Covalent Stabilization Research Articles

Enzyme-based biohybrid foams designed for continuous flow heterogeneous catalysis and biodiesel production

The one-pot synthesis and use of monolithic biohybrid foams in a continuous flow device reported in here presents the advantages of covalent stabilization of the enzymes, together with a low steric hindrance between proteins and substrates, optimized mass transport due to the interconnected macroporous network and simplicity with regard to the column in situ synthetic path. Those features, when applied to transesterification (biodiesel production) via enzyme catalysis, provide among the top enzymatic activities displayed by biohybrid catalysts bearing unprecedented endurance of continuous catalysis for a two month period.

Reactivity and Applications of New Amine Reactive Cross-Linkers for Mass Spectrometric Detection of Protein−Protein Complexes

Chemical cross-linking of proteins permits the stabilization of noncovalent complexes through introduction of covalent bonds. A crucial challenge is to find the fastest and most efficient cross-linkers in order to minimize reaction times and to handle delicate complexes. New cross-linkers were synthesized by introducing N-hydroxyphthalimide, hydroxybenzotriazole, and 1-hydroxy-7-azabenzotriazole as leaving groups instead of the commonly used N-hydroxysuccimidyl moiety. With the use of matrix-assisted laser desorption ionization (MALDI) mass spectrometry, these new cross-linkers were then compared with the commercially available disuccinimidyl suberate (DSS) for covalent stabilization of the gluthatione-S-transferase (GST) dimer and of an antibody-antigen complex. They showed a better efficiency, generated about 30% more cross-linked complex, and reacted about 10 times faster than DSS. The reaction with the GST dimer was utilized to get information about their reaction efficiency and kinetics. Their ability to stabilize only specific protein complexes was verified by incubating them with a mixture of the proteins GST and ubiquitin. Finally, the cross-linkers were incubated with synthetic peptides to study the selectivity of the binding with various amino acid side chains. Not only lysine but also tyrosine was found to react with the newly synthesized cross-linker containing 1-hydroxy-7-azabenzotriazole as the reactive group.

Functional Importance of a Structurally Distinct Homodimeric Complex of the Family B G Protein-Coupled Secretin Receptor

Oligomerization of G protein-coupled receptors has been described, but its structural basis and functional importance have been inconsistent. Here, we demonstrate that the agonist occupied wild-type secretin receptor is predominantly in a guanine nucleotide-sensitive high-affinity state and exhibits negative cooperativity, whereas the monomeric receptor is primarily in a guanine nucleotide-insensitive lower affinity state. We previously demonstrated constitutive homodimerization of this receptor through the lipid-exposed face of transmembrane (TM) IV. We now use cysteine-scanning mutagenesis of 14 TM IV residues, bioluminescence resonance energy transfer (BRET), and functional analysis to map spatial approximations and functional importance of specific residues in this complex. All, except for three helix-facing mutants, trafficked to the cell surface, where secretin was shown to bind and elicit cAMP production. Cells expressing complementary-tagged receptors were treated with cuprous phenanthroline to establish disulfide bonds between spatially approximated cysteines. BRET was measured as an indication of receptor oligomerization and was repeated after competitive disruption of oligomers with TM IV peptide to distinguish covalent from noncovalent associations. Although all constructs generated a significant BRET signal, this was disrupted by peptide in all except for single-site mutants replacing five residues with cysteine. Of these, covalent stabilization of receptor homodimers through positions of Gly(243), Ile(247), and Ala(250) resulted in a GTP-sensitive high-affinity state of the receptor, whereas the same procedure with Ala(246) and Phe(240) mutants resulted in a GTP-insensitive lower affinity state. We propose the existence of a functionally important, structurally specific high-affinity dimeric state of the secretin receptor, which may be typical of family B G protein-coupled receptors.

Denaturation and unfolding of human anaphylatoxin C3a: An unusually low covalent stability of its native disulfide bonds

The complement C3a anaphylatoxin is a major molecular mediator of innate immunity. It is a potent activator of mast cells, basophils and eosinophils and causes smooth muscle contraction. Structurally, C3a is a relatively small protein (77 amino acids) comprising a N-terminal domain connected by 3 native disulfide bonds and a helical C-terminal segment. The structural stability of C3a has been investigated here using three different methods: Disulfide scrambling; Differential CD spectroscopy; and Reductive unfolding. Two uncommon features regarding the stability of C3a and the structure of denatured C3a have been observed in this study. (a) There is an unusual disconnection between the conformational stability of C3a and the covalent stability of its three native disulfide bonds that is not seen with other disulfide proteins. As measured by both methods of disulfide scrambling and differential CD spectroscopy, the native C3a exhibits a global conformational stability that is comparable to numerous proteins with similar size and disulfide content, all with mid-point denaturation of [GdmCl] 1/2 at 3.4–5 M. These proteins include hirudin, tick anticoagulant protein and leech carboxypeptidase inhibitor. However, the native disulfide bonds of C3a is 150–1000 fold less stable than those proteins as evaluated by the method of reductive unfolding. The 3 native disulfide bonds of C3a can be collectively and quantitatively reduced with as low as 1 mM of dithiothreitol within 5 min. The fragility of the native disulfide bonds of C3a has not yet been observed with other native disulfide proteins. (b) Using the method of disulfide scrambling, denatured C3a was shown to consist of diverse isomers adopting varied extent of unfolding. Among them, the most extensively unfolded isomer of denatured C3a is found to assume beads-form disulfide pattern, comprising Cys 36–Cys 49 and two disulfide bonds formed by two pair of consecutive cysteines, Cys 22–Cys 23 and Cys 56–Cys 57, a unique disulfide structure of polypeptide that has not been documented previously.

Structure and Stability Changes of Human IgG1 Fc as a Consequence of Methionine Oxidation

The Fc region has two highly conserved methionine residues, Met 33 (C(H)3 domain) and Met 209 (C(H)3 domain), which are important for the Fc's structure and biological function. To understand the effect of methionine oxidation on the structure and stability of the human IgG1 Fc expressed in Escherichia coli, we have characterized the fully oxidized Fc using biophysical (DSC, CD, and NMR) and bioanalytical (SEC and RP-HPLC-MS) methods. Methionine oxidation resulted in a detectable secondary and tertiary structural alteration measured by circular dichroism. This is further supported by the NMR data. The HSQC spectral changes indicate the structures of both C(H)2 and C(H)3 domains are affected by methionine oxidation. The melting temperature (Tm) of the C(H)2 domain of the human IgG1 Fc was significantly reduced upon methionine oxidation, while the melting temperature of the C(H)3 domain was only affected slightly. The change in the C(H)2 domain T m depended on the extent of oxidation of both Met 33 and Met 209. This was confirmed by DSC analysis of methionine-oxidized samples of two site specific methionine mutants. When incubated at 45 degrees C, the oxidized Fc exhibited an increased aggregation rate. In addition, the oxidized Fc displayed an increased deamidation (at pH 7.4) rate at the Asn 67 and Asn 96 sites, both located on the C(H)2 domain, while the deamidation rates of the other residues were not affected. The methionine oxidation resulted in changes in the structure and stability of the Fc, which are primarily localized to the C(H)2 domain. These changes can impact the Fc's physical and covalent stability and potentially its biological functions; therefore, it is critical to monitor and control methionine oxidation during manufacturing and storage of protein therapeutics.

The Crystal Structure and Mutational Binding Analysis of the Extracellular Domain of the Platelet-activating Receptor CLEC-2

The human C-type lectin-like molecule CLEC-2 is expressed on the surface of platelets and signaling through CLEC-2 causes platelet activation and aggregation. CLEC-2 is a receptor for the platelet-aggregating snake venom protein rhodocytin. It is also a newly identified co-receptor for human immunodeficiency virus type 1 (HIV-1). An endogenous ligand has not yet been identified. We have solved the crystal structure of the extracellular domain of CLEC-2 to 1.6-A resolution, and identified the key structural features involved in ligand binding. A semi-helical loop region and flanking residues dominate the surface that is available for ligand binding. The precise distribution of hydrophobic and electrostatic features in this loop will determine the nature of any endogenous ligand with which it can interact. Major ligand-induced conformational change in CLEC-2 is unlikely as its overall fold is compact and robust. However, ligand binding could induce a tilt of a 3-10 helical portion of the long loop region. Mutational analysis and surface plasmon resonance binding studies support these observations. This study provides a framework for understanding the effects of rhodocytin venom binding on CLEC-2 and for understanding the nature of likely endogenous ligands and will provide a basis for rational design of drugs to block ligand binding.

A Unique Loop Extension in the Serine Protease Domain of Haptoglobin Is Essential for CD163 Recognition of the Haptoglobin-Hemoglobin Complex

Haptoglobin and haptoglobin-related protein are homologous hemoglobin-binding proteins consisting of a complement control repeat (alpha-chain) and a serine protease domain (beta-chain). Haptoglobin-hemoglobin complex formation promotes high affinity binding of hemoglobin to the macrophage scavenger receptor CD163 leading to endocytosis and degradation of the haptoglobin-hemoglobin complex. In contrast, complex formation between haptoglobin-related protein and hemoglobin does not promote high affinity interaction with CD163. To define structural components of haptoglobin important for CD163 recognition, we exploited this functional difference to design and analyze recombinant haptoglobin/haptoglobin-related protein chimeras complexed to hemoglobin. These data revealed that only the beta-chain of haptoglobin is involved in receptor recognition. Substitution of 4 closely spaced amino acid residues of the haptoglobin beta-chain (valine 259, glutamate 261, lysine 262, and threonine 264) abrogated the high affinity receptor binding. The 4 residues are encompassed by a part of the primary structure not present in other serine protease domain proteins. Structural modeling based on the well characterized serine protease domain fold suggests that this sequence represents a loop extension unique for haptoglobin and haptoglobin-related protein. A synthetic peptide representing the haptoglobin loop sequence exhibited a pronounced inhibitory effect on receptor binding of haptoglobin-hemoglobin.

Covalent stabilization of coiled coils of the HIV gp41 N region yields extremely potent and broad inhibitors of viral infection

Peptides from the N-heptad repeat region of the HIV gp41 protein can inhibit viral fusion, but their potency is limited by a low tendency to form a trimeric coiled-coil. Accordingly, stabilization of N peptides by fusion with the stable coiled-coil IZ yields nanomolar inhibitors [Eckert, D. M. & Kim, P. S. (2001) Proc. Natl. Acad. Sci. USA 98, 11187-11192]. Because the antiviral potency of IZN17 is limited by self-association equilibrium, we covalently stabilized the peptide by using interchain disulfide bonds. The resulting covalent trimer, (CCIZN17)3, has an extraordinary thermodynamic stability that translates into unprecedented antiviral potency: (CCIZN17)3 (i) inhibits fusion in a cell-cell fusion assay (IC50 = 260 pM); (ii) is the most potent fusion inhibitor described to date (IC50 = 40-380 pM) in a single-cycle infectivity assay against HIV(HXB2), HIV(NL4-3), and HIV(MN-1); (iii) efficiently neutralizes acute viral infection in peripheral blood mononuclear cells; and (iv) displays a broad antiviral profile, being able to neutralize 100% of a large panel of HIV isolates, including R5, X4, and R5/X4 strains. In all of these assays, the potency of N-peptide inhibitor (CCIZN17)3 was equal to or more than the C-peptide inhibitor in clinical use, DP178 (also known as Enfuvirtide and Fuzeon). More importantly, we show that the two inhibitors, which have different targets in gp41, synergize when used in combination. These features make (CCIZN17)3 an attractive lead to develop as an antiviral drug, alone or in combination with DP178, as well as a promising immunogen to elicit a fusion-blocking neutralizing antibody response.

Covalent stabilization of nanostructures: Robust block copolymer templates from novel thermoreactive systems

AbstractStructurally robust block copolymer templates with feature sizes of approximately 10 nm were prepared from functionalized poly(methyl methacrylate)‐b‐polystyrene block copolymers. By the inclusion of benzocyclobutene crosslinking groups in the polystyrene block, the covalent stabilization of thin films to both thermal treatment and solvent exposure became possible. In addition, the crosslinking of the poly(styrene‐benzocyclobutene) domains at 220 °C, followed by the removal of poly(methyl methacrylate), provided a robust, crosslinked nanostructure with greater processing and fabrication potential. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 1028–1037, 2005

Well‐Defined Carbon Nanoparticles Prepared from Water‐Soluble Shell Cross‐linked Micelles that Contain Polyacrylonitrile Cores

Carbon-based materials have traditionally played an important role in modern technologies, ranging from the manufacture of everyday-use products such as automobile tires (carbon fillers), through reinforcements in composites (carbon fibers), electrode materials for a variety of processes, to activated carbons widely used in separation techniques, to name just a few. In addition to those commodity materials, in recent decades there has been a dynamic growth in the area of advanced engineering carbon materials. Seminal events in this area include the development of synthetic diamond and highly oriented pyrolytic graphite in 1960. 2] In the last two decades, this field has been particularly reenergized by the discovery of fullerenes in 1985 and carbon nanotubes in 1991. In addition to generating tremendous fundamental interest, these nanostructured forms of carbon have found, or are expected to find, numerous applications such as advanced fillers, materials for energy and gas storage, templates, nanoprobes and sensors, and elements for molecular electronics devices. 11] Two main groups of strategies for the preparation of engineering carbon materials include: 1) pyrolysis of organic precursors (mostly polymeric) under inert atmosphere to yield large-scale engineering carbon materials and 2) physical/chemical vapor deposition techniques to produce well-defined nanostructured forms of carbon. Whereas techniques from the first group are applicable to large-scale production, they offer very limited control of the carbon (nano)structure. Techniques from the second group, while allowing for atomic-scale precision in nanostructure control, are relatively expensive, have limited yield, and require complex equipment. Recently, we developed a novel, low-cost route to welldefined nanostructured carbon materials based on the pyrolysis of block copolymer precursors that contain polyacrylonitrile (PAN) and a sacrificial block (e.g., poly(n-butyl acrylate). By this method, the carbon nanostructure is templated after the nanostructure of the PAN domains, which are formed through self-assembly and driven by phase separation between PAN and the sacrificial block. Upon pyrolysis, PAN domains are converted into increasingly graphitic carbon, whereas the sacrificial phase is volatilized. The prerequisite for this approach is the ability of the PAN domains to retain their nanostructure upon thermal treatment. The necessary stabilization is achieved through thermal treatment (heating in the presence of air to 230 8C), a process well-known in the field of carbon fibers, which causes the PAN precursor to form cyclic, ladder, and eventually crosslinked species. Nanostructured carbon materials derived through this novel route hold considerable promise in many areas such as: field emitters, supercapacitors, and photovoltaic cells. One appealing advantage of this approach is the possibility of combining it with currently used devicefabrication techniques that rely on thin-film processing and lithography. One of the potential issues here, however, is the processability of the precursors: PAN and its block copolymers are soluble in a narrow range of solvents such as: N,Ndimethylformamide (DMF), ethylene carbonate (EC), dimethylsulfoxide and N-methylpyrrolidone (NMP). As one of the possible ways of addressing this challenge, herein we present an alternative approach, which now relies on the use of covalently stabilized micellar precursors that are soluble in aqueous systems. These precursors belong to a class of shell cross-linked nanoparticles (shell cross-linked knedels, SCKs), formed by self-assembly and covalent stabilization of amphiphilic block copolymers. PAN block constitutes the core of the SCKs and the sacrificial block (e.g., poly(acrylic acid), PAA) forms a water-soluble shell, and some of the carboxylic acid groups from the PAA block within the shell layer are covalently cross-linked following the micellization process, which occurs in a mixture of DMF and water. The use of nanostructured amphiphilic block copolymers as a template to form inorganic nanostructures has numerous precedents. The use of SCKs rather than simple micelles in this study is motivated by their colloidal stability and their ability to retain their nanostructure upon processing into thin and ultrathin films, and, as demonstrated below, upon subsequent thermal treatment. Processability into thin films is of particular importance when nanostructures are to be patterned or organized into ordered arrays. [*] C. Tang, Prof. K. Matyjaszewski, Prof. T. Kowalewski Department of Chemistry Carnegie Mellon University 4400 Fifth Avenue, Pittsburgh, PA 15213 (USA) Fax: (+1)412-268-6897 E-mail: tomek@andrew.cmu.edu

Interactions and Reactions of Ferritin with DNA

Ferritin, normally considered a cytoplasmic iron-storage protein, is also found in the nuclei of some cells. There is no current agreement about its function(s) in this environment. Proposals include DNA protection, provision of iron to nuclear enzymes, and regulation of transcription initiation, but evidence for these functions is scanty. We have shown previously that H-ferritin subunits can be cross-linked to chromosomal DNA in vivo (Thompson, K. J., Fried, M. G., Ye, Z., Boyer, P., and Connor, J. R. (2002) J. Cell Sci. 115, 2165-2177). Here we describe systematic analyses of DNA binding and the covalent stability of DNA in the presence of ferritins from several different sources. Our data show that the H-subunit of human ferritin binds DNA, whereas neither the L-subunit nor the ferroxidase-deficient 222-mutant of the H-subunit has detectable binding activity. DNA binding is without significant preference for base composition, sequence, or the nature of DNA ends. H- and L-ferritins and ferritins of mixed subunit composition stimulate the conversion of superhelical plasmid DNA to the relaxed form. The sensitivity of this conversion to glycerol suggests that DNA is nicked by a free radical mechanism. The rate of nicking correlates with the iron content of the ferritin and is strongly inhibited by chelators. Ferritin-dependent nicking is characterized by a kinetic lag that is not seen in control reactions containing free iron species. These results suggest that the release of iron from ferritin is an important part of the nicking mechanism. The potential role of ferritin as a protector of the genome is discussed in the context of these results.

Cross-linking between chloroplast fructose-1,6-bisphosphatase and thioredoxinf

The interaction between chloroplast fructose-1,6-bisphosphatase (FBPase) and thioredoxin (Trx) f, two plant proteins involved in the Benson-Calvin cycle, is mainly of an electrostatic nature [Hermoso et al. (1996) Plant Mol Biol 30: 455–465; Reche et al. (1997) Physiol Plant 101: 463–470; Sahrawy et al. (1997) J Mol Biol 269: 623–630; Hermoso et al. (1999) Physiol Plant 105: 756–762], possibly involving carboxyl groups of the enzyme and amino groups of Trx f. We carried out the covalent stabilization of that ionic complex, for the purpose of studying the interaction between both proteins and the factors that influence it. We have used 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide, a reagent able to cross-link carboxyl and amino groups, which allows the formation of covalent bonds between the groups that, in solution, form ionic bonds. A stable functional complex between both proteins was formed. The efficiency in the formation of that complex depends on the redox state of Trx f, ionic strength and pH, showing a strong correlation with the Trx f-dependent enzyme activity. The complex also retains enzyme activity. This suggests that the formation of the covalent complex requires the previous stabilization of a specific functional ionic complex between both proteins, and that in this functional complex carboxyl groups of the enzyme and primary amines of Trx f are involved. This complex is not stable in a tetrameric structure of the enzyme. We could also detect covalent aggregates of FBPase subunits, which indicates the implication of ionic interactions in the stabilization of the tetrameric structure of the enzyme; besides, as molecular filtration experiments and electrophoresis suggest, hydrophobic forces would also be implicated in the enzyme structure.

Diblock copolymers, micelles, and shell‐crosslinked nanoparticles containing poly(4‐fluorostyrene): Tools for detailed analyses of nanostructured materials

AbstractAmphiphilic core–shell nanostructures containing 19F stable isotopic labels located regioselectively within the core domain were prepared by a combination of atom transfer radical polymerization (ATRP), supramolecular assembly, and condensation‐based crosslinking. Homopolymers and diblock copolymers containing 4‐fluorostyrene and methyl acrylate were prepared by ATRP, hydrolyzed, assembled into micelles, and converted into shell‐crosslinked nanoparticles (SCKs) by covalent stabilization of the acrylic acid residues in the shell. The ATRP‐based polymerizations, producing the homopolymers and diblock copolymers, were initiated by (1‐bromoethyl)benzene in the presence of CuBr metal and employed N,N,N′,N″,N″‐pentamethyldiethylenetriamine as the coordinating ligand for controlled polymerizations at 75–90 °C for 1–3 h. Number‐average molecular weights ranged from 2000 to 60,000 Da, and molecular weight distributions, generally less than 1.1 and 1.2, were achieved for the homopolymers and diblock copolymers, respectively. Methyl acrylate conversions as high as 70% were possible, without observable chain–chain coupling reactions or molecular weight distribution broadening, when bromoalkyl‐terminated poly(4‐fluorostyrene) was used as the macroinitiator. Poly(4‐fluorostyrene), incorporated as the second segment in the diblock copolymer synthesis, was initiated from a bromoalkyl‐terminated poly(methyl acrylate) macroinitiator. After hydrolysis of the poly(methyl acrylate) block segments, micelles were formed from the resulting amphiphilic block copolymers in aqueous solutions and were then stabilized by covalent intramicellar crosslinking throughout the poly(acrylic acid) shells to yield SCKs. The SCK nanostructures on solid substrates were visualized by atomic force microscopy and transmission electron microscopy. Dynamic light scattering was used to probe the effects of crosslinking on the resulting hydrodynamic diameters of nanoparticles in aqueous and buffered solutions. The presence of fluorine atoms in the diblock copolymers and resulting SCK nanostructures allowed for characterization by 19F NMR in addition to 1H NMR, 13C NMR, and IR spectroscopy. © 2001 John Wiley & Sons, Inc. J Polym Sci Part A: Polym Chem 39: 4152–4166, 2001

Membrane immunoglobulins are stabilized by interchain disulfide bonds occurring within the extracellular membrane-proximal domain.

Membrane-bound immunoglobulins have, in addition to the transmembrane and cytoplasmic portions, an extracellular membrane-proximal domain (EMPD), absent in the secretory forms. EMPDs of immunoglobulin isotypes alpha, gamma, and epsilon contain cysteines whose role has so far not been elucidated. Using a genetic strategy, we investigated the ability of these cysteines to form disulfide bridges. Shortened versions of human membrane immunoglobulins, depleted of cysteines known to form intermolecular disulfide bonds, were constructed and expressed on the surface of a B-cell line. The resulting membrane proteins contain a single chain fragment of variable regions (scFv) linked to the dimerizing domain from the immunoglobulin heavy chains (CH3 for alpha and gamma or CH4 for epsilon isotypes), followed by the corresponding EMPD and the transmembrane and cytoplasmic domains. The two functional membrane versions of the epsilon chain, containing the short and long EMPD, were analyzed. Our results show that the single cysteine within alpha1L and gamma1 EMPD and the short version of epsilon EMPD form an interchain disulfide bond. Conversely, the cysteine resident in the epsilon transmembrane domain remains unreacted. epsilon-long EMPD contains four cysteines; two are involved in interchain bonds while the remaining two are likely forming an intrachain bridge. Expression of a full-length membrane epsilon heavy chain mutant, in which Cys(121) and Cys(209) within domain CH2 (involved in interchain bridges) were mutated to alanines, confirmed that, within the complete IgE, EMPD cysteines form interchain disulfide bonds. In conclusion, we unveil evidence for additional covalent stabilization of membrane-bound immunoglobulins.

The antitumor agent ecteinascidin 743: characterization of its covalent DNA adducts and chemical stability.

Ecteinascidin 743 (Et 743), a natural product derived from the Caribbean tunicate Eteinascidia turbinata, is a potent antitumor agent currently in phase II clinical trials. Et 743 binds in the minor groove of DNA, forming covalent adducts by reacting with N2 of guanine. Although DNA is considered to be the macromolecular receptor for Et 743, the precise mechanism by which Et 743 exerts its remarkable antitumor activity has not yet been elucidated. The aim of this study is to provide a rationale for the antitumor activity of Et 743 by studying its fundamental interactions with DNA at the molecular level. First, DNA structural distortions induced by Et 743 were characterized using gel electrophoresis. Surprisingly, Et 743 bends DNA toward the major groove, a unique feature among DNA-interactive agents that occupy the minor groove. Second, in order to gain further insight into the molecular basis behind the apparent sequence selectivity of Et 743, the stability and structure of Et 743 adducts at different target sequences were determined. On the basis of this data, the overall stability of the Et 743-DNA adducts was found to be governed by the DNA target sequence, where the inability of Et 743 to form optimum bonding networks with its optimum recognition sites leads to the formation of an unstable adduct. Consequently, the reaction of Et 743 with DNA is reversible, and the rate of the reverse reaction is a function of the target and flanking sequences. The results from this study demonstrate that Et 743 differs from other DNA alkylating agents by its effects on DNA structure and sequence-dependent chemical stability. This information provides important insight into the underlying mechanisms for its unique profile of antitumor activity.

A Biomimetic Approach to Asymmetric Acyl Transfer Catalysis

Small peptide catalysts containing modified histidine residues are reported that effect enantioselective acylation reactions. The catalysts described include octapeptide β-hairpins (e.g., 11) that exhibit high selectivities (up to krel = 51), tetrapeptide β-turns (e.g., 7) that afford moderate selectivities (up to krel = 28), and several simple derivatives of the modified histidine amino acid that do not exhibit appreciable enantioselectivity. Supporting structural studies (1H NMR and X-ray) are presented which lead to the proposal of a model in which catalyst rigidity and structural complexity contribute to higher degrees of enantioselection. A covalently rigidified octapeptide (20) is prepared through solid-phase Ru-catalyzed ring-closing metathesis; kinetic evaluation of this peptide reveals that substituents along the peptide backbone may be more important than covalent stabilization of a structural motif. Detailed kinetics studies on the most selective peptide catalysts are presented that suggest the...

Packaging of DNA by shell crosslinked nanoparticles.

We demonstrate compaction of DNA with nanoscale biomimetic constructs which are robust synthetic analogs of globular proteins. These constructs are approximately 15 nm in diameter, shell crosslinked knedel-like (SCKs) nanoparticles, which are prepared by covalent stabilization of amphiphilic di-block co-polymer micelles, self-assembled in an aqueous solution. This synthetic approach yields size-controlled nanoparticles of persistent shape and containing positively charged functional groups at and near the particle surface. Such properties allow SCKs to bind with DNA through electrostatic interactions and facilitate reduction of the DNA hydrodynamic diameter through reversible compaction. Compaction of DNA by SCKs was evident in dynamic light scattering experiments and was directly observed by in situ atomic force microscopy. Moreover, enzymatic digestion of the DNA plasmid (pBR322, 4361 bp) by Eco RI was inhibited at low SCK:DNA ratios and prevented when [le]60 DNA bp were bound per SCK. Digestion by Msp I in the presence of SCKs resulted in longer DNA fragments, indicating that not all enzyme cleavage sites were accessible within the DNA/SCK aggregates. These results have implications for the development of vehicles for successful gene therapy applications.

Electronic structure and photoelectron spectra of nickel(II)-β-diketonate complexes

The photoelectron spectra of the nickel(II) complexes of acetylacetone, acetylacetoimine, ethylenebis(acetylacetoimine) have been measured and quantum chemical calculations by X α-DV method have been fulfilled. The spectra were interpreted, the nature of occupied molecular orbitals was established, and the effect of oxygen atoms substitution to –NR– groups was analyzed. From the average energies of even and odd group orbitals equality in the ligands structure (AA −) 2 and predominant contribution of even MOs in covalent bonding in Ni(AA) 2, the values of the complex covalent stabilization have been calculated. The results of Ni(AA) + 2 calculations with vacancies at different MOs allowed the appreciation the relaxation values of complex MOs. The ionization energies were obtained in two ways: one based on full energy differences E(Ni(AA) + 2)– E(Ni(AA) 2) and the other by the transition state method: IE i =− ε i ( n=1/2).

Modulation of type-1 plasminogen activator inhibitor-induced vitronectin multimerization

Summary We recently reported that the interaction of active type-1 plasminogen activator inhibitor (PAI-1) with native vitronectin (Vn) present in unfractionated plasma induced the generation of conformationally altered, multimeric Vn. Here, the role of disulfide bridging in Vn and the role of the somatomedin B (SMB) domain of Vn in PAI-1-induced multimerization were characterized. Blocking of free SH-groups in Vn with the simultaneous addition of active PAI-1 prevented disulfide-linked Vn multimerization. In contrast, conformational changes and non-covalent stabilized multimerization were not affected. Moreover, pretreatment of Vn with dithionitrobenzoic acid (DTNB), followed by extensive dialysis to remove SH-reactive compounds, blocked disulfide-linked Vn multimerization. These observations point to the importance of free reactive cysteine(s) already present in native plasma Vn for the covalent stabilization of PAI-1-induced Vn multimers. Moreover, non-covalently and covalently-stabilized Vn multimerization and conformational changes were specifically prevented by recombinant Vn polypeptides encompassing the N-terminal SMB domain. The latter polypeptides may be of importance in elucidating the importance of PAI-1-induced Vn multimerization in complex biological systems.

The Role of Cysteine-949 in the Binding of Transforming Growth Factor-β1 and Transforming Growth Factor-β2 to α 2-Macroglobulin

The reaction of α 2-macroglobulin (α 2M) with proteinases or methylamine causes a major conformational change in α 2M and cleavage of the α 2M thiol ester bonds. The resulting free Cys residues (Cys-949) contain the only free thiol groups in α 2M. In this investigation, we explored the role of Cys-949 in the binding of transforming growth factor-β1 (TGF-β1) and TGF-β2 to α 2M-methylamine. Modification of preformed α 2M-methylamine with iodoacetamide did not change the binding affinity of α 2M-methylamine for TGF-β1 or TGF-β2; the apparent K D values were 82 nM and 10 nM, respectively. TGF-β binding also remained unchanged when tested using an α 2M derivative prepared by simultaneous treatment of α 2M with methylamine and iodoacetamide. The slow thiol-disulfide exchange reaction that irreversibly stabilizes noncovalent growth factor-α 2M-methylamine complexes was completely inhibited by modification of Cys-949. These studies demonstrate that Cys-949 in α 2M is not essential for binding of TGF-β1 and TGF-β2 noncovalently; however, this residue plays a critical role in the covalent stabilization step of the reaction mechanism.