A new scaffold for amide ligation.

Highly chemoselective amide forming ligation reactions have facilitated the synthetic access to proteins and other amide-linked bioconjugates. In order to generalize this approach, a N(alpha)-2-phenyl ethanethiol scaffold has been developed to promote S to N acyl transfer in a manner analogous to native chemical ligation with N-terminal cysteine residues. Analysis of scaffold-mediated ligation reactions in aqueous solution indicate that the ligation rate at Xaa-Gly junctions is sufficient for the synthesis of large polypeptides. In addition, it was found that the ligation rate is independent of the stereocenter in the scaffold and S- to N-acyl transfer is rate limiting. These studies indicate that the N(alpha)-2-phenyl ethanethiol scaffold is a good candidate for the development of a ligation chemistry for the formation of Xaa-Gly peptides and other unhindered amides.     

Structural and kinetic aspects of chemical reactions in DNA duplexes. Information on DNA local structure obtained from chemical ligation data.

Chemical ligation of oligonucleotides in double-stranded helices has been considered in its structural-kinetic aspect. A study was made of (i) two series of DNA duplexes with various arrangements of reacting groups in the ligation junction induced by mispairing or by alteration of furanose structure (the replacement of dT unit with rU, aU, IU, xU, dxT ones) and of (ii) eight synthetic water-soluble carbodiimides with different substituents at N1 and N3 atoms. We assumed that some information on the local structure of modified sites in the duplex can be obtained from kinetic parameters of oligonucleotide coupling reaction. The ratio of kinetic constants k3/(k2 + k3) for productive and nonproductive decomposition of the activated phosphomonoester derivative apparently reflects the reaction site structure: for a given duplex this parameter is virtually independent of the condensing agent composition. Based on the analysis of the chemical ligation kinetics a suggestion has been made about the conformation of some modified units in the double helix.     

Biophysical and structural analysis of a novel heme B iron ligation in the flavocytochrome cellobiose dehydrogenase

The fungal extracellular flavocytochrome cellobiose dehydrogenase (CDH) participates in lignocellulose degradation. The enzyme has a cytochrome domain connected to a flavin-binding domain by a peptide linker. The cytochrome domain contains a 6-coordinate low spin b-type heme with unusual iron ligands and coordination geometry. Wild type CDH is only the second example of a b-type heme with Met-His ligation, and it is the first example of a Met-His ligation of heme b where the ligands are arranged in a nearly perpendicular orientation. To investigate the ligation further, Met65 was replaced with a histidine to create a bis-histidyl ligated iron typical of b-type cytochromes. The variant is expressed as a stable 90-kDa protein that retains the flavin domain catalytic reactivity. However, the ability of the mutant to reduce external one-electron acceptors such as cytochrome c is impaired. Electrochemical measurements demonstrate a decrease in the redox midpoint potential of the heme by 210 mV. In contrast to the wild type enzyme, the ferric state of the protoheme displays a mixed low spin/high spin state at room temperature and low spin character at 90 K, as determined by resonance Raman spectroscopy. The wild type cytochrome does not bind CO, but the ferrous state of the variant forms a CO complex, although the association rate is very low. The crystal structure of the M65H cytochrome domain has been determined at 1.9 A resolution. The variant structure confirms a bis-histidyl ligation but reveals unusual features. As for the wild type enzyme, the ligands have a nearly perpendicular arrangement. Furthermore, the iron is bound by imidazole N delta 1 and N epsilon 2 nitrogen atoms, rather than the typical N epsilon 2/N epsilon 2 coordination encountered in bis-histidyl ligated heme proteins. To our knowledge, this is the first example of a bis-histidyl N delta 1/N epsilon 2-coordinated protoporphyrin IX iron.     

Carbopeptides: chemoselective ligation of peptide aldehydes to an aminooxy-functionalized D-galactose template.

Multifunctional, topological template molecules such as linear and cyclic peptides have been used for the attachment of peptide strands to form novel protein models of, for example, 4-alpha-helix bundles. The concept of carbohydrates as templates for de novo design of potential protein models has been previously described and these novel chimeric compounds were termed carbopeptides. Here, a second generation strategy in which carbopeptides are synthesized by chemoselective ligation of a peptide aldehyde to an aminooxy-functionalized alpha-D-galactopyranoside is described. This template was prepared by per-O-acylation of methyl alpha-D-galactopyranoside with N,N-Boc2-aminooxyacetic acid to form a tetra-functionalized template, followed by treatment with TFA-CH2Cl2 to release the aminooxy functionality. The peptide aldehydes Fmoc-Ser-Gly-Gly-H and H-Ala-Leu-Ala-Lys-Leu-Gly-Gly-H were synthesized by a BAL strategy. Four identical copies of peptide aldehyde were smoothly attached to the template by chemoselective ligation to form a 2.1 and a 2.9 kDa carbopeptide, respectively.     

Interaction between prostanoids and nitric oxide in the control of tubular function in rats with chronic bile duct ligation.

Recent work indicates that both nitric oxide and cyclooxygenase products play an important role in the renal alterations of liver cirrhosis, although the interactions between them have not been completely established. The purpose of this study was to assess the effect of simultaneous blockade of nitric oxide synthase and cyclooxygenase in rats with chronic bile duct ligation and in control, sham-operated rats. Compared with control rats, chronic bile duct ligation rats, 23-25 days after surgery, showed a decreased mean arterial pressure, natriuresis, and kaliuresis, without differences in glomerular filtration rate, and an increased urinary nitrite excretion. Nitric oxide synthesis inhibition by administration of N(G)-nitro-L-arginine methyl ester induced, in control rats, an increase in mean arterial pressure, without significant changes in natriuresis or glomerular filtration rate. In chronic bile duct ligation rats, N(G)-nitro-L-arginine methyl ester induced an increase in mean arterial pressure, natriuresis, and kaliuresis, together with a reduction in urinary nitrite excretion and an increase in prostaglandin E2 excretion. Cyclooxygenase inhibition with indomethacin induced in both experimental groups a marked inhibition in urinary prostaglandin E2 excretion without significant changes in Na+ or K+ excretion, and a significant increase in urinary nitrite excretion in control rats. N(G)-Nitro-L-arginine methyl ester in addition to indomethacin prevented the indomethacin-induced increase in nitrite excretion and dramatically reduced sodium excretion in both experimental groups. Thus, the present study suggests that both nitric oxide and cyclooxygenase products interact in the control of urinary sodium excretion and that each system is activated in the absence of the other one.     

The active site structure of E. coli HPII catalase. Evidence favoring coordination of a tyrosinate proximal ligand to the chlorin iron.

E. coli produces 2 catalases known as HPI and HPII. While the heme prosthetic group of the HPII catalase has been established to be a dihydroporphyrin or chlorin, the identity of the proximal ligand to the iron has not been addressed. The magnetic circular dichroism (MCD) spectrum of native ferric HPII catalase is very similar to those of a 5-coordinate phenolate-ligated ferric chlorin complex, a model for tyrosinate proximal ligation, as well as of chlorin-reconstituted ferric horseradish peroxidase, a model for 5-coordinate histidine ligation. However, further MCD comparisons of chlorin-reconstituted myoglobin with parallel ligand-bound adducts of the catalase clearly rule out histidine ligation in the latter, leaving tyrosinate as the best candidate for the proximal ligand.     

DNA ligation catalyzed by human topoisomerase II alpha

The DNA ligation reaction of topoisomerase II is essential for genomic integrity. However, it has been impossible to examine many fundamental aspects of this reaction because ligation assays historically required the enzyme to cleave a DNA substrate before sealing the nucleic acid break. Recently, a cleavage-independent DNA ligation assay was developed for human topoisomerase IIalpha [Bromberg, K. D., Hendricks, C., Burgin, A. B., and Osheroff, N. (2002) J. Biol. Chem. 277, 31201-31206]. This assay overcomes the requirement for DNA cleavage by monitoring the ability of the enzyme to ligate a nicked oligonucleotide in which the 5'-terminal phosphate at the nick has been activated by covalent attachment to the tyrosine mimic, p-nitrophenol. The cleavage-independent ligation assay was used to more fully characterize the DNA ligation activity of human topoisomerase IIalpha. Results suggest that the active site tyrosine contributes little to the catalysis of DNA ligation beyond its primary role as an activating/leaving group. Although arginine 804 (the residue immediately N-terminal to the active site tyrosine) has been proposed to help anchor the 5'-DNA terminus during cleavage, conversion of this residue to alanine had only a modest effect on DNA ligation. Thus, it appears that arginine 804 does not play an essential role in DNA strand joining. In contrast, disruption of base pairing at the 5'-DNA terminus abrogated DNA ligation in the absence of a covalent enzyme-DNA bond. Therefore, it is proposed that base pairing represents a secondary mechanism for aligning the 5'-DNA termini for ligation. Finally, the human enzyme appears to ligate the two scissile bonds of a cleavage site in a nonconcerted fashion.     

Fast folding of cytochrome c.

Native iso-2 cytochrome c contains two residues (His 18, Met 80) coordinated to the covalently attached heme. On unfolding of iso-2, the His 18 ligand remains coordinated to the heme iron, whereas Met 80 is displaced by a non-native heme ligand, His 33 or His 39. To test whether non-native His-heme ligation slows folding, we have constructed a double mutant protein in which the non-native ligands are replaced by asparagine and lysine, respectively (H33N,H39K iso-2). The double mutant protein, which cannot form non-native histidine-heme coordinate bonds, folds significantly faster than normal iso-2 cytochrome c: gamma = 14-26 ms for H33N,H39K iso-2 versus gamma = 200-1,100 ms for iso-2. These results with iso-2 cytochrome c strongly support the hypothesis that non-native His-heme ligation results in a kinetic barrier to fast folding of cytochrome c. Assuming that the maximum rate of a conformational search is about 10(11) s-1, the results imply that the direct folding pathway of iso-2 involves passage through on the order of 10(9) or fewer partially folded conformers.     

Intrinsic intermolecular DNA ligation activity of eukaryotic topoisomerase II. Potential roles in recombination.

Drosophila melanogaster topoisomerase II is capable of joining phi X174 (+) strand DNA that it has cleaved to duplex oligonucleotide acceptor molecules by an intermolecular ligation reaction (Gale, K. C. and Osheroff, N. (1990) Biochemistry 29, 9538-9545). In order to investigate potential mechanisms for topoisomerase II-mediated DNA recombination, this intrinsic enzyme activity was further characterized. Intermolecular DNA ligation proceeded in a time-dependent fashion and was concentration-dependent with respect to oligonucleotide. The covalent linkage between phi X174 (+) strand DNA and acceptor molecules was confirmed by Southern analysis and alkaline gel electrophoresis. Topoisomerase II-mediated intermolecular DNA ligation required the oligonucleotide to contain a 3'-OH terminus. Moreover, the reaction was dependent on the presence of a divalent cation, was inhibited by salt, and was not affected by the presence of ATP. The enzyme was capable of ligating phi X174 (+) strand DNA to double-stranded oligonucleotides that contained 5'-overhang, 3'-overhand, or blunt ends. Single-stranded, nicked, or gapped oligonucleotides also could be used as acceptor molecules. These results demonstrate that the type II enzyme has an intrinsic ability to mediate illegitimate DNA recombination in vitro and suggests possible roles for topoisomerase II in nucleic acid recombination in vivo.     

Preparation of protected peptidyl thioester intermediates for native chemical ligation by Nalpha-9-fluorenylmethoxycarbonyl (Fmoc) chemistry: considerations of side-chain and backbone anchoring strategies, and compatible protection for N-terminal cysteine.

Native chemical ligation has proven to be a powerful method for the synthesis of small proteins and the semisynthesis of larger ones. The essential synthetic intermediates, which are C-terminal peptide thioesters, cannot survive the repetitive piperidine deprotection steps of N(alpha)-9-fluorenylmethoxycarbonyl (Fmoc) chemistry. Therefore, peptide scientists who prefer to not use N(alpha)-t-butyloxycarbonyl (Boc) chemistry need to adopt more esoteric strategies and tactics in order to integrate ligation approaches with Fmoc chemistry. In the present work, side-chain and backbone anchoring strategies have been used to prepare the required suitably (partially) protected and/or activated peptide intermediates spanning the length of bovine pancreatic trypsin inhibitor (BPTI). Three separate strategies for managing the critical N-terminal cysteine residue have been developed: (i) incorporation of N(alpha)-9-fluorenylmethoxycarbonyl-S-(N-methyl-N-phenylcarbamoyl)sulfenylcysteine [Fmoc-Cys(Snm)-OH], allowing creation of an otherwise fully protected resin-bound intermediate with N-terminal free Cys; (ii) incorporation of N(alpha)-9-fluorenylmethoxycarbonyl-S-triphenylmethylcysteine [Fmoc-Cys(Trt)-OH], generating a stable Fmoc-Cys(H)-peptide upon acidolytic cleavage; and (iii) incorporation of N(alpha)-t-butyloxycarbonyl-S-fluorenylmethylcysteine [Boc-Cys(Fm)-OH], generating a stable H-Cys(Fm)-peptide upon cleavage. In separate stages of these strategies, thioesters are established at the C-termini by selective deprotection and coupling steps carried out while peptides remain bound to the supports. Pilot native chemical ligations were pursued directly on-resin, as well as in solution after cleavage/purification.     

Chemical ligation of oligodeoxyribonucleotides on circular DNA templates.

We report the use of small circular DNA as a triplex-directing template for the highly efficient chemical ligation of oligodeoxyribonucleotides (ODNs) using cyanogen bromide (BrCN). These investigations compared the use of a linear homopyrimidine DNA template (17mer) and a circular pyrimidine-rich DNA template (44mer) for directing the chemical ligation of two homopurine ODNs (6mer + 11mer). The effects of substrate/template ratio, buffer, salt, ionic strength, pH and temperature have been examined in the BrCN activated ligation reactions. The optimal yield of 51% for ligation on the linear template was at pH 6.0, 200 mM MgCl2, 4 degreesC. In contrast, near quantitative ligation on the circular template occurred at higher pH, higher temperature, and showed less dependence on Mg2+concentration (97% yield, pH 7.5, 200 mM MgCl2, 25 degreesC). The relative observed rate of the ligation reaction was a minimum of 35 times faster on the circular DNA template relative to the linear template at pH 7.5, 200 mM MgCl2, 4 degreesC. These investigations reveal that chemical ligation of short ODNs on circularized DNA templates through triplex formation is a highly efficient process over a broad range of conditions.     

Incorporation of 1,N6-ethenoadenosine into the 3' terminus of tRNA using T4 RNA ligase. 1. Preparation of yeast tRNAPhe derivatives

The 3'-terminal A-C-C-A sequence of yeast tRNAPhe has been modified by replacing either adenosine 76 or 73 with the fluorescent analogues 1,N6-ethenoadenosine (epsilon A) or 2-aza-1,N6-ethenoadenosine (aza-epsilon A). T4 RNA ligase was used to join the nucleoside 3',5'-bisphosphates to the 3' end of the tRNA which was shortened by one [tRNAPhe(-A)] or four [tRNAPhe(-ACCA)] nucleotides. It was found that the base-paired 3'-terminal cytidine 72 in tRNAPhe(-ACCA) is a more efficient acceptor in the ligation reaction than the unpaired cytidine 75 at the A-C-C terminus of tRNAPhe(-A). This finding indicates that the mobility of the accepting nucleoside substantially influences the ligation reaction, the efficiency being higher the lower the mobility. This conclusion is corroborated by the observation that the ligation reaction with the double-stranded substrate exhibits a positive temperature dependence rather than a negative one as found for single-stranded acceptors. The replacement of the 3'-terminal adenosine 76 with epsilon A and aza-epsilon A leads to moderately fluorescent tRNAPhe derivatives, which are inactive in the aminoacylation reaction. A number of other tRNAs (Met, Ser, Glu, Lys and Leu-specific tRNAs both from yeast and Escherichia coli) are also inactivated by epsilon A incorporation. Replacement of adenosine 73 followed by repair of the C-C-A end using nucleotidyl transferase leads to tRNAPhe derivatives which are fully active in the aminoacylation reaction and in polyphenylalanine synthesis. The fluorescence of epsilon A and aza-epsilon A at position 73 is virtually completely quenched, suggesting a stacked arrangement of bases around this position. There is no fluorescence increase when the epsilon A-labeled tRNAPhe is complexed with phenylalanyl-tRNA synthetase, elongation factor Tu, or ribosomes. These observations indicate that the stacked conformation of the 3' terminus is not changed appreciably in these complexes.     

Anchorage of synthetic peptides onto liposomes via hydrazone and alpha-oxo hydrazone bonds. preliminary functional investigations

Synthetic peptidoliposomes have been designed and prepared according to a chemoselective ligation. Two aldehyde-functionalized lipidic anchors were synthesized and incorporated into the lipidic bilayers of unilamellar liposomes during their preparation. Complementary hydrazino acetyl peptides were synthesized on the solid phase using N,N',N'-tri(tert-butyloxycarbonyl)-hydrazino acetic acid and further coupled to the aldehyde groups displayed at the surface of the vesicles. Coupling yields were measured by amino acid hydrolysis following total acid hydrolysis. The ligation methodology proved superior to the simple insertion of lipopeptides, which was performed for comparison in terms of yields, implementation, and reproducibility. To check whether the grafted-peptides were accessible and functional, cytoplasmic sequences of LAMP protein (lysosomal associated membrane protein), which is involved in intracellular membrane trafficking, have been selected. Using this model, we demonstrated in vitro the specific interaction of the synthetic LAMP-peptidoliposomes with the cytoplasmic adaptor protein AP-3, a result that contributes to the understanding of protein sorting in cells. Thus, these results clearly indicate the usefulness of such peptidoliposomes, easily prepared by hydrazone chemoselective ligation, as a tool for biological investigation.     

Nonenymatic ligation of double-helical DNA by alternate-strand triple helix formation.

Nonenzymatic ligation of double-stranded DNA has been performed using an alternate-strand binding oligodeoxyribonucleotide template to juxtapose the duplex termini in a triple helical complex. The template associates with the duplex termini by Hoogsteen hydrogen bonding to alternate strands on opposite sides of the ligation site. Intermolecular and intramolecular ligation of linearized plasmid DNA are observed in the reaction, which depends on the template oligodeoxyribonucleotide and a condensing agent, N-cyanoimidazole. Intramolecular ligation products include those in which both strands are covalently closed in a circle. Ligation of the two strands is sequential and occurs at comparable rates for the first and second strands ligating. The covalent linkages formed in the reaction can be cleaved by the restriction endonuclease Stu I, supporting their identification as phosphodiesters.     

Sterilization: Canadian choices.

Significant differences in cost and safety between vasectomy and tubal ligation have been reported. For this reason the incidence of these two procedures between 1976 and 1986 was studied to obtain information upon which future policy decisions might be based. Although tubal ligation predominated in almost every province and year its rate declined by 27.6% over the study period, whereas the rate of vasectomy increased by 39.1%. When projected to 1988 the national rates for the two procedures became nearly equal; those for Quebec had become equal by 1986. Provincial differences were most marked in eastern Canada, where neighbouring provinces had the highest and the lowest rates of sterilization in the country. Given the relative economic and surgical disadvantages of tubal ligation, policymakers may wish to consider fostering an increased acceptance of vasectomy, particularly in areas where such acceptance continues to be slow.     

Protein splicing of a Pyrococcus abyssi intein with a C-terminal glutamine

Protein splicing involves the excision of an intervening polypeptide sequence, the intein, from a precursor protein and the concomitant ligation of the flanking polypeptides, the exteins, by a peptide bond. Most reported inteins have a C-terminal asparagine residue, and it has been shown that cyclization of this residue is coupled to peptide bond cleavage between the intein and C-extein. We show that the intein interrupting the DNA polymerase II DP2 subunit in Pyrococcus abyssi, which has a C-terminal glutamine, is capable of facilitating protein splicing. Substitution of an asparagine for the C-terminal glutamine moderately improves the rate and extent of protein splicing. However, substitution of an alanine for the penultimate histidine residue, with either asparagine or glutamine in the C-terminal position, prevents protein splicing and facilitates cleavage at the intein N terminus. The intein facilitates in vitro protein splicing only at temperatures above 30 degrees C and can be purified as a nonspliced precursor. This temperature dependence has enabled us to characterize the optimal in vitro splicing conditions and determine the rate constants for splicing as a function of temperature.     

Expressed protein ligation. Method and applications.

The introduction of noncanonical amino acids and biophysical probes into peptides and proteins, and total or segmental isotopic labelling has the potential to greatly aid the determination of protein structure, function and protein-protein interactions. To obtain a peptide as large as possible by solid-phase peptide synthesis, native chemical ligation was introduced to enable synthesis of proteins of up to 120 amino acids in length. After the discovery of inteins, with their self-splicing properties and their application in protein synthesis, the semisynthetic methodology, expressed protein ligation, was developed to circumvent size limitation problems. Today, diverse expression vectors are available that allow the production of N- and C-terminal fragments that are needed for ligation to produce large amounts and high purity protein(s) (protein alpha-thioesters and peptides or proteins with N-terminal Cys). Unfortunately, expressed protein ligation is still limited mainly by the requirement of a Cys residue. Of course, additional Cys residues can be introduced into the sequence by site directed mutagenesis or synthesis, however, those mutations may disturb protein structure and function. Recently, alternative ligation approaches have been developed that do not require Cys residues. Accordingly, it is theoretically possible to obtain each modified protein using ligation strategies.     

Chemical synthesis of circular proteins

Circular proteins, once thought to be rare, are now commonly found in plants. Their chemical synthesis, once thought to be difficult, is now readily achievable. The enabling methodology is largely due to the advances in entropic chemical ligation to overcome the entropy barrier in coupling the N- and C-terminal ends of large peptide segments for either intermolecular ligation or intramolecular ligation in end-to-end cyclization. Key elements of an entropic chemical ligation consist of a chemoselective capture step merging the N and C termini as a covalently linked O/S-ester intermediate to permit the subsequent step of an intramolecular O/S-N acyl shift to form an amide. Many ligation methods exploit the supernucleophilicity of a thiol side chain at the N terminus for the capture reaction, which makes cysteine-rich peptides ideal candidates for the entropy-driven macrocyclization. Advances in desulfurization and modification of the thiol-containing amino acids at the ligation sites to other amino acids add extra dimensions to the entropy-driven ligation methods. This minireview describes recent advances of entropy-driven ligation to prepare circular proteins with or without a cysteinyl side chain.     

Human topoisomerase IIalpha possesses an intrinsic nucleic acid specificity for DNA ligation. Use of 5' covalently activated oligonucleotide substrates to study enzyme mechanism

Despite the importance of topoisomerase II-mediated DNA ligation to the essential physiological functions of the enzyme, the mechanistic details of this important reaction are poorly understood. Because topoisomerase II normally does not release cleaved DNA molecules prior to ligation, it is not known whether all of the nucleic acid specificity of its cleavage/ligation cycle is embodied in DNA cleavage or whether ligation also contributes specificity to the enzyme. All currently available ligation assays require that topoisomerase II cleave the initial DNA substrate before rejoining can be monitored. Consequently, it has been impossible to examine the specificity of DNA ligation separately from that of scission. To address this issue, a cleavage-independent topoisomerase II DNA ligation assay was developed. This assay utilizes a nicked oligonucleotide whose 5'-phosphate terminus at the nick has been activated by covalent attachment to the tyrosine mimic, p-nitrophenol. Human topoisomerase IIalpha and enzymes with active-site mutations that abrogated cleavage activity ligated the activated nick by catalyzing the direct attack of the terminal 3'-OH on the activated 5'-phosphate. Results with different DNA sequences indicate that human topoisomerase IIalpha possesses an intrinsic nucleic acid specificity for ligation that parallels its specificity for DNA cleavage.     

Solid phase synthesis of peptide-selenoesters

The synthesis of proteins by native chemical ligation greatly enhances the application of chemistry to complex molecules such as proteins. The essential building blocks for this approach traditionally have been peptide-thioester segments that are linked chemoselectively in consecutive reactions. By using peptide selenoesters instead of thioesters, the ligation rate can be significantly accelerated permitting couplings at difficult sites and potentially enabling new ligation strategies. To facilitate the routine synthesis of selenoester peptides, a general and straightforward procedure has been developed that generates a suitably functionalized resin from which the desired selenoester peptide can be readily synthesized. This simple approach utilizes readily available and cheap chemical agents and enables production of peptide selenoesters of excellent quality in short time and with high recovery. In addition, the stability of peptide selenoesters was examined under different native chemical ligation conditions and compared to thioesters. Selenoesters are slightly more reactive and more susceptible to hydrolysis and aminolysis than thioesters but sufficiently stable under mildly acidic conditions (pH 6.5). Under these conditions, rapid selenoester-mediated ligation is kinetically favoured.