glycoproteins involved in calcium-dependent cell-cell adhesion. Calcium ions are coordinated at the interface between successive modules of the cadherin ectodomain and are thought to regulate the adhesive interactions of cadherins when present at millimolar concentrations. It is widely accepted that calcium plays a critical role in cadherin-mediated cell-cell adhesion, but the nature of cadherin-calcium binding remains a matter of debate. We investigated the parameters of noncovalent cadherin-calcium binding, using the two N-terminal modules of E-cadherin (E/EC12) with a native N-terminal end and nondenaturing electrospray ionization mass spectrometry. By directly visualizing the molecular complexes, we demonstrated that E/EC12 binds three calcium ions, with an average KD of 20 +/- 0.7 microM. These calcium ions bound cooperatively to E/EC12 in its monomeric state, and these properties were not modified by an N-terminal extension consisting of a single methionine residue. This binding induced specific structural changes, as shown by assessments of protease sensitivity, circular dichroism, and mass spectrometry. Furthermore, the D103A mutation (a residue involved in E-cadherin adhesive function) modified calcium binding and led to a loss of cooperativity and the absence of structural changes, despite calcium binding. As the amino acids involved in calcium binding are found within the cadherin consensus motif, our findings may be relevant to other members of the cadherin family.

Endogenous plasma Peptide detection and identification in the rat by a combination of fractionation methods and mass spectrometry.

Mass spectrometry-based analyses are essential tools in the field of biomarker research. However, detection and characterization of plasma low abundance and/or low molecular weight peptides is challenged by the presence of highly abundant proteins, salts and lipids. Numerous strategies have already been tested to reduce the complexity of plasma samples. The aim of this study was to enrich the low molecular weight fraction of rat plasma. To this end, we developed and compared simple protocols based on membrane filtration, solid phase extraction, and a combination of both. As assessed by UV absorbance, an albumin depletion >99% was obtained. The multistep fractionation strategy (including reverse phase HPLC) allowed detection, in a reproducible manner (CV < 30%-35%), of more than 450 peaks below 3000 Da by MALDI-TOF/MS. A MALDI-TOF/MS-determined LOD as low as 1 fmol/muL was obtained, thus allowing nanoLC-Chip/MS/MS identification of spiked peptides representing ~10(-6)% of total proteins, by weight. Signal peptide recovery ranged between 5%-100% according to the spiked peptide considered. Tens of peptide sequence tags from endogenous plasma peptides were also obtained and high confidence identifications of low abundance fibrinopeptide A and B are reported here to show the efficiency of the protocol. It is concluded that the fractionation protocol presented would be of particular interest for future differential (high throughput) analyses of the plasma low molecular weight fraction.

Oligomerization behavior of the archaeal Sm2-type protein from Archaeoglobus fulgidus.

As part of a functional analysis of archaeal Sm-related proteins, we have studied the oligomerization behavior of the Sm-2 type protein from the euryarchaeon Archaeoglobus fulgidus using gel filtration chromatography and noncovalent mass spectrometry. Our experiments show that the oligomeric state of the protein depends on the pH and presence of RNA. The protein forms a hexamer at acidic pH in the absence of RNA. The addition of RNA (oligo U10) induces the formation of a heptamer over the whole pH range studied. The stability of both the hexamer and the RNA-bound heptamer increases at lower pH.

Monitoring ligand-mediated nuclear receptor-coregulator interactions by noncovalent mass spectrometry.

Retinoid receptors are ligand-dependent transcription factors belonging to the nuclear receptor superfamily. Retinoic acid (RARalpha, beta, gamma) and retinoid X (RXRalpha, beta, gamma) receptors mediate the retinoid/rexinoid signal to the transcriptional machineries by interacting at the first level with coactivators or corepressors, which leads to the recruitment of enzymatically active noncovalent complexes at target gene promoters. It has been shown that the interaction of corepressors with nuclear receptors involves conserved LXXI/HIXXXI/L consensus sequences termed corepressor nuclear receptor (CoRNR) boxes. Here we describe the use of nondenaturing electrospray ionization mass spectrometry (ESI-MS) to determine the characteristics of CoRNR box peptide binding to the ligand binding domains of the RARalpha-RXRalpha heterodimer. The stability of the RARalpha-RXRalpha-CoRNR ternary complexes was monitored in the presence of different types of agonists or antagonists for the two receptors, including inverse agonists. These results show unambiguously the differential impact of distinct retinoids on corepressor binding. We show that ESI-MS is a powerful technique that complements classical methods and allows one to: (a) obtain direct evidence for the formation of noncovalent NR complexes; (b) determine ligand binding stoichiometries and (c) monitor ligand effects on these complexes.

Respiratory adaptations to the deep-sea hydrothermal vent environment: the case of Segonzacia mesatlantica, a crab from the Mid-Atlantic Ridge.

Segonzacia mesatlantica (Crustacea; Decapoda; Brachyura) is the only endemic crab species known from the Mid-Atlantic Ridge (MAR) hydrothermal vents. Known from all explored sites in the Atlantic, its wide distribution makes this species a model to study physiological adaptation, and specifically respiratory strategies. Native haemocyanin (Hc) comprises four non-covalent associations in equilibrium formed by monomers, hexamers, dodecamers and octadecamers made up of approximately 75 kDa polypeptide chains. Four different amino acid chains are observed with a molecular mass ranging from 75,234 to 75,972 Da. Experiments carried-out under pressure suggested that the percentage of monomer increased in the haemolymph under hypoxic condition. We have also observed a shift of the proportion of the two dodecamer series, suggesting a rapid modification of the Hc phenotype between hypoxic and hyperoxic conditions. Native Hc possesses a high oxygen affinity ( P50 = 2.2 Torr at 15 degrees C and pH 7.5), a large Bohr effect (Deltalog P50 / DeltapH approximately -2.7) and a slightly reverse temperature effect (DeltaH = +17.19 kJ mol(-1). The composition of Segonzacia haemolymph is similar to that of other littoral species except for the large enrichment in free copper and zinc. As for other species from hydrothermal vent sites, Segonzacia haemolymph possesses a higher buffer capacity than littoral species. Moreover, species from the hydrothermal vent decapods from Pacific hydrothermal vent that encounter higher CO2 content in their environment have a higher buffer capacity than Atlantic vent species. The results presented are discussed in relation with the physico-chemical characteristics of the hydrothermal vent environment.

All-trans retinoic acid is a ligand for the orphan nuclear receptor ROR beta.

Retinoids regulate gene expression through binding to the nuclear retinoic acid receptors (RARs) and retinoid X receptors (RXRs). In contrast, no ligands for the retinoic acid receptor-related orphan receptors beta and gamma (ROR beta and gamma) have been identified, yet structural data and structure-function analyses indicate that ROR beta is a ligand-regulated nuclear receptor. Using nondenaturing mass spectrometry and scintillation proximity assays we found that all-trans retinoic acid (ATRA) and several retinoids bind to the ROR beta ligand-binding domain (LBD). The crystal structures of the complex with ATRA and with the synthetic analog ALRT 1550 reveal the binding modes of these ligands. ATRA and related retinoids inhibit ROR beta but not ROR alpha transcriptional activity suggesting that high-affinity, subtype-specific ligands could be designed for the identification of ROR beta target genes. Our results identify ROR beta as a retinoid-regulated nuclear receptor, providing a novel pathway for retinoid action.

Unfolding of rabbit muscle creatine kinase induced by acid. A study using electrospray ionization mass spectrometry, isothermal titration calorimetry, and fluorescence spectroscopy.

Electrospray ionization mass spectrometry, isothermal titration calorimetry (ITC), fluorescence spectroscopy, and glutaraldehyde cross-linking SDS-PAGE have been used to study the unfolding of rabbit muscle creatine kinase (MM-CK) induced by acid. The mass spectrometric experiments show that MM-CK is unfolded gradually when titrated with acid. MM-CK is a dimer (the native state) at pH 7.0 and becomes an equilibrium mixture of the dimer and a partially folded monomer (the intermediate) between pH 6.7 and 5.0. The dimeric protein becomes an equilibrium mixture of the intermediate and an unfolded monomer (the unfolded state) between pH 5.0 and 3.0 and is almost fully unfolded at pH 3.0 reached. The results from a "phase diagram" method of fluorescence show that the conformational transition between the native state and the intermediate of MM-CK occurs in the pH range of 7.0-5.2, and the transition between the intermediate and the unfolded state of the protein occurs between pH 5.2 and 3.0. The intrinsic molar enthalpy changes for formation of the unfolded state of MM-CK induced by acid at 15.0, 25.0, 30.0, and 37.0 degrees C have been determined by ITC. A large positive molar heat capacity change of the unfolding, 8.78 kcal mol-1 K-1, at all temperatures examined indicates that hydrophobic interaction is the dominant driving force stabilizing the native structure of MM-CK. Combining the results from these four methods, we conclude that the acid-induced unfolding of MM-CK follows a "three-state" model and that the intermediate state of the protein is a partially folded monomer.

Comparative ESI-MS study of approximately 2.2 MDa native hemocyanins from deep-sea and shore crabs: from protein oligomeric state to biotope.

In the past years, the potential of electrospray ionization mass spectrometry (ESI-MS) for the observation of intact weak interactions, such as non-covalent protein-ligand, protein-protein, protein-DNA complexes, has spread out. The coupling of ESI with time-of-flight (TOF) and quadrupole-time-of-flight (Q-TOF) analyzers has even enabled the detection of larger complexes with molecular weights greatly higher than 200 kDa. In this paper, we report a comparative ESI-MS study on the protein quaternary structure of native hemocyanins (Hc) from crabs living in different biotopes: a shore crab (Carcinus maenas) and two deep-sea crabs (Segonzacia mesatlantica and Bythograea thermydron). Hc is an extracellular blood protein, composed of several protein chains which can associate in large multimers. The goal of this study is to point out that the oligomerization state of native Hcs is biotope-dependent. Depending on the crab, ESI-MS analyses under non-denaturing conditions reveal different oligomeric forms present in equilibrium in solution. Molecular weights up to 2,235 kDa were measured for the associations of 30 subunits of the Bythograea thermydron Hc. Thanks to ESI-MS analyses, it could be concluded for the first time that the oligomerization state of native Hcs is dependent on the crab environment. The investigation of these different non-covalent self-assemblies is very important for the life history of crabs, since they are directly related with different oxygen binding abilities and thus, with their ability to colonize habitats with different oxygen contents.