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PubMed1999

Relationships between alterations in glutathione metabolism and the disposition of inorganic mercury in rats: effects of biliary ligation and chemically induced modulation of glutathione status.

Influences of biliary ligation and systemic depletion of glutathione (GSH) or modulation of GSH status on the disposition of a low, non-nephrotoxic i.v. dose of inorganic mercury were evaluated in rats in the present study. Renal and hepatic disposition, and the urinary and fecal excretion, of inorganic mercury were assessed 24 h after the injection of a 0.5-micromol/kg dose of mercuric chloride in control rats and rats pretreated with acivicin (two 10-mg/kg i.p. doses in 2 ml/kg normal saline, 90 min apart, 60 min before mercuric chloride), buthionine sulfoximine (BSO; 2 mmol/kg i.v. in 4 ml/kg normal saline, 2 h before mercuric chloride) or diethylmaleate (DEM; 3.37 mmol/kg i.p. in 2 ml/kg corn oil, 2 h before mercuric chloride) that either underwent or did not undergo acute biliary ligation prior to the injection of mercury. Among the groups that did not undergo biliary ligation, the pretreatments used to alter GSH status systemically had varying effects on the disposition of inorganic mercury in the kidneys, liver, and blood. Biliary ligation caused the net renal accumulation of mercury to decrease under all pretreatment conditions. By contrast, biliary ligation caused significant increases in the hepatic burden of mercury in all pretreatment groups except in theacivicin-pretreated group. Blood levels of mercury also increased as a result of biliary ligation, regardless of the type of pretreatment used. The present findings indicate that biliary ligation combined with methods used to modulate GSH status systemically have additive effects with respect to causing reductions in the net renal accumulation of mercury. Additionally, the findings indicate that at least some fraction of the renal accumulation of inorganic mercury is linked mechanistically to the hepato-biliary system.

Animals↗
PubMed2003

Recombinogenic flap ligation mediated by human topoisomerase I.

Aberration of eukaryotic topoisomerase I catalysis leads to potentially recombinogenic pathways by allowing the joining of heterologous DNA strands. Recently, a new ligation pathway (flap ligation) was presented for vaccinia virus topoisomerase I, in which blunt end cleavage complexes ligate the recessed end of duplex acceptors having a single-stranded 3'-tail. This reaction was suggested to play an important role in the repair of topoisomerase I-induced DNA double-strand breaks. Here, we characterize flap ligation mediated by human topoisomerase I. We demonstrate that cleavage complexes containing the enzyme at a blunt end allow invasion of a 3'-acceptor tail matching the scissile strand of the donor, which facilitates ligation of the recessed 5'-hydroxyl end. However, the reaction was strictly dependent on the length of double-stranded DNA of the donor complexes, and longer stretches of base-pairing inhibited strand invasion. The stabilization of the DNA helix was most probably provided by the covalently bound enzyme itself, since deleting the N-terminal domain of human topoisomerase I stimulated flap ligation. We suggest that stabilization of the DNA duplex upon enzyme binding may play an important role during normal topoisomerase I catalysis by preventing undesired strand transfer reactions. For flap ligation to function in a repair pathway, factors other than topoisomerase I, such as helicases, would be necessary to unwind the DNA duplex and allow strand invasion.

Base Sequence↗
PubMed2001

Tubal ligation and risk of ovarian cancer in carriers of BRCA1 or BRCA2 mutations: a case-control study.

BACKGROUND: In several case-control and prospective studies, tubal ligation has been associated with a decreased risk of invasive epithelial ovarian cancer. We aimed to assess the potential of tubal ligation in reducing the risk of ovarian cancer in women who carry predisposing mutations in the BRCA1 or BRCA2 genes. METHODS: We did a matched case-control study among women from Canada, the USA, and the UK who had undergone genetic testing and who carried a pathogenic mutation in BRCA1 or BRCA2. Cases were 232 women with a history of invasive ovarian cancer, and controls were 232 women without ovarian cancer, and who had both ovaries intact. Cases and controls were matched for year of birth, country of residence, and mutation (BRCA1 or BRCA2). The odds ratio for developing ovarian cancer was estimated for tubal ligation, adjusting for oral contraceptive use, parity, history of breast cancer, and ethnic group. FINDINGS: In an unadjusted analysis among BRCA1 carriers, significantly fewer cases than controls had ever had tubal ligation (30 of 173 [18%] vs 60 of 173 [35%], odds ratio 0.37 [95% CI 0.21-0.63]; p=0.0003). After adjustment for oral contraceptive use, parity, history of breast cancer and ethnic group, the odds ratio was 0.39 (p=0.002). Combination of tubal ligation and past use of an oral contraceptive was associated with an odds ratio of 0.28 (0.15-0.52). No protective effect of tubal ligation was seen among carriers of the BRCA2 mutation. INTERPRETATION: Tubal ligation is a feasible option to reduce the risk of ovarian cancer in women with BRCA1 mutations who have completed childbearing.

Adult↗
PubMed2002

A new technique to prevent self-ligation of DNA.

The most widely used technique for preventing self-ligation (self-circularization and concatenation) of DNA is dephosphorylation of the 5'-end, which stops DNA ligase from catalyzing the formation of phosphodiester bonds between the 3'-hydroxyl and 5'-phosphate residues at the DNA ends. The 5'-dephosphorylation technique cannot be applied to both DNA species to be ligated and thus, the untreated DNA species remains capable of self-ligation. To prevent this self-ligation, we replaced the 2'-deoxyribose at the 3'-end of the untreated DNA species with a 2',3'-dideoxyribose. Self-ligation was prevented at the replaced 3'-end, while the 5'-phosphate remaining at the 5'-end permitted ligation with the 3'-hydroxyl end of the 5'-dephosphorylated DNA strand. We successfully applied this 3'-replacement technique to gene cloning, adapter-mediated polymerase chain reaction and messenger RNA fingerprinting. The 3'-replacement technique is simple and not restricted by sequence or conformation of the DNA termini and is thus applicable to a wide variety of methods involving ligation.

Cloning, Molecular↗
PubMed1991

Experimental resolution of cooperative free energies for the ten ligation species of cobalt(II)/iron(II)-CO hemoglobin.

Cooperative free energies have been determined for the 10 ligation species of human hemoglobin in the Co(II)/Fe(II)-CO system. In this system, subunits containing unligated cobaltous hemes coexist in the same tetramer with naturally occurring ferrous hemes that are ligated with carbon monoxide. Tetramers comprising the 10 structurally unique combinations of ligated and unligated subunits were characterized in terms of their dimer-tetramer assembly free energies. By use of the thermodynamic linkage between assembly and ligation, the experimentally resolved values were used to obtain the corresponding cooperative free energies (i.e., the differences between actual free energies of ligation and the summed contributions of intrinsic values). The results obtained are in general accord with previous findings on this same system (Imai et al., 1980). The present study extends this earlier work by resolving the cooperative properties of each configurational isomer of the doubly ligated tetramers. The 10 ligation species were found to distribute into 5 discrete cooperative free energy levels according to a combinatorial code which includes, as a special case, the code found previously with cyanomethemoglobin and manganese-substituted hemoglobin (Smith et al., 1987; Daugherty et al., 1991). This distribution exhibits additional characteristics found in the oxygenation of normal ferrous hemoglobin including the quaternary enhancement effect (Mills & Ackers, 1979a,b). These results, and those of the following paper (Doyle et al., 1991), strongly support the premise that a common set of qualitative rules governs the cooperative interactions in hemoglobin irrespective of

Carbon Monoxide↗
PubMed2004

Effects of deletion and site-directed mutations on ligation steps of NAD+-dependent DNA ligase: a biochemical analysis of BRCA1 C-terminal domain.

DNA strand joining entails three consecutive steps: enzyme adenylation to form AMP-ligase, substrate adenylation to form AMP-DNA, and nick closure. In this study, we investigate the effects on ligation steps by deletion and site-directed mutagenesis of the BRCA1 C-terminal (BRCT) domain using NAD(+)-dependent DNA ligase from Thermus species AK16D. Deletion of the BRCT domain resulted in substantial loss of ligation activity, but the mutant was still able to form an AMP-ligase intermediate, suggesting that the defects caused by deletion of the entire BRCT domain occur primarily at steps after enzyme adenylation. The lack of AMP-DNA accumulation by the domain deletion mutant as compared to the wild-type ligase indicates that the BRCT domain plays a role in the substrate adenylation step. Gel mobility shift analysis suggests that the BRCT domain and helix-hairpin-helix subdomain play a role in DNA binding. Similar to the BRCT domain deletion mutant, the G617I mutant showed a low ligation activity and lack of accumulation of AMP-DNA intermediate. However, the G617I mutant was only weakly adenylated, suggesting that a point mutation in the BRCT domain could also affect the enzyme adenylation step. The significant reduction of ligation activity by G634I appears to be attributable to a defect at the substrate adenylation step. The greater ligation of mismatched substrates by G638I is accountable by accelerated conversion of the AMP-DNA intermediate to a ligation product at the final nick closure step. The mutational effects of the BRCT domain on ligation steps in relation to protein-DNA and potential protein-protein interactions are discussed.

Amino Acid Sequence↗
PubMed1999

Engineering cytochrome c peroxidase into cytochrome P450: a proximal effect on heme-thiolate ligation.

In an effort to investigate factors required to stabilize heme-thiolate ligation, key structural components necessary to convert cytochrome c peroxidase (CcP) into a thiolate-ligated cytochrome P450-like enzyme have been evaluated and the H175C/D235L CcP double mutant has been engineered. The UV-visible absorption, magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) spectra for the double mutant at pH 8.0 are reported herein. The close similarity between the spectra of ferric substrate-bound cytochrome P450cam and those of the exogenous ligand-free ferric state of the double mutant with all three techniques support the conclusion that the latter has a pentacoordinate, high-spin heme with thiolate ligation. Previous efforts to prepare a thiolate-ligated mutant of CcP with the H175C single mutant led to Cys oxidation to cysteic acid [Choudhury et al. (1994) J. Biol. Chem. 267, 25656-25659]. Therefore it is concluded that changing the proximal Asp235 residue to Leu is critical in forming a stable heme-thiolate ligation in the resting state of the enzyme. To further probe the versatility of the CcP double mutant as a ferric P450 model, hexacoordinate low-spin complexes have also been prepared. Addition of the neutral ligand imidazole or of the anionic ligand cyanide results in formation of hexacoordinate adducts that retain thiolate ligation as determined by spectral comparison to the analogous derivatives of ferric P450cam. The stability of these complexes and their similarity to the analogous forms of P450cam illustrates the potential of the H175C/D235L CcP double mutant as a model for ferric P450 enzymes. This study marks the first time a stable cyanoferric complex of a model P450 has been made and demonstrates the importance of the environment around the primary coordination ligands in stabilizing metal-ligand ligation.

Aspartic Acid↗
PubMed2003

Tandem ligation of multipartite peptides with cell-permeable activity.

To prepare multipartite peptides with several functional cargoes including a cell-permeable sequence or transportant for intracellular delivery, tandem ligation of peptides is a convenient convergent approach with the fewest synthetic steps. It links three or four unprotected segments forming two or more regiospecific bonds consecutively without a deprotection step. This paper describes a tandem ligation strategy to prepare multipartite peptides with normal and branched architectures carrying a novel transportant peptide that is rich in arginine and proline to permit their cargoes to be translocated across membranes to affect their biological functions in cytoplasm. Our strategy consists of three ligation methods specific for amino terminal cysteine (Cys), serine/threonine (Ser/Thr), and N(alpha)-chloroacetylated amine to afford Xaa-Cys, Xaa-OPro (oxaproline) and Xaa-psiGly (pseudoglycine) at the ligation sites, respectively. Assembly of single-chain peptides from three different segments was achieved by the tandem Cys/OPro ligation to form two amide bonds, an Xaa-Cys and then an Xaa-OPro. Assembly of two- and three-chain peptides with branched architectures from four different segments was accomplished by tandem Cys/psiGly/OPro ligation. These NT-specific tandem ligation strategies were successful in generating cell-permeable multipartite peptides with one-, two-, and three-chain architectures, ranging in size from 52 to 75 residues and without the need of a protection or deprotection step. In addition, our results show that there is considerable flexibility in architectural design to obtain cell-permeable multipartite peptides containing a transportant sequence.

Biological Transport↗
PubMed2004

Destabilizing universal linkers for signal amplification in self-ligating probes for RNA.

Recent studies have established the utility of oligonucleotide ligation methods in the detection of DNAs and RNAs in solution and in cellular imaging. Notably, the ligated full-length oligonucleotide products commonly bind to the target nucleic acid much more tightly than do the two starting half-probes, which effectively limits the resulting signals to one per target. Here, we report on a molecular strategy for destabilizing ligated products in template-promoted self-ligation reactions, thus yielding multiple signals per target. A new universal linker design is described in which a dabsyl leaving group is placed on a short alkane tether. This allows the placement of an electrophile at the end of any DNA sequence, in contrast to earlier ligation strategies, and it also speeds reaction rates by a factor of 4-5. This new class of molecular linker/activator yields as much as 92-fold amplification of signals in DNA and RNA detection, and proceeds without enzymes, added reagents, or thermal cycling. The linker is shown to destabilize the ligation product without destabilizing the transition state for ligation. This lowers product inhibition, and the target DNA or RNA thus becomes a catalyst for isothermally generating multiple signals for its detection. This enhanced signal generation is demonstrated in solution experiments and in solid supported assays.

Cross-Linking Reagents↗
PubMed1996

Kinetic and mechanistic analysis of nonenzymatic, template-directed oligoribonucleotide ligation.

The role of divalent cations in the mechanism of pyrophosphate-activated, template-directed oligoribonucleotide ligation has been investigated. The dependence of the reaction rate on Mg2+ concentration suggests a kinetic scheme in which a Mg2+ ion must bind before ligation can proceed. Mn2+, Ca2+, Sr2+, and Ba2+ can also catalyze the reaction. Although Pb2+ and Zn2+ do not catalyze the reaction in the absence of other divalent ions, they significantly modulate the reaction rate when added in the presence of Mg2+, with Pb2+ stimulating the reaction (up to 65-fold) and Zn2+ inhibiting the reaction. The logarithm of the ligation rate increases linearly, with slope of 0.95, as a function of pH, indicating that the reaction involves a single critical deprotonation step. The ligation rates observed with the different divalent metal ion catalysts (Mn2+ > Mg2+ > Ca2+ > Sr2+ = Ba2+) vary inversely with the pKa values of their bound water molecules. The pH profile and these relative ligation rates suggest a mechanism in which a metal-bound hydroxide ion located near the ligation junction promotes catalysis, most likely by deprotonation of the hydroxl nucleophile. The effects of changing either the leaving group or the attacking hydroxyl, together with the large delta S(++) value for oligonucleotide ligation (about -20 eu), are consistent with an associative transition state.

Catalysis↗
PubMed2002

A cassette ligation strategy with thioether replacement of three Gly-Gly peptide bonds: total chemical synthesis of the 101 residue protein early pregnancy factor [psi(CH(2)S)28-29,56-57,76-77].

The 101 residue protein "early pregnancy factor" (EPF), also known as human chaperonin 10, was synthesized from four functionalized, but unprotected, peptide segments by a sequential thioether ligation strategy. The approach exploits the differential reactivity of a peptide-NHCH(2)CH(2)SH thiolate with XCH(2)CO-peptides, where X = Cl or I/Br. Initial model studies with short functionalized (but unprotected) peptides showed a significantly faster reaction of a peptide-NHCH(2)CH(2)SH thiolate with a BrCH(2)CO-peptide than with a ClCH(2)CO-peptide, where thiolate displacement of the halide leads to chemoselective formation of a thioether surrogate for the Gly-Gly peptide bond. This rate difference was used as the basis of a novel sequential ligation approach to the synthesis of large polypeptide chains. Thus, ligation of a model bifunctional N(alpha)-chloroacetyl, C-terminal thiolated peptide with a second N(alpha)-bromoacetyl peptide demonstrated chemoselective bromide displacement by the thiol group. Further investigations showed that the relatively unreactive N(alpha)-chloroacetyl peptides could be "activated" by halide exchange using saturated KI solutions to yield the highly reactive N(alpha)-iodoacetyl peptides. These findings were used to formulate a sequential thioether ligation strategy for the synthesis of EPF, a 101 amino acid protein containing three Gly-Gly sites approximately equidistantly spaced within the peptide chain. Four peptide segments or "cassettes" comprising the EPF protein sequence (BrAc-[EPF 78-101] 12, ClAc-[EPF 58-75]-[NHCH(2)CH(2)SH] 13, ClAc-[EPF 30-55]-[NHCH(2)CH(2)SH] 14, and Ac-[EPF 1-27]-[NHCH(2)CH(2)SH] 15) of EPF were synthesized in high yield and purity using Boc SPPS chemistry. In the stepwise sequential ligation strategy, reaction of peptides 12 and 13 was followed by conversion of the N-terminal chloroacetyl functional group to an iodoacetyl, thus activating the product peptide for further ligation with peptide 14. The process of ligation followed by iodoacetyl activation was repeated to yield an analogue of EPF (EPF psi(CH(2)S)(28)(-)(29,56)(-)(57,76)(-)(77)) 19 in 19% overall yield.

Amino Acid Sequence↗
PubMed2006

Staudinger ligation of peptides at non-glycyl residues.

The Staudinger ligation provides a means to form an amide bond between a phosphinothioester and azide. This reaction holds promise for the ligation of peptides en route to the total chemical synthesis of proteins. (Diphenylphosphino)methanethiol is the most efficacious of known reagents for mediating the Staudinger ligation of peptides, providing high (> 90%) isolated yields for equimolar couplings in which a glycine residue is at the nascent junction. Surprisingly, the yields are lower (< 50%) for non-glycyl couplings due to an aza-Wittig reaction that diverts the reaction toward a phosphonamide byproduct. Here, the partitioning of the reaction toward Staudinger ligation (and away from the aza-Wittig reaction) is shown to increase with increasing electron density on phosphorus. This electron density can be tuned either by installing functional groups on the phenyl substituents of (diphenylphosphino)methanethiol or by changing the polarity of the solvent. Installing p-methoxy groups and using a solvent of low polarity (such as toluene or dioxane) provide especially high (> 80%) isolated yields for the ligation of two non-glycyl residues. These conditions retain the high chemoselectivity of the reaction and do not lead to a substantial change in reaction rate. The traceless Staudinger ligation is now poised to enable the iterative ligation of peptides with little regard for their sequence, as well as the synthesis of amide bonds for other purposes.

Amides↗
PubMed1999

The fidelity of template-directed oligonucleotide ligation and the inevitability of polymerase function.

The first living systems may have employed template-directed oligonucleotide ligation for replication. The utility of oligonucleotide ligation as a mechanism for the origin and evolution of life is in part dependent on its fidelity. We have devised a method for evaluating ligation fidelity in which ligation substrates are selected from random sequence libraries. The fidelities of chemical and enzymatic ligation are compared under a variety of conditions. While reaction conditions can be found that promote high fidelity copying, departure from these conditions leads to error-prone copying. In particular, ligation reactions with shorter oligonucleotide substrates are less efficient but more faithful. These results support a model for origins in which there was selective pressure for template-directed oligonucleotide ligation to be gradually supplanted by mononucleotide polymerization.

Base Sequence↗
PubMed2003

Histopathological changes in ovary and endometrium after tubal ligation: a rat model.

BACKGROUND: To evaluate the histopathological effects of tubal ligation on ovary and endometrium in a rat model. METHODS: Twenty-four female Wistar albino rats weighing 220-260 g were used. The rats were assigned randomly into tubal ligation and control groups. While tubal ligation was applied to the first group of rats, only a laparotomy was performed in the second group. Six weeks later, a second laparotomy was performed and uterine horns and ovaries of the rats in the two groups were excised for histopathological assessment. A pathologist blinded to the groups made histopathological examination including quantification of endometrial phases, presence of endometrial inflammation and counting the number of tertiary follicles and corpora lutea in each ovary. RESULTS: We found no significant difference between tubal ligation and control groups related to the number of tertiary follicles and corpora lutea (p > 0.05). However, in the tubal ligation group, endometrial inflammatory infiltration was significantly higher than in the control group (p < 0.05). CONCLUSION: Tubal ligation does not affect ovarian histology as an indicator of ovarian function. However, endometrial inflammation may occur after tubal ligation and lead to menstrual irregularities as an early complication.

Animals↗
PubMed2005

Frictional evaluations of dental typodont models using four self-ligating designs and a conventional design.

Abstract: After a previous study using typodonts and three standardized archwire (AW) sizes, the frictional evaluations of four self-ligating brackets were directed toward the optimal AW-bracket system. Four participating manufacturers suggested three AWs, which were a representation of the three stages of orthodontic treatment, to be coupled with their respective self-ligating design. Four replicated typodont models were mounted with a self-ligating design, and a fifth model was mounted with a conventional design that served as a control. The first experiment evaluated the manufacturer-suggested AWs against the respective self-ligating design. Because no third-stage AWs could engage their respective designs, a second experiment was implemented to gain more detailed analyses of the designs. This experiment included any successful manufacturer-suggested AWs from the first experiment against the four self-ligating designs and the control design. All self-ligating designs performed with the efficiency and reproducibility associated with expectations. Specifically, self-ligation outperformed the conventional brackets when coupled with up to 0.020- x 0.020-inch wires. The clearance of the various AW sizes and alloys changed with malocclusion. Furthermore, the parameter that best correlated with drawing forces was the bending stiffness of the AW, which was directly associated with the nominal dimension of each wire. The best AW-bracket system can be selected, when taking into account the stiffness (elastic modulus and size of the AW) along with the amount of malocclusion present, once the treatment plan is determined.

Dental Alloys↗
PubMed1985

Experimental resolution of cooperative free energies for the ten ligation states of human hemoglobin.

Tetrameric human hemoglobin can assume ten molecular forms that differ in the number and configuration of ligands bound at the four heme sites. For each of these species we have determined the cooperative free energy--i.e., the deviation in free energy of ligation from that which would obtain for the same sites binding as independent alpha and beta subunits. These cooperative free energies were resolved from measurements on the dissociation into dimers of tetramers in which each subunit is either unligated (Fe2+ deoxy) or "ligated" by conversion into the cyanomet form (Fe3+ CN). The results indicate that each hemoglobin tetramer acts as a three-level molecular switch. During the course of ligation, the total cooperative free energy (6 kcal/mol over all four binding steps) is expended in two transitions that are synchronized with particular ligation steps. Whether a cooperative energy transition occurs or not depends upon how the ligation step changes both the number and configuration of ligated subunits. The hemoglobin tetramer is thus a "combinatorial switch." The finding of three distinct free energy levels for the ten ligation states suggests the existence of three major structural forms of the hemoglobin tetramer.

Hemoglobins↗
PubMed1989

Mechanism of mercury(II) reductase and influence of ligation on the reduction of mercury(II) by a water soluble 1,5-dihydroflavin.

The nature and rate of reduction of Hg2+ to Hg0 by 1,5-dihydro-3,(3-sulfopropyl)lumiflavin (FIH2) in buffered aqueous solutions (pH 4.7) is dependent on the ligation of Hg2+. In the presence of N,N-bis(2-hydroxyethyl)glycine or when ligated to ethylenediaminetetraacetic acid, the reduction is first order in Hg2+ and FIH2. The apparent second-order rate constant with N,N-bis(2-hydroxyethyl)glycine (2.2 x 10(6) M-1.s-1) is much greater than that in the presence of ligating ethylenediaminetetraacetic acid (1.5 x 10(2) M-1.s-1). When ligated by mercaptoethanesulfonate, reduction of Hg2+ by FIH2 is characterized by a pronounced lag phase, which is dependent on the concentration of mercaptoethanesulfonate. The rate decreases with increase in mercaptoethanesulfonate, and with an excess of 10 equivalents, Hg2+ is not reduced by FIH2. These observations show that bis-ligation by thiolate greatly decreases the reducibility of Hg2+ and that further ligation by thiolate further retards the reaction. Comparison of oxidation-reduction potentials at various pH values shows that bis-ligation (or greater) of Hg2+ by thiolate substantially lowers the reduction potential of Hg2+ below that of 3(3-sulfopropyl)lumiflavin (FIox). Thus, the ease of reduction of Hg2+ complexes by FIH2 decreases with increasing thermodynamic stability of the complex. These results do not support the proposed role of the thiol functionalities in facilitating the mercury(II) reductase (Hg:NADP+ oxidoreductase, EC 1.16.1.1)-catalyzed reduction of Hg2+ through tris- or tetraligation of Hg2+.

Edetic Acid↗
PubMed1999

Protein synthesis by native chemical ligation: expanded scope by using straightforward methodology.

The total chemical synthesis of proteins has great potential for increasing our understanding of the molecular basis of protein function. The introduction of native chemical ligation techniques to join unprotected peptides next to a cysteine residue has greatly facilitated the synthesis of proteins of moderate size. Here, we describe a straightforward methodology that has enabled us to rapidly analyze the compatibility of the native chemical ligation strategy for X-Cys ligation sites, where X is any of the 20 naturally occurring amino acids. The simplified methodology avoids the necessity of specific amino acid thioester linkers or alkylation of C-terminal thioacid peptides. Experiments using matrix-assisted laser-desorption ionization MS analysis of combinatorial ligations of LYRAX-C-terminal thioester peptides to the peptide CRANK show that all 20 amino acids are suitable for ligation, with Val, Ile, and Pro representing less favorable choices because of slow ligation rates. To illustrate the method's utility, two 124-aa proteins were manually synthesized by using a three-step, four-piece ligation to yield a fully active human secretory phospholipase A(2) and a catalytically inactive analog. The combination of flexibility in design with general access because of simplified methodology broadens the applicability and versatility of chemical protein synthesis.

Amino Acid Sequence↗