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PubMed2004

Supramolecular photomagnetic materials: photoinduced dimerization of ferrocene-based polychlorotriphenylmethyl radicals.

New ferrocenyl Schiff-base polychlorotriphenylmethyl radicals have been synthesized and characterized. The imino group of one such radical undergoes an irreversible trans to cis structural isomerization induced by light. Such photoinduced isomerization has been monitored by UV/Vis and ESR spectroscopy and also monitored by HPLC. ESR frozen solution experiments at low temperature revealed that the cis isomer dimerizes, showing a strong antiferromagnetic interaction. Although numerous photochromic supramolecular systems have been described, such a photoinduced self-assembly process represents the first example of a one-way photoswitchable magnetic system in which a conversion between a doublet and a singlet ground state species is promoted by a photoinduced dimerization process driven by the formation of hydrogen bonds. DFT calculations on the minimized structure and on the rotational barriers have been performed to establish the origin of such behavior. The effect of the substituents and the media polarity on the photoisomerization of this imine chromophore have also been studied. It has been observed that the efficiency of the process is markedly dependent on the presence and characteristics of electron-donor and electron-acceptor substituents of the ferrocenyl Schiff-base polychlorotriphenylmethyl radicals as well as on the polarity of the solvent.

Journal Article↗
PubMed2004

Ligand design for alkali-metal-templated self-assembly of unique high-nuclearity CuII aggregates with diverse coordination cage units: crystal structures and properties.

The construction of two unique, high-nuclearity Cu(II) supramolecular aggregates with tetrahedral or octahedral cage units, [(mu(3)-Cl)[Li subset Cu(4)(mu-L(1))(3)](3)](ClO(4))(8)(H(2)O)(4.5) (1) and [[Na(2) subset Cu(12)(mu-L(2))(8)(mu-Cl)(4)](ClO(4))(8)(H(2)O)(10)(H(3)O(+))(2)](infinity) (2) by alkali-metal-templated (Li(+) or Na(+)) self-assembly, was achieved by the use of two newly designed carboxylic-functionalized diazamesocyclic ligands, N,N'-bis(3-propionyloxy)-1,4-diazacycloheptane (H(2)L(1)) or 1,5-diazacyclooctane-N,N'-diacetate acid (H(2)L(2)). Complex 1 crystallizes in the trigonal R3c space group (a = b = 20.866(3), c = 126.26(4) A and Z = 12), and 2 in the triclinic P1 space group (a = 13.632(4), b = 14.754(4), c = 19.517(6) A, alpha = 99.836(6), beta = 95.793(5), gamma = 116.124(5) degrees and Z = 1). By subtle variation of the ligand structures and the alkali-metal templates, different polymeric motifs were obtained: a dodecanuclear architecture 1 consisting of three Cu(4) tetrahedral cage units with a Li(+) template, and a supramolecular chain 2 consisting of two crystallographically nonequivalent octahedral Cu(6) polyhedra with a Na(+) template. The effects of ligand functionality and alkali metal template ions on the self-assembly processes of both coordination supramolecular aggregates, and their magnetic behaviors are discussed in detail.

Journal Article↗
PubMed2003

tert-Butylphosphonic acid: from the bulk to the gas phase.

The structure of tert-butylphosphonic acid in the solid, in solution, and in the gas phase was studied by single-crystal X-ray diffraction, (1)H and (31)P NMR spectroscopic studies in solution, solid-state (31)P NMR spectroscopy, and electrospray ionization mass spectrometry. In addition, density functional theory (DFT) calculations at the B3LYP/6-31G*, B3LYP/6-31+G*, and B3LYP/6-311+G* level of theory for a large number of H-bonded aggregates of the type (tBuPO(3)H(2))(n) (C(n), P(n); n=1-7) support the experimental work. Crystallization of tBuPO(3)H(2) from polar solvents such as CH(3)CN or THF gives the H-bonded one-dimensional polymer 2, whereas crystallization from the less polar solvent CDCl(3) favors the formation of the H-bonded cluster (tBuPO(3)H(2))(6).CDCl(3) (1). In CDCl(3) the hexamer (tBuPO(3)H(2))(6) (C(6)) is replaced by smaller aggregates down to the monomer with decreasing concentration. DFT calculations and natural bond orbital (NBO) analyses for the clusters C(1)-C(7) and the linear arrays P(1)-P(7) reveal the hexamer C(6) to be the energetically favored structure resulting from cooperative strengthening of the hydrogen bonds in the H-bonded framework. However, the average hydrogen bond strengths calculated for C(6) and P(2) do not differ significantly (42-43 kJ mol(-1)). The average distances r(O.O), r(Obond;H), r(Pdbond;O), and r(Pbond;OH) in C(1)-C(7) and P(1)-P(7) are closely related to the hydrogen bond strength. Electrospray ionization mass spectrometry shows the presence of different anionic species of the type [(tBuPO(3)H(2))(n)-H](-) (A(1)-A(7), n=1-7) depending on the instrumental conditions. DFT calculations at the B3LYP/6-31G* level of theory were carried out for A(1)-A(6). We suggest the dimer [(tBuPO(3)H(2))(2)-H](-) (A(2)) and the trimer [(tBuPO(3)H(2))(3)-H](-) (A(3)) are the energetically favored anionic structures. A hydrogen bond energy of approximately 83 kJ mol(-1) was calculated for A(2). Electrospray ionization mass spectrometry is not suitable to study the assembling process of neutral H-bonded tert-butylphosphonic acid since the removal of a proton from the neutral aggregates has a large influence on the hydrogen bond strength and the cluster structure.

Journal Article↗
PubMed2005

From discrete particles to spherical aggregates: a simple approach to the self-assembly of Au colloids.

Here we demonstrate a simple, template-free approach to the formation of spherical gold aggregates through the reduction of HAuCl4 by NaBH4, in the presence of cysteine (Cys) as a capping agent. The resulting aggregates are quite stable in solution. The pH of the solution and the molar ratio of Au:Cys are two key empirical factors in the formation of such highly ordered aggregates. At slightly alkaline pH (7-10) and with Au:Cys ratios ranging from 1:0.5 to 1:2, spherical Au aggregates of 30-80 nm are formed. At lower Cys ratios (Au:Cys> or =1:0.5) very loosely linked aggregates are formed; however, at very high Cys ratios (Au:Cys< or =1:3), highly dispersed Au particles of 2-4 nm are obtained, which are virtually indistinguishable from the original colloidal form. Aggregate size is influenced markedly by component concentration; a 3-fold increase in standard levels resulted in Au spherical aggregates of a larger size, 200-500 nm. In addition, we used a combination of Cys and lysine (Lys) as a capping agent/cross-linker and found that the morphology of the Au colloid aggregates can be easily manipulated from a linear to a spherical form by adjusting the proportions of Cys and Lys in the capping agent/cross-linker mixture. The introduction of mercapto (SH)-containing organic acids reduced the cross-linking ability of Cys, especially in the case of long-chain acids. Complete disruption of the spherical aggregates highlights the importance of Cys per se. An explanation of this ordered self-assembly process is proposed, in the context of the known surface chemistry of Au colloids.

Journal Article↗
PubMed2002

Polyproline II structure in proteins: identification by chiroptical spectroscopies, stability, and functions.

In the last years polyproline II (PPII) structure has been demonstrated to be essential to biological activities such as signal transduction, transcription, cell motility, and immune response. The polyproline left-handed helical structure was nearly unknown until now and often confused with unordered, disordered, irregular, unstructured, extended, or random coil conformations because it is neither alpha-helical nor beta-turn nor beta-sheet, i.e., a classical structure. In spite of the regularity of the PPII structure and, more precisely, its well-defined dihedral angle values, a typical feature of PPII structure is the absence of any intramolecular hydrogen bonds that renders the PPII structure indistinguishable from an irregular backbone structure by (1)H-NMR spectroscopy. The only way to unambiguously reveal PPII structure in solution is to use spectroscopies based on optical activity, such as circular dichroism (CD), vibrational circular dichroism (VCD), and Raman optical activity (ROA). Herein we focus on the identification of PPII structure by CD, widely considered to be the most reliable methodology. Then we report on VCD and ROA spectroscopies as tools in the identification of PPII structure. A third section is dedicated to the analysis of the stabilization of PPII conformation in aqueous solution. Finally, the significance of PPII in self-assembly processes, in elasticity of elastomeric proteins, and in proteins-(peptides) proteins molecular recognition processes are considered.

Circular Dichroism↗
PubMed2005

Dynamics of Z-band based proteins in developing skeletal muscle cells.

During myofibril formation, Z-bodies, small complexes of alpha-actinin and associated proteins, grow in size, fuse and align to produce Z-bands. To determine if there were changes in protein dynamics during the assembly process, Fluorescence Recovery after Photobleaching was used to measure the exchange of Z-body and Z-band proteins with cytoplasmic pools in cultures of quail myotubes. Myotubes were transfected with plasmids encoding Yellow, Green, or Cyan Fluorescent Protein linked to the Z-band proteins: actin, alpha-actinin, cypher, FATZ, myotilin, and telethonin. Each Z-band protein showed a characteristic recovery rate and mobility. All except telethonin were localized in both Z-bodies and Z-bands. Proteins that were present both early in development in Z-bodies and later in Z-bands had faster exchange rates in Z-bodies. These results suggest that during myofibrillogenesis, molecular interactions develop between the Z-band proteins that decrease their mobility and increase the stability of the Z-bands. A truncated construct of alpha-actinin, which localized in Z-bands in myotubes and exhibited a very low rate of exchange, led to disruption of myofibrils, suggesting the importance of dynamic, intact alpha-actinin molecules for the formation and maintenance of Z-bands. Our experiments reveal the Z-band to be a much more dynamic structure than its appearance in electron micrographs of cross-striated muscle cells might suggest.

Animals↗
PubMed1994

A conserved region in the tail domain of vimentin is involved in its assembly into intermediate filaments.

Although the head and rod domains of intermediate filament (IF) proteins are known to play significant roles in filament assembly, the role of the tail domain in this function is unclear and the available information supports contradictory conclusions. We examined this question by comparing transfection of the same cDNA constructs, encoding vimentins with modified tail domains, into cell lines that do and do not contain endogenous IF proteins. By this approach, we were able to distinguish between the ability of a mutant IF protein to initiate assembly de novo, from that of incorporating into existing filament networks. Vimentins with modifications at or near a highly conserved tripeptide, arg-asp-gly (RDG), of the tail domain incorporated into existing IF networks in vimentin-expressing (vim+) cells, but were assembly-incompetent in cells that did not express IF proteins (vim-). The failure of the RDG mutant vimentins to assemble into filament arrays in vim- cells was reversible by re-introducing a wild-type vimentin cDNA, whereupon both wild-type and mutant vimentins coassembled into one and the same IF network. We conclude that the function of the tail domain of type III IF proteins, and possibly of keratins K8 and K18, in IF assembly is distinct from those of other domains; a region encompassing the RDG tripeptide appears to be important in the assembly process.

Adrenal Gland Neoplasms↗
PubMed1989

Surface expression of the T cell receptor complex requires charged residues within the alpha chain transmembrane region.

The T cell receptor (TcR) complex is a multi-subunit glycoprotein comprising at least five transmembrane polypeptides. An unusual characteristic of each of the transbilayer domains is the presence of charged amino acids. To examine the importance of these residues for the association and consequent surface expression of the components of the complex, a TcR alpha chain containing either charged or neutral residues within its transbilayer segment was introduced into the human T cell line MOLT-4, and the appearance of the TcR complex at the cell surface was assayed. Surface expression was observed only in MOLT-4 cells transfected with the alpha chain containing charged transbilayer residues. Thus, these residues most probably play a crucial role in the assembly process.

Amino Acids↗
PubMed1993

Analysis of the six additional chemicals for in vitro assays of the European Economic Communities' EEC aneuploidy programme using Saccharomyces cerevisiae D61.M and the in vitro porcine brain tubulin assembly assay.

We tested six additional chemicals (acetaldehyde, benomyl, diethylstilboestrol, diethylstilboestrol dipropionate, griseofulvin, and mercaptoethanol) for in vitro systems of the coordinated programme to study aneuploidy induction sponsored by the Commission of the European Communities in two in vitro test systems. Using Saccharomyces cerevisiae D61.M (mitotic chromosomal malsegregation assay), benomyl showed a dose-dependent increase in the frequency of chromosomal malsegregation with a lowest effective dose tested (LEDT) of 30 micrograms/ml (0.1 mM). Diethylstilboestrol (DES) showed solvent-dependent effects. DES dissolved in ethanol induced an increase in chromosomal malsegregation as well as in the frequency of total resistant colonies (mutations and recombinations) with a LEDT around 13 micrograms/ml (0.048 mM). Using dimethylsulfoxide as the solvent, no increases were observed with DES up to 333 micrograms/ml (1.24 mM). Acetaldehyde induced an increase in chromosomal malsegregation with the cold treatment protocol (LEDT: 1.25 microliters/ml (21 mM) and 0.75 microliters/ml (13 mM), respectively) but no increase with the overnight protocol (highest dose tested (HDT): 1.75 microliters/ml; 30 mM). Concerning the frequency of total cycloheximide-resistant colonies (mutations and recombinations) increases were obtained with both protocols. The other three compounds were negative when tested up to toxic doses (survival below 10%), up to the maximum solubility in the solvent used or up to heavy precipitation in the incubation mix. The HDT were 333 micrograms/ml (0.88 mM) for diethylstilboestrol dipropionate, 1,600 micrograms/ml (4.5 mM) for griseofulvin and 0.5 microliters/ml (7 mM) for mercaptoethanol. Concerning effects on porcine brain tubulin assembly in vitro, diethylstilboestrol and griseofulvin inhibited the assembly process. The IC30% (30% inhibition concentration) values were 12.5 microM and 100 microM for DES and griseofulvin, respectively. Mercaptoethanol showed no effects up to 50 mM.

Acetaldehyde↗
PubMed1984

The mechanism of self-assembly of the multi-enzyme complex tryptophan synthase from Escherichia coli.

The alpha subunit is bound with negative cooperativity to the holo beta 2 subunit of tryptophan synthase in phosphate buffer. Thus it is feasible to measure separately the rates of formation both of the stable alpha beta 2 subcomplex from beta 2, and of the mature alpha 2 beta 2 complex from alpha beta 2, using stopped-flow techniques. Addition of each alpha subunit proceeds in two steps; an initial alpha beta protomer is formed rapidly, which subsequently isomerizes slowly to the equilibrium state. The rates of dissociation of both the alpha beta 2 and alpha 2 beta 2 complexes were measured by trapping released alpha subunit with enzymically inactive reduced beta 2 subunit. The reversal of the slow isomerization both determines the rate of dissociation, and accounts for the high overall affinity of the beta protomer for the alpha subunit. The data fit to a sequential assembly mechanism consisting of seven protein species and yields values for most of the rate constants and all of the microscopic equilibrium constants. Negative cooperativity arises from a weaker initial binding of the second alpha subunit, as expressed by its larger off-constant, possibly due to steric hindrance. The kinetics of binding of L-serine and indolepropanol phosphate during the assembly process shows that the beta protomer is already partially activated in the initial alpha beta complex. Full activation is achieved in the slow isomerization reaction. In contrast, the alpha subunit gains high affinity for indolepropanol phosphate only in the isomerization reaction. These observations indicate that the isomerization involves synchronous conformation changes of both alpha and beta protomers.

Bacterial Proteins↗
PubMed1984

Assembly of transfected DNA into chromatin: structural changes in the origin-promoter-enhancer region upon replication.

Chimeric SV40 DNA containing only the early region, or plasmid DNA harboring the origin-promoter-enhancer region of SV40, when introduced into CV-1 or Cos-1 monkey cells by DEAE-dextran mediated transfer are rapidly assembled in a typical chromatin structure revealed by the generation of a regular 190 bp repeat ladder after micrococcal nuclease digestion. DNA replication is not required for this assembly process. Chromatin-specific DNase I hypersensitive sites are observed in the enhancer region of these minichromosomes. The pattern of the sites differs between non-replicating and post-replicated chromatin. The latter is identical to that observed in the lytic cycle. The presence of large T antigen is not sufficient for the shift in the structure of the chromatin. These experiments suggest that replication can modulate protein-DNA interactions during viral infection or upon cell differentiation.

Animals↗
PubMed1986

Both hydrophobic domains of M13 procoat are required to initiate membrane insertion.

M13 procoat protein has two hydrophobic domains, one in the leader peptide and one which anchors the mature coat protein in the membrane. Disruption of the membrane anchor region by insertion of arginyl residues does not yield periplasmic coat protein. Instead, the rate of membrane assembly is slowed greater than 100-fold (t1/2 less than 5 s for wild-type, t1/2 greater than 10 min for mutant). The hydrophobic region of mature coat protein not only functions as a membrane anchor, but has an important role in the membrane assembly process per se.

Amino Acid Sequence↗
PubMed1989

Isolation of scid pre-B cells that rearrange kappa light chain genes: formation of normal signal and abnormal coding joins.

Consistent with an ordered immunoglobulin (Ig) gene assembly process during precursor (pre-) B cell differentiation, we find that most Abelson murine leukemia virus (A-MuLV)-transformed pre-B cells derived from scid (severe combined immune deficient) mice actively form aberrant rearrangements of their Ig heavy chain locus but do not rearrange endogenous kappa light chain variable region gene segments. However, we have identified several scid A-MuLV transformants that transcribe the germline Ig kappa light chain constant region and actively rearrange the kappa variable region gene locus. In one case progression to the stage of kappa light chain gene rearrangement did not require expression of Ig mu heavy chains; furthermore, this progression could not be efficiently induced following expression of mu heavy chains from an introduced vector. As observed in pre-B cell lines from normal mice, attempted V kappa-to-J kappa rearrangements in scid transformants occur by inversion at least as frequently as by deletion. The inverted rearrangements result in retention of both products of the recombination event in the chromosome, thus allowing their examination. scid kappa coding sequence joins are aberrant and analogous in structure to previously described scid heavy chain coding joins. In contrast, the recognition signals that flank involved coding segments frequently are joined precisely back-to-back in normal fashion. The scid VDJ recombinase defect therefore does not significantly impair recognition of, site-specific cutting at, or juxtaposition and appropriate ligation of signal sequences. Our finding that the scid defect prevents formation of correct coding but not signal joins distinguishes these events mechanistically.

Abelson murine leukemia virus↗
PubMed1994

The influenza virus hemagglutinin cytoplasmic tail is not essential for virus assembly or infectivity.

The influenza A virus hemagglutinin (HA) glycoprotein contains a cytoplasmic tail which consists of 10-11 amino acids, of which five residues re conserved in all subtypes of influenza A virus. As the cytoplasmic tail is not needed for intracellular transport to the plasma membrane, it has become virtually dogma that the role of the cytoplasmic tail is in forming protein-protein interactions necessary for creating an infectious budding virus. To investigate the role of the HA cytoplasmic tail in virus replication, reverse genetics was used to obtain an influenza virus that lacked an HA cytoplasmic tail. The rescued virus contained the HA of subtype A/Udorn/72 in a helper virus (subtype A/WSN/33) background. Biochemical analysis indicated that only the introduced tail- HA was incorporated into virions and these particles lacked a detectable fragment of the helper virus HA. The tail- HA rescued virus assembled and replicated almost as efficiently as virions containing wild-type HA, suggesting that the cytoplasmic tail is not essential for the virus assembly process. Nonetheless, a revertant virus was isolated, suggesting that possession of a cytoplasmic tail does confer an advantage.

Amino Acid Sequence↗
PubMed1995

In vivo iodination of a misfolded proinsulin reveals co-localized signals for Bip binding and for degradation in the ER.

The signal for degradation of proteins in the endoplasmic reticulum (ER) is thought to be the exposure of internal domains which are buried when the protein has adopted its correct conformation and which are also exposed in assembly intermediates. This raises the question of why the intermediates are not degraded. We developed a system based on the peroxidase-catalyzed iodination of tyrosine residues which continuously monitors the exposure of internal domains of proinsulin. In CHO cells this system discriminated between assembly intermediates of wild type (wt) proinsulin and misfolded proinsulin, as shown by the exclusive iodination of a misfolded mutant which was finally degraded in the ER. Iodination in vitro showed that the assembly intermediates of wt proinsulin also exposed internal domains. This iodination was inhibited by the addition of the molecular chaperone Bip which was co-immunoprecipitated with proinsulin in CHO cells. The results obtained with the mutant proinsulin support the assumption that exposed internal domains represent the signal for degradation in the ER. Observations of wt proinsulin show that Bip masks internal domains of normal assembly intermediates during the entire assembly process, thereby suppressing their degradation. We propose that internal domains contain co-localized signals for Bip binding and for degradation.

Adenosine Triphosphate↗
PubMed1995

A domain sharing model for active site assembly within the Mu A tetramer during transposition: the enhancer may specify domain contributions.

The functional configuration of Mu transposase (A protein) is its tetrameric form. We present here a model for the organization of a functional Mu A tetramer. Within the tetramer, assembly of each of the two active sites for Mu end cleavage requires amino acid contributions from the central and C-terminal domains (domains II and III respectively) of at least two Mu A monomers in a trans configuration. The Mu enhancer is likely to function in this assembly process by specifying the two monomers that provide their C-terminal domains for strand cleavage. The Mu B protein is not required in this step. Each of the two active sites for the strand transfer reaction is also organized by domain sharing (but in the reverse mode) between Mu A monomers; i.e. a donor of domain II (also the recipient of domain III) during cleavage is a recipient of domain II (and the donor of domain III) during strand transfer. The function of the Mu B protein (which is required at the strand transfer step) and that of the enhancer element may be analogous in that their interactions with Mu A (domain III and domain I alpha respectively) promote conformations of Mu A conducive to strand cleavage or strand transfer.

Bacteriophage mu↗
PubMed1976

The oral apparatus of Tetrahymena pyriformis, strain WH-6. II. Cytochalasin B inhibition of oral apparatus morphogenesis.

The effects of cytochalasin B on oral apparatus morphogenesis and cell division were studied in synchronized Tetrahymena pyriformis, strain WH-6 syngen 1. Cytochalasin B brought about the rapid arrest of oral apparatus primordium development when added prior to the completion of oral apparatus membranelle differentiation. Cells arrested in development did not divide. When cytochalasin B was added after this transition point, oral apparatus morphogenesis and cell division were completed. The effects of cytochalasin B could be reversed by washing it from the medium. Even though cytochalasin B (at 400 mug/ml) reduced protein synthesis by 30%, the data are consistent with the interpretation that cytochalasin B prevents an assembly process during the membranelle differentiation phase of oral apparatus development.

Animals↗
PubMed1995

Reassembly of the 66 kD neurofilament protein in vitro following isolation and purification from bovine spinal cord.

NF-66, also known as alpha-internexin, has been characterized as a 66 kD mammalian neurofilament (NF) protein whose expression in developing rat brain precedes that of the low molecular weight NF protein (NF-L). NF-66 is thought to assemble into 10 nm diameter intermediate filaments in vitro, although the precise nature of the assembly process remains obscure. Likewise, the ability of NF-66 to polymerize with the low (NF-L), middle (NF-M), and high (NF-H) M(r)NF proteins has not been defined. This investigation describes the reassembly of bovine NF-66 regarding its formation into 10 nm diameter filaments as well as its potential for polymerization with other type IV intermediate filaments. NF-66 and the NF triplet proteins were isolated from bovine spinal cord using established biochemical extraction and isolation procedures (Balin et al., Brain Res 556:181-195, 1991), and purified by a combination of high performance liquid chromatography (HPLC) (DEAE anion exchange and hydroxylapatite column chromatography) and gel elution strategies. In vitro reassembly experiments revealed that NF-66 formed approximately 10 nm diameter filaments of varying length; immunoelectron microscopy demonstrated labeling of these filaments by a monoclonal antibody to intermediate filament antigen (IFA), a polyclonal antibody against rat NF-66 and by a monoclonal antibody generated against the core region of NF-M but cross-reactive with NF-66. This report is the first investigation to look at the in vitro interaction between NF-66 and other type IV intermediate filament proteins (NF-H, -M, and -L) and establishes that NF-66 forms heteropolymeric filaments with these other neurofilament proteins, as confirmed by double immunolabeling. These studies suggest that NF-66 could provide a nucleation site for the polymerization of later-expressed proteins during neuronal development.

Animals↗