Peptide Ligation Research Articles

Refolding the unfoldable: A systematic approach for renaturation of Bacillus circulans xylanase.

Xylanases are important polysaccharide-cleaving catalysts for the pulp and paper, animal feeds and biofuels industries. They have also proved to be valuable model systems for understanding enzymatic catalysis, with one of the best studied being the GH11 xylanase from Bacillus circulans (Bcx). However, proteins from this class are very recalcitrant to refolding in vitro. This both limits their high level expression in heterologous hosts, and prevents experimental approaches, such as peptide ligation or chemical modifications, to probe and engineer their stability and function. To solve this problem, a systematic screening approach was employed to identify suitable buffer conditions for renaturing Bcx in vitro. The fractional factorial screen employed identified starting conditions for refolding, which were then refined and developed into a generic protocol for renaturing preparative amounts of active Bcx in a 50-60% yield from inclusion bodies. The method is robust and proved equally proficient at refolding circularly permuted versions that carry cysteine mutations. This general approach should be applicable to related GH11 xylanases, as well as proteins adopting a similar β-jellyroll fold, that are otherwise recalcitrant to refolding in vitro.

Omniligase‐1: A Powerful Tool for Peptide Head‐to‐Tail Cyclization

AbstractStrategies for the efficient synthesis of peptide macrocycles have been a long‐standing goal. In this paper, we demonstrate the use of the peptide ligase termed omniligase‐1 as a versatile and broadly applicable enzymatic tool for peptide cyclization. Several head‐to‐tail (multi)cyclic peptides have been synthesized, including the cyclotide MCoTI‐II. Cyclization and oxidative folding of the cyclotide MCoTI‐II were efficiently performed in a one‐pot reaction on a 1‐gram scale. The native cyclotide was isolated and the correct disulfide bonding pattern was confirmed by NMR structure determination. Furthermore, compatibility of chemo‐enzymatic peptide synthesis (CEPS) using omniligase‐1 with methods such as chemical ligation of peptides onto scaffolds (CLIPS) was successfully demonstrated by synthesizing a kinase‐inhibitor derived tricyclic peptide. Our studies indicate that the minimal ring size for omniligase‐1 mediated cyclization is 11 amino acids, whereas the cyclization of peptides longer than 12 amino acids proceeds with remarkable efficiency. In addition, several macrocycles containing non‐peptidic backbones (e.g., polyethylene glycol), isopeptide bonds (amino acid side‐chain attachment) as well as d‐amino acids could be efficiently cyclized.magnified image

Revisiting ligation at selenomethionine: Insights into native chemical ligation at selenocysteine and homoselenocysteine

Selenomethionine (Sem) has been incorporated recombinantly into proteins many times to elucidate their structure and function. In this paper, we revisit incorporation via chemical protein synthesis to shed light on the mechanism of native chemical ligation. The effect of chalcogen position on ligation is investigated, and selenium-containing peptide ligation is optimized. Additionally, selective methylation is performed on selenolates in a peptide in the presence of unprotected thiols.

Accelerated Protein Synthesis via One-Pot Ligation-Deselenization Chemistry

Peptide ligation chemistry has revolutionized protein science by facilitating access to synthetic proteins. Here, we describe the development of additive-free ligation-deselenization chemistry at β-selenoaspartate and γ-selenoglutamate that enables the generation of native polypeptide products on unprecedented timescales. The deselenization step is chemoselective in the presence of unprotected selenocysteine, which is highlighted in the synthesis of selenoprotein K. The power of the methodology is also showcased through the synthesis of three tick-derived thrombin-inhibiting proteins, each of which were assembled, purified, and isolated for biological assays within a few hours. The methodology described here should serve as a powerful means of accessing synthetic proteins, including therapeutic leads, in the future.

Segmental isotopic labeling of a single-domain globular protein without any refolding step by an asparaginyl endopeptidase.

Asparaginyl endopeptidases (AEPs) catalyze head-to-tail backbone cyclization of naturally occurring cyclic peptides such as cyclotides, and have become an important peptide-engineering tool for macrocyclization and peptide ligation. Here, we report efficient protein ligation in trans by mimicking efficient backbone cyclization by an AEP without any excess of reactants. We demonstrate a practical application of segmental isotopic labeling for NMR studies of a single-domain globular protein without any refolding step using the recombinant AEP prepared from Escherichia coli. This simple protein ligation approach using an AEP could be applied for incorporation of various biophysical probes into proteins as well as post-translational production of full-length proteins.

Removable Backbone Modification Method for the Chemical Synthesis of Membrane Proteins.

Chemical synthesis can produce water-soluble globular proteins bearing specifically designed modifications. These synthetic molecules have been used to study the biological functions of proteins and to improve the pharmacological properties of protein drugs. However, the above advances notwithstanding, membrane proteins (MPs), which comprise 20-30% of all proteins in the proteomes of most eukaryotic cells, remain elusive with regard to chemical synthesis. This difficulty stems from the strong hydrophobic character of MPs, which can cause considerable handling issues during ligation, purification, and characterization steps. Considerable efforts have been made to improve the solubility of transmembrane peptides for chemical ligation. These methods can be classified into two main categories: the manipulation of external factors and chemical modification of the peptide. This Account summarizes our research advances in the development of chemical modification especially the two generations of removable backbone modification (RBM) strategy for the chemical synthesis of MPs. In the first RBM generation, we install a removable modification group at the backbone amide of Gly within the transmembrane peptides. In the second RBM generation, the RBM group can be installed into all primary amino acid residues. The second RBM strategy combines the activated intramolecular O-to-N acyl transfer reaction, in which a phenyl group remains unprotected during the coupling process, which can play a catalytic role to generate the activated phenyl ester to assist in the formation of amide. The key feature of the RBM group is its switchable stability in trifluoroacetic acid. The stability of these backbone amide N-modifications toward TFA can be modified by regulating the electronic effects of phenol groups. The free phenol group is acylated to survive the TFA deprotection step, while the acyl phenyl ester will be quantitatively hydrolyzed in a neutral aqueous solution, and the free phenol group increases the electron density of the benzene ring to make the RBM labile to TFA. The transmembrane peptide segment bearing RBM groups behaves like a water-soluble peptide during fluorenylmethyloxycarbonyl based solid-phase peptide synthesis (Fmoc SPPS), ligation, purification, and characterization. The quantitative removal of the RBM group can be performed to obtain full-length MPs. The RBM strategy was used to prepare the core transmembrane domain Kir5.1[64-179] not readily accessible by recombinant protein expression, the influenza A virus M2 proton channel with phosphorylation, the cation-specific ion channel p7 from the hepatitis C virus with site-specific NMR isotope labels, and so on. The RBM method enables the practical engineering of small- to medium-sized MPs or membrane protein domains to address fundamental questions in the biochemical, biophysical, and pharmaceutical sciences.

Structure and specificity of a new class of Ca2+-independent housekeeping sortase from Streptomyces avermitilis provide insights into its non-canonical substrate preference

Surface proteins in Gram-positive bacteria are incorporated into the cell wall through a peptide ligation reaction catalyzed by transpeptidase sortase. Six main classes (A-F) of sortase have been identified of which class A sortase is meant for housekeeping functions. The prototypic housekeeping sortase A (SaSrtA) from Staphylococcus aureus cleaves LPXTG-containing proteins at the scissile T-G peptide bond and ligates protein-LPXT to the terminal Gly residue of the nascent cross-bridge of peptidoglycan lipid II precursor. Sortase-mediated ligation ("sortagging") of LPXTG-containing substrates and Gly-terminated nucleophiles occurs in vitro as well as in cellulo in the presence of Ca2+ and has been applied extensively for protein conjugations. Although the majority of applications emanate from SaSrtA, low catalytic efficiency, LPXTG specificity restriction, and Ca2+ requirement (particularly for in cellulo applications) remain a drawback. Given that Gram-positive bacteria genomes encode a variety of sortases, natural sortase mining can be a viable complementary approach akin to engineering of wild-type SaSrtA. Here, we describe the structure and specificity of a new class E sortase (SavSrtE) annotated to perform housekeeping roles in Streptomyces avermitilis Biochemical experiments define the attributes of an optimum peptide substrate, demonstrate Ca2+-independent activity, and provide insights about contrasting functional characteristics of SavSrtE and SaSrtA. Crystal structure, substrate docking, and mutagenesis experiments have identified a critical residue that dictates the preference for a non-canonical LAXTG recognition motif over LPXTG. These results have implications for rational tailoring of substrate tolerance in sortases. Besides, Ca2+-independent orthogonal specificity of SavSrtE is likely to expand the sortagging toolkit.

Peptide oligomers from ultra-short peptides using sortase

Sortase A catalyzed ligation of ultra-short peptides leads to inter/intra-molecular transpeptidation to form either linear or cyclic oligomers dependent upon the peptide length. Cyclic peptides were the main products for peptides with more than 15aa. However, for ultra-short (<15aa) peptides, cyclic oligomers became predominant in prolonged reactions. Peptides with 1–3 aminoglycines were equally active but peptide oligomers from peptide containing more than one aminoglycine were prone to hydrolysis.

Manganese‐Catalyzed C−H Alkynylation: Expedient Peptide Synthesis and Modification

AbstractManganese(I)‐catalyzed C−H alkynylations with organic halides occurred with unparalleled substrate scope, and thus enabled step‐economical C−H functionalizations with silyl, aryl, alkenyl, and alkyl haloalkynes. The versatility of the manganese(I) catalysis manifold enabled C−H couplings with haloalkynes featuring, among others, fluorescent labels, steroids, and amino acids, thereby setting the stage for peptide ligation as well as the efficient molecular assembly of acyclic and cyclic peptides. A plausible catalytic cycle was proposed.

Manganese-Catalyzed C-H Alkynylation: Expedient Peptide Synthesis and Modification.

Manganese(I)-catalyzed C-H alkynylations with organic halides occurred with unparalleled substrate scope, and thus enabled step-economical C-H functionalizations with silyl, aryl, alkenyl, and alkyl haloalkynes. The versatility of the manganese(I) catalysis manifold enabled C-H couplings with haloalkynes featuring, among others, fluorescent labels, steroids, and amino acids, thereby setting the stage for peptide ligation as well as the efficient molecular assembly of acyclic and cyclic peptides. A plausible catalytic cycle was proposed.

Bioorthogonal Diversification of Peptides through Selective Ruthenium(II)‐Catalyzed C–H Activation

AbstractMethods for the chemoselective modification of amino acids and peptides are powerful techniques in biomolecular chemistry. Among other applications, they enable the total synthesis of artificial peptides. In recent years, significant momentum has been gained by exploiting palladium‐catalyzed cross‐coupling for peptide modification. Despite major advances, the prefunctionalization elements on the coupling partners translate into undesired byproduct formation and lengthy synthetic operations. In sharp contrast, we herein illustrate the unprecedented use of versatile ruthenium(II)carboxylate catalysis for the step‐economical late‐stage diversification of α‐ and β‐amino acids, as well as peptides, through chemo‐selective C−H arylation under racemization‐free reaction conditions. The ligand‐accelerated C−H activation strategy proved water‐tolerant and set the stage for direct fluorescence labelling as well as various modes of peptide ligation with excellent levels of positional selectivity in a bioorthogonal fashion. The synthetic utility of our approach is further demonstrated by twofold C−H arylations for the complexity‐increasing assembly of artificial peptides within a multicatalytic C−H activation manifold.

Bioorthogonal Diversification of Peptides through Selective Ruthenium(II)-Catalyzed C-H Activation.

Methods for the chemoselective modification of amino acids and peptides are powerful techniques in biomolecular chemistry. Among other applications, they enable the total synthesis of artificial peptides. In recent years, significant momentum has been gained by exploiting palladium-catalyzed cross-coupling for peptide modification. Despite major advances, the prefunctionalization elements on the coupling partners translate into undesired byproduct formation and lengthy synthetic operations. In sharp contrast, we herein illustrate the unprecedented use of versatile ruthenium(II)carboxylate catalysis for the step-economical late-stage diversification of α- and β-amino acids, as well as peptides, through chemo-selective C-H arylation under racemization-free reaction conditions. The ligand-accelerated C-H activation strategy proved water-tolerant and set the stage for direct fluorescence labelling as well as various modes of peptide ligation with excellent levels of positional selectivity in a bioorthogonal fashion. The synthetic utility of our approach is further demonstrated by twofold C-H arylations for the complexity-increasing assembly of artificial peptides within a multicatalytic C-H activation manifold.

One-pot native chemical ligation by combination of two orthogonal thioester precursors.

We developed a one-pot peptide ligation method using two orthogonal thioester precursors and a protecting group for the ligation reaction between Asp and Cys. Combination of the two precursors facilitated the one-pot operation and yielded the entire polypeptide. The usefulness of this method was successfully demonstrated by the total synthesis of histone H4.

Singlet oxygen-mediated one-pot chemoselective peptide–peptide ligation

We here describe a furan oxidation based site-specific chemical ligation approach using unprotected peptide segments. This approach involves two steps: after photooxidation of a furan-containing peptide, ligation is achieved by reaction of the unmasked keto-enal with C- or N-terminal α-nucleophilic moieties of the second peptide such as hydrazine or hydrazide to form a pyridazinium or pyrrolidinone linkage respectively.

Engineering a Diverse Ligase Toolbox for Peptide Segment Condensation

AbstractThe substrate profile of peptiligase, a stable enzyme designed for peptide ligation in aqueous environments, was mapped using six different peptide libraries. The most discriminating substrate binding pocket proved to be the first nucleophile binding subsite (S1′), which is crucial for the peptide ligation yield. Two important amino acids shaping the S1′ pocket are M213 and L208. A site‐saturation library of the M213 position yielded two variants with a significantly broadened substrate profile, i.e., M213G and M213P. Next, examination of two libraries with M213G+L208X and M213P+L208X (with X being any proteinogenic amino acid) resulted in a toolbox of enzymes which can accommodate any proteinogenic amino acid in the S1′ pocket, except proline. The applicability of a particular enzyme variant in chemoenzymatic peptide synthesis was demonstrated by coupling at the gram scale of two peptide segments to yield exenatide, a 39‐mer therapeutic peptide used in the treatment of diabetes type II. The overall yield of 43% is at least 2‐fold higher than yields reported for conventional syntheses of exenatide by full solid‐phase peptide synthesis; large‐scale production costs are expected to be significantly reduced if the enzymatic coupling process is employed to manufacture this peptide.magnified image

The C-Terminal O-S Acyl Shift Pathway under Acidic Condition to Propose Peptide-Thioesters.

Peptide-thioester is a pivotal intermediate for peptide ligation and N-, C-terminal cyclization. In this study, desired pathway and the side products of two C-terminal handles, hydroxyethylthiol (HET) and hydroxypropylthiol (HPT) are described in different conditions as well as kinetic studies. In addition, a new mechanism of C-terminal residue racemization is proposed on the basis of differentiation of products derived from the two C-terminal handles in preparing peptide thioesters through an acid-catalyzed tandem thiol switch, first by an intramolecular O-S acyl shift, and then by an intermolecular S-S exchange.

Photoprotected Peptide α‐Ketoacids and Hydroxylamines for Iterative and One‐Pot KAHA Ligations: Synthesis of NEDD8

The convergent synthesis of proteins by multiple ligations requires segments protected at the N‐ and/or C‐terminus with masking groups that are orthogonal to the acid‐ and base‐labile protecting groups used in Fmoc‐SPPS. They must be stable to solid‐phase peptide synthesis, HPLC purification, and ligation conditions and easily removed in the presence of unprotected side chains. In this report, we document photolabile protecting groups for both α‐ketoacids and hydroxylamines, the key functional groups employed in the α‐ketoacid–hydroxylamine (KAHA) ligation. The novel photoprotected α‐ketoacid is easily installed onto numerous different C‐terminal peptide α‐ketoacids and removed by UV light under aqueous conditions. These advances were applied to the one‐pot synthesis of NEDD8, an important modifier protein, by three different convergent routes. These new protecting groups provide greater flexibility on the order of fragment assembly and reduce the number of reaction and purification steps needed for protein synthesis with the KAHA ligation.

HF-Free Boc Synthesis of Peptide Thioesters for Ligation and Cyclization.

We have developed a convenient method for the direct synthesis of peptide thioesters, versatile intermediates for peptide ligation and cyclic peptide synthesis. The technology uses a modified Boc SPPS strategy that avoids the use of anhydrous HF. Boc in situ neutralization protocols are used in combination with Merrifield hydroxymethyl resin and TFA/TMSBr cleavage. Avoiding HF extends the scope of Boc SPPS to post‐translational modifications that are compatible with the milder cleavage conditions, demonstrated here with the synthesis of the phosphorylated protein CHK2. Peptide thioesters give easy, direct, access to cyclic peptides, illustrated by the synthesis of cyclorasin, a KRAS inhibitor.

HF‐Free Boc Synthesis of Peptide Thioesters for Ligation and Cyclization

AbstractWe have developed a convenient method for the direct synthesis of peptide thioesters, versatile intermediates for peptide ligation and cyclic peptide synthesis. The technology uses a modified Boc SPPS strategy that avoids the use of anhydrous HF. Boc in situ neutralization protocols are used in combination with Merrifield hydroxymethyl resin and TFA/TMSBr cleavage. Avoiding HF extends the scope of Boc SPPS to post‐translational modifications that are compatible with the milder cleavage conditions, demonstrated here with the synthesis of the phosphorylated protein CHK2. Peptide thioesters give easy, direct, access to cyclic peptides, illustrated by the synthesis of cyclorasin, a KRAS inhibitor.

Butelase-mediated cyclization and ligation of peptides and proteins.

Enzymes that catalyze efficient macrocyclization or site-specific ligation of peptides and proteins can enable tools for drug design and protein engineering. Here we describe a protocol to use butelase 1, a recently discovered peptide ligase, for high-efficiency cyclization and ligation of peptides and proteins ranging in size from 10 to >200 residues. Butelase 1 is the fastest known ligase and is found in pods of the common medicinal plant Clitoria ternatea (also known as butterfly pea). It has a very simple C-terminal-specific recognition motif that requires Asn/Asp (Asx) at the P1 position and a dipeptide His-Val at the P1' and P2' positions. Substrates for butelase-mediated ligation can be prepared by standard Fmoc (9-fluorenylmethyloxycarbonyl) chemistry or recombinant expression with the minimal addition of this tripeptide Asn-His-Val motif at the C terminus. Butelase 1 achieves cyclizations that are 20,000 times faster than those of sortase A, a commonly used enzyme for backbone cyclization. Unlike sortase A, butelase is traceless, and it can be used for the total synthesis of naturally occurring peptides and proteins. Furthermore, butelase 1 is also useful for intermolecular ligations and synthesis of peptide or protein thioesters, which are versatile activated intermediates necessary for and compatible with many chemical ligation methods. The protocol describes steps for isolation and purification of butelase 1 from plant extract using a four-step chromatography procedure, which takes ∼3 d. We then describe steps for intramolecular cyclization, intermolecular ligation and butelase-mediated synthesis of protein thioesters. Butelase reactions are generally completed within minutes and often achieve excellent yields.