Molar Eq Research Articles

Design of Lipid-Protein Conjugates Using Amphiphilic Peptide Substrates of Microbial Transglutaminase.

Lipid modification of proteins plays a significant role in regulating the cellular environment. Mimicking natural lipidated proteins is a key technique for assessing the function of proteins modified with lipids and also to render self-assembly of lipids to a target protein. Herein, we report a facile method of conjugating proteins with lipid-fused peptides under homogeneous physiological conditions by using the microbial transglutaminase (MTG) reaction. MTG catalyzes the cross-linking reaction between a specific glutamine (Q) in a protein and a lysine (K) in newly designed lipid-fused peptides. The water-soluble peptide substrates for lipid modification, C14-X-MRHKGS, were newly synthesized, where C14, X, and MRHKGS represent myristic acid, linker peptides composed of G, P, or S, and MTG-reactive K surrounded with basic amino acids, respectively. The MTG-mediated cross-linking reaction between a protein fused with LLQG at the C-terminus and C14-X-MRHKGS (5 molar eq) dissolved in a phosphate saline solution resulted in lipid-protein conjugates with yields of 70 to 100%. The anchoring ability of the obtained lipid-protein conjugates to cell membranes was dependent on the number of G residues in the GnS linker, suggesting that self-assembly and hydrophobicity of the GnS motif serves to enhance membrane anchoring of lipid-protein conjugates.

Modification by isolevuglandins, highly reactive γ-ketoaldehydes, deleteriously alters high-density lipoprotein structure and function

Cardiovascular disease risk depends on high-density lipoprotein (HDL) function, not HDL-cholesterol. Isolevuglandins (IsoLGs) are lipid dicarbonyls that react with lysine residues of proteins and phosphatidylethanolamine. IsoLG adducts are elevated in atherosclerosis. The consequences of IsoLG modification of HDL have not been studied. We hypothesized that IsoLG modification of apoA-I deleteriously alters HDL function. We determined the effect of IsoLG on HDL structure-function and whether pentylpyridoxamine (PPM), a dicarbonyl scavenger, can preserve HDL function. IsoLG adducts in HDL derived from patients with familial hypercholesterolemia (n = 10, 233.4 ± 158.3 ng/mg) were found to be significantly higher than in healthy controls (n = 7, 90.1 ± 33.4 pg/mg protein). Further, HDL exposed to myeloperoxidase had elevated IsoLG-lysine adducts (5.7 ng/mg protein) compared with unexposed HDL (0.5 ng/mg protein). Preincubation with PPM reduced IsoLG-lysine adducts by 67%, whereas its inactive analogue pentylpyridoxine did not. The addition of IsoLG produced apoA-I and apoA-II cross-links beginning at 0.3 molar eq of IsoLG/mol of apoA-I (0.3 eq), whereas succinylaldehyde and 4-hydroxynonenal required 10 and 30 eq. IsoLG increased HDL size, generating a subpopulation of 16-23 nm. 1 eq of IsoLG decreased HDL-mediated [3H]cholesterol efflux from macrophages via ABCA1, which corresponded to a decrease in HDL-apoA-I exchange from 47.4% to only 24.8%. This suggests that IsoLG inhibits apoA-I from disassociating from HDL to interact with ABCA1. The addition of 0.3 eq of IsoLG ablated HDL's ability to inhibit LPS-stimulated cytokine expression by macrophages and increased IL-1β expression by 3.5-fold. The structural-functional effects were partially rescued with PPM scavenging.

Structure and Activity of the Peptidyl-Prolyl Isomerase Domain from the Histone Chaperone Fpr4 toward Histone H3 Proline Isomerization

The FK506-binding protein (FKBP) family of peptidyl-prolyl isomerases (PPIases) is characterized by a common catalytic domain that binds to the inhibitors FK506 and rapamycin. As one of four FKBPs within the yeast Saccharomyces cerevisiae, Fpr4 has been described as a histone chaperone, and is in addition implicated in epigenetic function in part due to its mediation of cis-trans conversion of proline residues within histone tails. To better understand the molecular details of this activity, we have determined the solution structure of the Fpr4 C-terminal PPIase domain by using NMR spectroscopy. This canonical FKBP domain actively increases the rate of isomerization of three decapeptides derived from the N terminus of yeast histone H3, whereas maintaining intrinsic cis and trans populations. Observation of the uncatalyzed and Fpr4-catalyzed isomerization rates at equilibrium demonstrate Pro(16) and Pro(30) of histone H3 as the major proline targets of Fpr4, with little activity shown against Pro(38). This alternate ranking of the three target prolines, as compared with affinity determination or the classical chymotrypsin-based fluorescent assay, reveals the mechanistic importance of substrate residues C-terminal to the peptidyl-prolyl bond.

Ferredoxin Competes with Bacterial Frataxin in Binding to the Desulfurase IscS*

The bacterial iron-sulfur cluster (isc) operon is an essential machine that is highly conserved from bacteria to primates and responsible for iron-sulfur cluster biogenesis. Among its components are the genes for the desulfurase IscS that provides sulfur for cluster formation, and a specialized ferredoxin (Fdx) whose role is still unknown. Preliminary evidence suggests that IscS and Fdx interact but nothing is known about the binding site and the role of the interaction. Here, we have characterized the interaction using a combination of biophysical tools and mutagenesis. By modeling the Fdx·IscS complex based on experimental restraints we show that Fdx competes for the binding site of CyaY, the bacterial ortholog of frataxin and sits in a cavity close to the enzyme active site. By in vivo mutagenesis in bacteria we prove the importance of the surface of interaction for cluster formation. Our data provide the first structural insights into the role of Fdx in cluster assembly.

Chemo-enzymatic preparation of new bio-based bis- and trisphenols: new versatile building blocks for polymer chemistry

Immobilized Candida antartica lipase B (aka CAL-B) was used to perform solvent-free and in solvent transesterifications of a derivative from ferulic acid (1), ethyl dihydroferulate (2), with various bio-based polyols (isosorbide, 1,3-propanediol, 1,4-butanediol and glycerol). The use of 1.5 molar eq of ethyl dihydroferulate (2) per alcohol functionality and CAL-B (10% by weight relative to the total weight of polyol and 2) led to the corresponding poly-ferulates (3–6) in very good yields and high purity. These poly-ferulates were then efficiently transformed through one-step processes into the corresponding acrylates (7–10) and allyl ethers (11–14) to demonstrate their potential use as biorenewable macromonomers in a wide range of polymerization systems.

Chemo-enzymatic functionalization of gallic and vanillic acids: synthesis of bio-based epoxy resins prepolymers

The chemo-enzymatic epoxidation of allylated gallic and vanillic acids with lipase B from Candida antarctica and aqueous hydrogen peroxide in the presence of caprylic acid was investigated. The use of 1 molar eq of caprylic acid and 1.8–2 molar eq of H2O2 per allylic double bond led to a high degree of conversion to epoxides (89% yield for allylated gallic acid and 87% yield for allylated vanillic acid) comparatively to the hazardous peracid mCPBA (1.5–3.2 molar eq per double bond allowed 75–85% yield). The epoxidized products obtained could be used in the formulation of novel bio-based epoxy resins.

E2 Conjugating Enzyme Selectivity and Requirements for Function of the E3 Ubiquitin Ligase CHIP

The transfer of ubiquitin (Ub) to a substrate protein requires a cascade of E1 activating, E2 conjugating, and E3 ligating enzymes. E3 Ub ligases containing U-box and RING domains bind both E2∼Ub conjugates and substrates to facilitate transfer of the Ub molecule. Although the overall mode of action of E3 ligases is well established, many of the mechanistic details that determine the outcome of ubiquitination are poorly understood. CHIP (carboxyl terminus of Hsc70-interacting protein) is a U-box E3 ligase that serves as a co-chaperone to heat shock proteins and is critical for the regulation of unfolded proteins in the cytosol. We have performed a systematic analysis of the interactions of CHIP with E2 conjugating enzymes and found that only a subset bind and function. Moreover, some E2 enzymes function in pairs to create products that neither create individually. Characterization of the products of these reactions showed that different E2 enzymes produce different ubiquitination products, i.e. that E2 determines the outcome of Ub transfer. Site-directed mutagenesis on the E2 enzymes Ube2D1 and Ube2L3 (UbcH5a and UbcH7) established that an SPA motif in loop 7 of E2 is required for binding to CHIP but is not sufficient for activation of the E2∼Ub conjugate and consequent ubiquitination activity. These data support the proposal that the E2 SPA motif provides specificity for binding to CHIP, whereas activation of the E2∼Ub conjugate is derived from other molecular determinants.

ChemInform Abstract: Reductive Amination of Carboxylic Acids and [11C]Magnesium Halide Carboxylates.

The reductive amination of carboxylic acids was shown to be promoted by 2-chloropyridine hydrochloride (3 eq). It allowed the one-pot preparation of N-alkylamines in yields up to 93% from carboxylic acid (1 eq), amine (1 eq) and sodium borohydride (5 molar eq). The reaction, carried out with [11C]magnesium halide carboxylates (11C, β+, t1/2∶20 min), led to N-[11C]alkylamines in 20–25% radiochemical yields (decay corrected to the end of bombardment, 30 min preparation time from [11C]CO2). In this case, the addition of pyridinium salts led only to the corresponding [11C]carboxylic acids.

Use of Biotin Derivatives to Probe Conformational Changes in Proteins

Sulfosuccinimidyl-6-(biotinamido) hexanoate and derivatives thereof covalently bind to the epsilon-amino group of lysine residues. Our observation that access of the biotin derivative to specific lysine residues depends on conformational properties of the entire polypeptide chain prompted us to investigate whether differential biotinylation patterns of a protein can be used as indicators for conformational changes. Bovine serum albumin is a soluble protein with characteristic unfolding kinetics upon exposure to high temperature. First, we show that biotinylation patterns of proteins are highly reproducible. Second, we demonstrate by mass spectrometry and tandem mass spectrometry that unfolding of the protein correlates with the accessibility of the biotin derivative to specific lysine residues. We have applied this experimental strategy to the analysis of a cell-surface protein, viz. the human band 3 anion exchanger of erythrocytes infected with the malaria parasite Plasmodium falciparum. We found that Lys(826) in a highly flexible loop can be biotinylated in non-infected (but not infected) erythrocytes, confirming earlier observations (Winograd, E., and Sherman, I. W. (2004) Mol. Biochem. Parasitol. 138, 83-87) based on epitope-specific monoclonal antibodies suggesting that this region undergoes a conformational change upon infection.

Calcium-dependent and -independent Binding of Soybean Calmodulin Isoforms to the Calmodulin Binding Domain of Tobacco MAPK Phosphatase-1

The recent finding of an interaction between calmodulin (CaM) and the tobacco mitogen-activated protein kinase phosphatase-1 (NtMKP1) establishes an important connection between Ca(2+) signaling and the MAPK cascade, two of the most important signaling pathways in plant cells. Here we have used different biophysical techniques, including fluorescence and NMR spectroscopy as well as microcalorimetry, to characterize the binding of soybean CaM isoforms, SCaM-1 and -4, to synthetic peptides derived from the CaM binding domain of NtMKP1. We find that the actual CaM binding region is shorter than what had previously been suggested. Moreover, the peptide binds to the SCaM C-terminal domain even in the absence of free Ca(2+) with the single Trp residue of the NtMKP1 peptides buried in a solvent-inaccessible hydrophobic region. In the presence of Ca(2+), the peptides bind first to the C-terminal lobe of the SCaMs with a nanomolar affinity, and at higher peptide concentrations, a second peptide binds to the N-terminal domain with lower affinity. Thermodynamic analysis demonstrates that the formation of the peptide-bound complex with the Ca(2+)-loaded SCaMs is driven by favorable binding enthalpy due to a combination of hydrophobic and electrostatic interactions. Experiments with CaM proteolytic fragments showed that the two domains bind the peptide in an independent manner. To our knowledge, this is the first report providing direct evidence for sequential binding of two identical peptides of a target protein to CaM. Discussion of the potential biological role of this interaction motif is also provided.

Vertebrate Myosin VIIb Is a High Duty Ratio Motor Adapted for Generating and Maintaining Tension

Kinetic adaptation of muscle and non-muscle myosins plays a central role in defining the unique cellular functions of these molecular motor enzymes. The unconventional vertebrate class VII myosin, myosin VIIb, is highly expressed in polarized cells and localizes to highly ordered actin filament bundles such as those found in the microvilli of the intestinal brush border and kidney. We have cloned mouse myosin VIIb from a cDNA library, expressed and purified the catalytic motor domain, and characterized its actin-activated ATPase cycle using quantitative equilibrium and kinetic methods. The myosin VIIb steady-state ATPase activity is slow (approximately 1 s(-1)), activated by very low actin filament concentrations (K(ATPase) approximately 0.7 microm), and limited by ADP release from actomyosin. The slow ADP dissociation rate constant generates a long lifetime of the strong binding actomyosin.ADP states. ADP and actin binding is uncoupled, which enables myosin VIIb to remain strongly bound to actin and ADP at very low actin concentrations. In the presence of 2 mm ATP and 2 microm actin, the duty ratio of myosin VIIb is approximately 0.8. The enzymatic properties of actomyosin VIIb are suited for generating and maintaining tension and favor a role for myosin VIIb in anchoring membrane surface receptors to the actin cytoskeleton. Given the high conservation of vertebrate class VII myosins, deafness phenotypes arising from disruption of normal myosin VIIa function are likely to reflect a loss of tension in the stereocilia of inner ear hair cells.

SREBP-1 Dimerization Specificity Maps to Both the Helix-Loop-Helix and Leucine Zipper Domains: USE OF A DOMINANT NEGATIVE

The mammalian SREBP family contains two genes that code for B-HLH-ZIP proteins that bind sequence-specific DNA to regulate the expression of genes involved in lipid metabolism. We have designed a dominant negative (DN), termed A-SREBP-1, that inhibits the DNA binding of either SREBP protein. A-SREBP-1 consists of the dimerization domain of B-SREBP-1 and a polyglutamic acid sequence that replaces the basic region. A-SREBP-1 heterodimerizes with either B-SREBP-1 or B-SREBP-2, and both heterodimers are more stable than B-SREBP-1 bound to DNA. Circular dichroism thermal denaturation studies show that the B-SREBP-1.A-SREBP-1 heterodimer is -9.8 kcal mol(-1) dimer(-1) more stable than the B-SREBP-1 homodimer. EMSA assays demonstrate that A-SREBP-1 can inhibit the DNA binding of either B-SREBP-1 or B-SREBP-2 in an equimolar competition but does not inhibit the DNA binding of the three B-HLH-ZIP proteins MAX, USF, or MITF, even at 100 molar eq. Chimeric proteins containing the HLH domain of SREBP-1 and the leucine zipper from either MAX, USF, or MITF indicate that both the HLH and leucine zipper regions of SREBP-1 contribute to its dimerization specificity. Transient co-transfection studies demonstrate that A-SREBP-1 can inhibit the transactivation of SREBP-1 and SREBP-2 but not USF. A-SREBP-1 may be useful in metabolic diseases where SREBP family members are overexpressed.

The Structure of the Zinc Finger Domain from Human Splicing Factor ZNF265 Fold

Identification of the protein domains that are responsible for RNA recognition has lagged behind the characterization of protein-DNA interactions. However, it is now becoming clear that a range of structural motifs bind to RNA and their structures and molecular mechanisms of action are beginning to be elucidated. In this report, we have expressed and purified one of the two putative RNA-binding domains from ZNF265, a protein that has been shown to bind to the spliceosomal components U1-70K and U2AF35 and to direct alternative splicing. We show that this domain, which contains four highly conserved cysteine residues, forms a stable, monomeric structure upon the addition of 1 molar eq of Zn(II). Determination of the solution structure of this domain reveals a conformation comprising two stacked beta-hairpins oriented at approximately 80 degrees to each other and sandwiching the zinc ion; the fold resembles the zinc ribbon class of zinc-binding domains, although with one less beta-strand than most members of the class. Analysis of the structure reveals a striking resemblance to known RNA-binding motifs in terms of the distribution of key surface residues responsible for making RNA contacts, despite a complete lack of structural homology. Furthermore, we have used an RNA gel shift assay to demonstrate that a single crossed finger domain from ZNF265 is capable of binding to an RNA message. Taken together, these results define a new RNA-binding motif and should provide insight into the functions of the >100 uncharacterized proteins in the sequence data bases that contain this domain.

The synthesis and studies towards the self-replication of bis(capped porphyrins).

Connecting two facially-protected porphyrins was expected to lead to an equal mixture of laterally-bridged doubly-protected bis-porphyrins; one in which the two porphyrin units were protected on the same face (syn) and one with the two prophyrin units protected on opposite faces (anti). Addition of a co-factor (bidentate ligand) was expected to lead predominantly to the syn-bis-porphyrin by a templated self-replication process. This concept was explored using Baldwin's capped porphyrin. Bis(capped porphyrins) were synthesised in several steps starting from zinc(II) capped porphyrin 2. Nitration of 2 followed by reduction and photo-oxidation yields a mixture of zinc(II) porphyrindiones 7 and 8 that can separated by HPLC. The condensation of 2 molar eq. of zinc(II) porphyrin-7,8-dione 8 with 1,2,4,5-benzenetetramine leads to the formation of a 1:1 mixture of syn- and anti-dizinc(II) bis(7,8-capped porphyrins), 11 and 12, respectively, that have almost identical spectroscopic properties. These two geometric isomers were distinguished by significant differences in their molecular recognition properties. Likewise the syn- and anti-dizinc(II) bis(2,3-capped porphyrins), 9 and 10, respectively, are synthesised from the related zinc(II) capped porphyrin-2,3-dione 7, and were also identified using molecular recognition studies. The molecular recognition properties of these bis(capped porphyrins) were utilised in studies of self-replicating porphyrin systems. The results show that tetraazaanthraceno-bis-porphyrins 9-12 can catalyse their own formation but self-replication was not observed. These results highlight the potential that these interesting hosts have as templates in supramolecular chemistry, synthesis and catalysis.

Differential Regulation of a Hyperthermophilic α-Amylase with a Novel (Ca,Zn) Two-metal Center by Zinc

The crystal structure of the alpha-amylase from the hyperthermophilic archaeon Pyrococcus woesei was solved in the presence of three inhibitors: acarbose, Tris, and zinc. In the absence of exogenous metals, this alpha-amylase bound 1 and 4 molar eq of zinc and calcium, respectively. The structure reveals a novel, activating, two-metal (Ca,Zn)-binding site and a second inhibitory zinc-binding site that is found in the -1 sugar-binding pocket within the active site. The data resolve the apparent paradox between the zinc requirement for catalytic activity and its strong inhibitory effect when added in molar excess. They provide a rationale as to why this alpha-amylase, in contrast to commercially available alpha-amylases, does not require the addition of metal ions for full catalytic activity, suggesting it as an ideal target to maximize the efficiency of industrial processes like liquefaction of starch.

Refolding and purification of yeast carboxypeptidase Y expressed as inclusion bodies in Escherichia coli.

The genes encoding carboxypeptidase Y (CPY) and CPY propeptide (CPYPR) from Saccharomyces cerevisiae were cloned and expressed in Escherichia coli. Six consecutive histidine residues were fused to the C-terminus of the CPYPR for facilitated purification. High-level expression of CPY and CPYPR-His(6) was achieved but most of the expressed proteins were present in the form of inclusion bodies in the bacterial cytoplasm. The CPY and CPYPR-His(6) produced as inclusion bodies were separated from the cells and solubilized in 6 and 3 M guanidinium chloride, respectively. The denatured CPYPR-His(6) was refolded by dilution 1:30 into the renaturation buffer (50 mM Tris-HCl containing 0.5 M NaCl and 3 mM EDTA, pH 8.0), and the refolded CPYPR-His(6) was further purified to 90% purity by single-step immobilized metal ion affinity chromatography. The denatured CPY was refolded by dilution 1:60 into the renaturation buffer containing CPYPR-His(6) at various concentrations. Increasing the molar ratio of CPYPR-His(6) to CPY resulted in an increase in the CPY refolding yield, indicating that the CPYPR-His(6) plays a chaperone-like role in in vitro folding of CPY. The refolded CPY was purified to 92% purity by single-step p-aminobenzylsuccinic acid affinity chromatography. When refolding was carried out in the presence of 10 molar eq CPYPR-His(6), the specific activity, N-(2-furanacryloyl)-l-phenylalanyl-l-phenylalanine hydrolysis activity per milligram of protein, of purified recombinant CPY was found to be about 63% of that of native S. cerevisiae CPY.

Reductive amination of carboxylic acids and [11C]magnesium halide carboxylates

The reductive amination of carboxylic acids was shown to be promoted by 2-chloropyridine hydrochloride (3 eq). It allowed the one-pot preparation of N-alkylamines in yields up to 93% from carboxylic acid (1 eq), amine (1 eq) and sodium borohydride (5 molar eq). The reaction, carried out with [11C]magnesium halide carboxylates (11C, β+, t1/2∶20 min), led to N-[11C]alkylamines in 20–25% radiochemical yields (decay corrected to the end of bombardment, 30 min preparation time from [11C]CO2). In this case, the addition of pyridinium salts led only to the corresponding [11C]carboxylic acids.

Mechanism of Formation of N2-Benzylguanine in the Reaction of 2-Amino-6-chloropurine with Sodium Benzyl Oxide, and Benzylation of Nucleic Acid Bases.

The mechanism of formation of N2-benzylguanine in the reaction of 2-amino-6-chloropurine with a large excess (12-13 molar eq) of sodium benzyl oxide in benzyl alcohol at 130°C was studied, N2, O6-Dibenzylguanine, a reaction intermediate, was isolated and a possible mechanism for its formation is discussed. Furthermore, using this sodium benzyl oxide system, benzylation at the amino group of nucleic acid bases was facilitated.

Thione-Thiol Rearrangement of Xanthates Catalyzed by Pyridine N-Oxides. Remarkably Enhanced Reactivity of 4-Dialkylaminopyridine N-Oxides.

Pyridine N-oxides bearing electron-donating substituents (III) are efficient catalysts for rearrangement of O-alkyl S-methyl dithiocarbonates (xanthates) (I) to the corresponding S-alkyl S-methyl dithiocarbonates (dithiolcarbonates) (II). Of the catalysts tested, 4-piperidinopyridine N-oxide (IIIh) is the best from the viewpoints of catalytic activity and solubility in I. Heating of I in the presence of catalytic amounts (0.02-0.05 molar eq) of IIIh gave II together with the symmetric S, S-dialkyl and S, S-dimethyl dithiocarbonates in good yields. The catalytic behavior of donor-substituted pyridine N-oxides is discussed on the basis of kinetic and molecular orbital calculation data. The complete calculation of the perturbation equation on the initial stage of the reaction was consistent with the experimentally observed activity of the catalysts.

SYNTHESIS AND STRUCTURE OF SOME PHOSPHONYLATED OXIMES RELATED TO ORGANOPHOSPHATE NERVE AGENTS

Abstract Oximes, and in particular oximate salts, can be useful nucleophiles for the treatment of organophosphate nerve agent poisoning and decontamination of chemical warfare agents. In this paper, the reactions of phosphonochloridate analogues of the chemical warfare nerve agents VX, GB, and GA and the oximes 2-butanone oxime, 2,3-butanedione monoxime, and its potassium salt (KD), are examined. Under controlled conditions. (0°C, 1 molar eq. KD), the major product is the O-(O-alkyl phosphonyl)oxime; with excess oximate KD, the intermediate phosphonylated oximes containing an α-carbonyl undergo a “second-order” Beckmann rearrangement to give (E)-mono(O-acetyloxime)-2,3-butanedione.