Linker Chains Research Articles

Identification of potential cellular targets of aloisine A by affinity chromatography

Affinity chromatography was used to identify potential cellular targets of aloisine A (7- n-butyl-6-(4′-hydroxyphenyl)- 5H-pyrrolo[2,3 b]pyrazine), a potent inhibitor of cyclin-dependent kinases. This technique is based on the immobilization of the drug on a solid matrix, followed by identification of specifically bound proteins. To this end, both aloisine A and the protein-kinase inactive control N-methyl aloisine, bearing extended linker chains have been synthesized. We present the preparation of such analogues having the triethylene glycol chain at different positions of the molecule, as well as their immobilization on an agarose-based matrix. Affinity chromatography of various biological extracts on the aloisine matrices allowed the identification of both protein kinases and non-kinase proteins as potential cellular targets of aloisine.

Synthesis and cellular uptake properties of guanidine-containing molecular transporters built on the sucrose scaffold

Novel sucrose-based G7 molecular transporters show different patterns of intracellular localization depending on the nature of the linker chains as well as the fluorescent dyes.

Novel Guanidine-Containing Molecular Transporters Based on Lactose Scaffolds: Lipophilicity Effect on the Intracellular Organellar Selectivity

We have synthesized two lactose-based molecular transporters, each containing seven guanidine residues attached to the lactose scaffold through omega-aminocarboxylate linker chains of two different lengths, and have examined their cellular uptakes and intracellular and organellar localizations in HeLa cells, as well as their tissue distributions in mice. Both molecular transporters showed higher cellular uptake efficiencies than Arg8, and wide tissue distributions including the brain. Mitochondrial localization is of special interest because of its potential relevance to "mitochondrial diseases". Interestingly, it has been found that the intracellular localization sites of the G7 molecular transporters-namely either mitochondria or lysosomes and endocytic vesicles-are largely determined by the linker chain lengths, or their associated lipophilicities.

Polyfluoroalkyl, Polyethylene Glycol, 1,4-Bismethylenebenzene, or 1,4-Bismethylene-2,3,5,6-Tetrafluorobenzene Bridged Functionalized Dicationic Ionic Liquids: Synthesis and Properties as High Temperature Lubricants

A new class of geminal dicationic ionic liquids was formed with a bridging moiety, such as a polyalkyl ether, polyfluoroalkyl, 1,4-bismethylenebenzene, or 1,4-bismethylene-2,3,5,6-tetrafluorobenzene link, between alkyl-substituted imidazolium rings. Their properties were modified by varying the linker chains and/or alkyl substituents on the imidazolium ring. The polyfluoroalkyl bridged dicationic ionic liquids exhibit the highest densities and viscosities. Although melting points are directly proportional to the length of the alkyl substituent, the densities decrease concomitantly. With 1,4-bismethylene-2,3,5,6-tetrafluorobenzene as the linking chain and with longer alkyl substituents on the imidazolium rings, the nonpolar character of the ionic liquid greatly increases, e.g., the solubilities in toluene of dicationic ionic liquids 36 and 37, where the ring substituents are C10H21 and C14H29, increase markedly. Some of the salts exhibit higher conductivities than an equimolar tetrabutylammonium iodide solution in acetonitrile/toluene. These new ionic liquids (except with PF6− as anion) display outstanding tribological properties in temperature ramp tests by performing very well at 300 °C, thus meeting one criterion for high-temperature lubricants.

Interaction of giant extracellular Glossoscolex paulistus hemoglobin (HbGp) with ionic surfactants: A MALDI-TOF-MS study

The giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) is constituted by approximately 144 subunits containing heme groups with molecular masses in the range of 16–19 kDa forming a monomer ( d) and a trimer ( abc), and around 36 non-heme structures, named linkers ( L). Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF-MS) analysis was performed recently, to obtain directly information on the molecular masses of the different subunits from HbGp in the oxy-form. This technique demonstrated structural similarity between HbGp and the widely studied hemoglobin of Lumbricus terrestris (HbLt). Indeed, two major isoforms ( d 1 and d 2) of identical proportions with masses of 16,355 ± 25 and 16,428 ± 24 Da, respectively, and two minor isoforms ( d 3 and d 4) with masses around 16.6 kDa were detected for monomer d of HbGp. In the present work, the effects of anionic sodium dodecyl sulfate (SDS) and cationic cethyltrimethylammonium chloride (CTAC) on the oligomeric structure of HbGp have been studied by MALDI-TOF-MS in order to evaluate the interaction between ionic surfactants and HbGp. The data obtained with this technique show an effective interaction of cationic surfactant CTAC with the two isoforms of monomer d, d 1 and d 2, both in the whole protein as well as in the pure isolated monomer. The results show that up to 10 molecules of CTAC are bound to each isoform of the monomer. Differently, the mass spectra obtained for SDS–HbGp system showed that the addition of the anionic surfactant SDS does not originate any mass increment of the monomeric subunits, indicating that SDS–HbGp interaction is, probably, significantly less effective as compared to CTAC–HbGp one. The acid p I of the protein around 5.5 is, probably, responsible for this behavior. The results of this work suggest also some interaction of both surfactants with linker chains as well as with trimers, as judged from observed mass increments. Our data are consistent with a recent spectroscopic study showing a strong interaction between CTAC and HbGp at physiological pH [P.S.Santiago, et al, Biochim. Biophys. Acta 1770 (2007) 506-517.].

Synthesis and cellular uptake of cell delivering 2,6-pyridinediylbisalkanamide submicron-sized sheets in HeLa cells

2,6-Pyridinediylbisalkanamides were synthesized, using diaminopyridine (DAP) as a linker and alkyl chains of varying lengths, that upon self-assembly form submicron-sized sheets and their uptake into the cytoplasm of HeLa cells was studied by confocal microscopy.

Dicationic molten salts (ionic liquids) as re-usable media for the controlled pyrolysis of cellulose to anhydrosugars

Molten dicationic imidazolium chloride or bromide salts, with short alkyl linker chains, have been found to be stable, re-usable media for the controlled pyrolysis of cellulose without acid pre-treatment to give anhydrosugars, primarily levoglucosenone, at a substantially lower temperature (180 °C) than most previously reported methods (typically > 300 °C).

Partial characterization of giant extracellular hemoglobin of Glossoscolex paulistus: A MALDI-TOF-MS study

In this work, MALDI-TOF-MS analysis was performed to obtain information on the molecular mass of the different subunits from the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) in the oxy-form. Experiments were performed for the whole protein at pH 7.0, for the partially dissociated protein at pH 9.0, and for the fraction obtained from gel filtration in Sephadex G-200, at pH 9.0, corresponding to the isolated monomer d. Besides that, experiments were performed for the whole protein treated with 2-mercaptoethanol in order to monitor the effects of reduction of the disulfide bonds, which are expected to maintain the trimer ( abc) in the native molecule. The results are compared to those reported for the homologous hemoglobin of Lumbricus terrestris (HbLt) and some tentative assignments are made for the observed polypeptides. The monomer d is found to exist in, at least, two major forms of identical proportions with masses of 16,355 ± 25 and 16,428 ± 24 Da, respectively. Two minor forms were also observed around 16 kDa for the monomers. Upon disulfide bonds reduction the peak associated to the trimer is absent in the mass spectrum, and new peaks assigned tentatively to the monomers a, b and c on the basis of comparison with Lumbricus terrestris hemoglobin literature data are observed. Their molecular masses were 18,258 ± 30, 16,492 ± 24 and 17,363 ± 17 Da, respectively. Two linker chains for HbGp were also observed at 25,817 ± 50 and 26,761 ± 16 Da, and this result is different from HbLt, where four linker chains were reported in the range 24–32 kDa. Finally, trimers ( abc) were observed at 51–52 kDa. This partial characterization, performed for the first time, is an important step in the characterization of subunits of this giant extracellular hemoglobin.

Transport Properties of Solid Polymer Electrolytes Prepared from Oligomeric Fluorosulfonimide Lithium Salts Dissolved in High Molecular Weight Poly(ethylene oxide)

Transport properties such as ionic conductivity, lithium transference number, and apparent salt diffusion coefficient are reported for solid polymer electrolytes (SPEs) prepared using several oligomeric bis[(perfluoroalkyl)sulfonyl]imide (fluorosulfonimide) lithium salts dissolved in high molecular weight poly(ethylene oxide) (PEO). The salt series consists of polyanions in which two discrete fluorosulfonimide anions are linked together by [(perfluorobutylene)disulfonyl]imide linker chains. The restricted diffusion technique was used to measure the apparent salt diffusion coefficients in SPEs, and cationic transference numbers were determined using both potentiostatic polarization and electrochemical impedance spectroscopy methods. A general trend of diminished salt diffusion coefficient with increasing anion size was observed and is opposite to the trend observed in ionic conductivity. This unexpected finding is rationalized in terms of the cumulative effects of charge carrier concentration, anion mobility, ion pairing, host plasticization by the anions, and salt phase segregation on the conductivity.

Observation of Non-Förster-Type Energy-Transfer Behavior in Quantum Dot−Phthalocyanine Conjugates

The effect of linker chain length on the energy transfer from CdSe quantum dots (QDs) to silicon phthalocyanine (Pc) photodynamic therapy agents was investigated by steady-state and femtosecond time-resolved spectroscopy with 500 nm light for the specific excitation of the QD energy donor. The conjugation between the QD and the Pc was achieved with linker chains varying from 4 to 9 bond lengths by incorporating 1-6 methylene groups into the axial ligand of the Pc. With increasing chain length, the energy-transfer efficiency increased, which appears to be opposed to a purely Förster-type resonance energy-transfer behavior that is commonly discussed for the energy transfer in QD conjugates. The obtained results provide strong evidence for a capping-layer-mediated energy transfer in the QD-based donor-acceptor conjugates.

Gene Structure and Molecular Phylogeny of the Linker Chains from the Giant Annelid Hexagonal Bilayer Hemoglobins

Giant extracellular hexagonal bilayer hemoglobin (HBL-Hb), found only in annelids, is an approximately 3500-kDa heteropolymeric structure involved in oxygen transport. The HBL-Hbs are comprised of globin and linker chains, the latter being required for the assembly of the quaternary structure. The linker chains, varying in size from 225 to 283 amino acids, have a conserved cysteine-rich domain within their N-terminal moiety that is homologous to the cysteine-rich modules constituting the ligand binding domain of the low-density lipoprotein receptor (LDLR) protein family found in many metazoans. We have investigated the gene structure of linkers from Arenicola marina, Alvinella pompejana, Nereis diversicolor, Lumbricus terrestris, and Riftia pachyptila. We found, contrary to the results obtained earlier with linker genes from N. diversicolor and L. terrestris, that in all of the foregoing cases, the linker LDL-A module is flanked by two phase 1 introns, as in the human LDLR gene, with two more introns in the 3' side whose positions varied with the species. In addition, we obtained 13 linker cDNAs that have been determined experimentally or found in the EST database LumbriBASE. A molecular phylogenetic analysis of the linker primary sequences demonstrated that they cluster into two distinct families of linker proteins. We propose that the common gene ancestor to annelid linker genes exhibited a four-intron and five-exon structure and gave rise to the two families subsequent to a duplication event.

When Size Matters

Erythrocruorins are very large extracellular hemoglobins found in some invertebrates. In this issue of Structure, Royer et al. (2006) report the structure of the 3.6 MDa, 180-mer of Lumbricus (earthworm) hemoglobin consisting of 144 globin and 36 linker chains.

Linker chains of the gigantic hemoglobin of the earthworm Lumbricus terrestris: Primary structures of linkers L2, L3, and L4 and analysis of the connectivity of the disulfide bonds in linker L1

The extracellular hemoglobin (Hb) of the earthworm, Lumbricus terrestris, has four major kinds of globin chains: a, b, c, and d, present in equimolar proportions, and additional non-heme, non-globin scaffolding chains called linkers that are required for the calcium-dependent assembly of the full-sized molecule. The amino acid sequences of all four of the globin chains and one of the linkers (L1) have previously been determined. The amino acid sequences via cDNA of each of the three remaining linkers, L2, L3, and L4, have been determined so that the sequences of all constituent polypeptides of the hemoglobin are now known. Each linker has a highly conserved cysteine-rich segment of approximately 40 residues that is homologous with the seven ligand-binding repeats of the human low-density lipoprotein receptor (LDLR). Analysis of linker L1 shows that the connectivity of the three disulfide bonds is exactly the same as in the LDLR ligand-binding repeats. The presence of a calcium-binding site comprising one glutamyl and three aspartyl residues in both the LDLR repeats and in the linkers supports the suggestion that calcium is required for the folding and disulfide connectivity of the linkers as in the LDLR repeats. Linker L2 is markedly heterogeneous and contains unusual glycine-rich sequences near the NH2-terminus and a polar zipper-like sequence with imperfect repeats of Asp-Asp-His at the carboxyl terminus. Similar Asp-Asp-His repeats have been found in a protein homologous to superoxide dismutase in the hemolymph of certain mussels. These repeats may function as metal-binding sites.

Adhesion of PDMS Elastomers to Functional Substrates

ABSTRACT The JKR technique was applied to study the influence of interfacial reactions on the adhesion between functional elastomer gels and functional solid substrates. The gelation chemistry of poly(dimethyl siloxane) (PDMS) gels cured by hydrosilylation reactions was purposely adjusted to produce an excess of either silane or vinyl functionality. Hemispherical lenses of these materials were then contacted under load with a variety of functionalized solid substrates: poly(styrene-b-butadiene) copolymers with vinyl functionality, vinyl-terminated trimethoxysilane self-assembled monolayers, and α,ω-functional PDMS brushes terminated with either monomethoxy or hydroxyl groups. To rule out chain interpenetration effects, the molecular weights were kept below the entanglement molecular weight or immiscible polymers were employed on opposite sides of the interface. Significant adhesion enhancement was observed for most systems, indicating that a variety of different interfacial reactions can occur across the interface between PDMS elastomers and solid polymeric substrates. The overall nature of the adhesion enhancement found is consistent with the predictions of the Lake–Thomas theory for failure of elastomers, increasing linearly with the length and areal density of covalent linker chains that span the interface.

The sulfide binding function of annelid hemoglobins: relic of an old biosystem?

Invertebrates living in sulfide-rich environments have developed different strategies of coping with sulfide toxicity. Some bivalves and annelids have hemoglobins that are capable of binding sulfide for detoxification and/or transporting it to internal bacterial symbionts. Annelids living in the sulfide-rich environments have giant (∼3.6 MDa) hemoglobin, consisting of 144 globin chains arranged in a hexagonal bilayer structure held together by 36 nonglobin linker chains. Some globin chains contain either a free cysteine residue at positions Cys + 1 or at position Cys + 11 relative to the E7 distal residue in the E helix and EF interhelical region, respectively, which bind sulfide. The hexagonal bilayer hemoglobins of annelids living in environments lacking sulfide, do not have the corresponding free cysteine residues and cannot bind sulphide. Given that the early stages of life occurred under anoxic conditions in the presence of sulfide, it is possible that the sulfide binding function from modern annelid globins inhabiting sulphide rich habitats is an evolutionary relic. This proposal seems supported by the recent finding of “protoglobins” which also have a corresponding cysteine residue in Archea known to exist in hyperthermophilic and sulfide-rich environments.

Optically Active Tropocoronands Having Amino Acid Residues in Linker Chains: Syntheses, Metal Coordination Properties, and Their Abilities as an Asymmetric Catalyst

Optically active tropocoronands (7) having L-amino acid moieties were synthesized by stepwise and one-pot reactions of tropolone derivatives with amino acid linker chains. The coordination styles of their Ni(II) and Pd(II) complexes and the abilities as an asymmetric catalyst were investigated.

An electrospray ionization mass spectrometric study of the subunit structure of the giant hemoglobin from the leech Nephelopsis oscura

The subunit structure of the giant, extracellular hexagonal bilayer (HBL) hemoglobin (Hb) from the leech Nephelopsis oscura was investigated by electrospray ionization mass spectrometry (ESI-MS) employing a maximum entropy deconvolution of its complex, multiply charged ESI spectra. The denatured unreduced Hb consisted of three monomer globin chains (M), a1 = 16,535 Da, a2 = 17,171 Da and a3 = 17,315 Da, five nonglobin linker chains, L1 = 24,512 Da, L2 = 24,586 Da, L3 = 24,979 Da, L4 = 25,006 Da, and L5 = 25,566 Da and two subunits of 32,950 Da and 33,125 Da. ESI-MS of the denatured, reduced Hb showed that the latter were disulfide-bonded heterodimers (D) of globin chains b1 = 16,322 Da and b2 = 16,499 Da with chain c = 16,632 Da. Time-of-flight ESI-MS of the Hb at pH 3.8, 4.5, 5.0, 5.8 and 7.0 revealed a distribution of charge states from 32 + to 37 + with masses decreasing from 211 to 208.5 kDa with increase in cone voltage from 60 to 160 V, indicating the presence of a subassembly comprising 12 globin chains. The subunit composition 6M + 3D + 12h, where M = 16993 Da and D = 33004 Da are the weighted masses and h = 616.5 Da, provides a calculated mass, 208.37 kDa that is closest to 208.5 kDa. Our experimental findings are consistent with the bracelet model of HBL Hbs, verified by the recent low-resolution crystal structure of Lumbricus Hb, wherein an HBL arrangement of 12 globin dodecamer subassemblies is tethered to a central complex of 36 linker chains for a total mass of 208.37 × 12 + 24.94 × 36 = 3398 kDa.

The stoichiometry of the four linker subunits of Lumbricus terrestris hemoglobin suggests an asymmetric distribution

The extracellular, giant (∼3.6 MDa) hexagonal bilayer hemoglobin of the earthworm Lumbricus terrestris consists of 12 dodecamers of globin chains tethered to a central complex of 36 non-globin, linker chains (24–32 kDa). Four types of linker chains L1–L4 have been detected by electrospray ionization (ESI) and by matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) and isolated by reversed phase high pressure liquid chromatography (HPLC). Deconvolution of the HPLC elution profile and of the MS spectra provided the following individual linker contents, expressed as percent of the sum of the four linker peak areas: HPLC-21% L1, 37% L2, 23% L3 and 19% L4, MALDI-47% L1, 29% L2, 16% L3 and 8% L4; ESI-24% L1, 16% L2, 40% L3 and 20% L4; respectively. Comparison with electrophoretic results revealed a surprising lack of overall agreement between all the methods. The calculated mean values of the available linker contents were found to be 32±12% L1, 28±9% L2, 27±10% L3 and 13±7% L4, suggesting the following relative stoichiometry: L1: L2: L3: L4 ≈1: 1: 1: 0.5. With a total of 36 linkers, a hexagonally symmetric distribution of each of the four linker chains is impossible. Thus, the asymmetric linker distribution provides an explanation for the existence of a large dipole moment of Lumbricus terrestris hemoglobin, 17300±2300 Da ( Takashima et al., 1999).

Mass distributions of a macromolecular assembly based on electrospray ionization mass spectrometric masses of the constituent subunits.

Macromolecular assemblies containing multiple protein subunits and having masses in the megadalton (MDa) range are involved in most of the functions of a living cell. Because of variation in the number and masses of subunits, macromolecular assemblies do not have a unique mass, but rather a mass distribution. The giant extracellular erythrocruorins (Ers), approximately 3.5 MDa, comprised of at least 180 polypeptide chains, are one of the best characterized assemblies. Three-dimensional reconstructions from cryoelectron microscopic images show them to be hexagonal bilayer complexes of 12 subassemblies, each comprised of 12 globin chains, anchored to a subassembly of 36 nonglobin linker chains. We have calculated the most probable mass distributions for Lumbricus and Riftia assemblies and their globin and linker subassemblies, based on the Lumbricus Er stoichiometry and using accurate subunit masses obtained by electrospray ionization mass spectrometry. The expected masses of Lumbricus and Riftia Ers are 3.517 MDa and 3.284 MDa, respectively, with a possible variation of approximately 9% due to the breadth of the mass distributions. The Lumbricus Er mass is in astonishingly good agreement with the mean of 23 known masses, 3.524 +/- 0.481 MDa.

Revisiting the structure of Alvinella pompejana hemoglobin at 20 Å resolution by cryoelectron microscopy

The hemoglobin of the polychaete worm Alvinella pompejana was reconstructed at 20 Å resolution from frozen-hydrated samples observed by electron microscopy according to the random conical tilt series method. This three-dimensional reconstruction was mirror-inverted with respect to a previous volume published by de Haas et al. in 1996. In order to explain this handedness discrepancy, various 3D reconstructions using different reference volumes were carried out showing that the choice of the first volume was the keystone during the refinement process. The 3D reconstruction volume of A. pompejana Hb presented structural features characteristic of annelid Hbs with two hexagonal layers each comprising six hollow globular subassemblies and a complex of non-heme linker chains. Moreover, the eclipsed conformation of the two hexagonal layers and a HGS architecture similar to that described for Arenicola marina Hb led to the conclusion that A. pompejana Hb belonged to the architectural type II according to the definition of Jouan et al. (2001). A comparison between this cryo-EM volume and X-ray crystallography density maps of Lumbricus terrestris type-I Hb (Royer et al., 2000) showed that the triple stranded coiled coil structures of linker chains were different. Based on this observation, a model was proposed to explain the eclipsed conformation of the two hexagonal layers of type-II Hbs.