RESUMO
Chemical protein synthesis offers a powerful way to access otherwise-difficult-to-obtain proteins such as mirror-image proteins. Although a large number of proteins have been chemically synthesized to date, the acquisition to proteins containing hydrophobic peptide fragments has proven challenging. Here, we describe an approach that combines the removable backbone modification strategy and the peptide hydrazide-based native chemical ligation for the chemical synthesis of a 28 kDa full-length PET degrading enzyme IGGC (a higher depolymerization efficiency of variant leaf-branch compost cutinase (LCC)) containing hydrophobic peptide segments. The synthetic ICCG exhibits the enzymatic activity and will be useful in establishing the corresponding mirror-image version of ICCG.
Assuntos
Polietilenotereftalatos , Hidrolases/química , Fragmentos de Peptídeos , Peptídeos/química , Polietilenotereftalatos/químicaRESUMO
D-peptide ligands can be screened for therapeutic potency and enzymatic stability using synthetic mirror-image proteins (D-proteins), but efficient acquisition of these D-proteins can be hampered by the need to accomplish their in vitro folding, which often requires the formation of correctly linked disulfide bonds. Here, we report the finding that temporary installation of natural O-linked-ß-N-acetyl-D-glucosamine (O-GlcNAc) groups onto selected D-serine or D-threonine residues of the synthetic disulfide-bonded D-proteins can facilitate their folding in vitro, and that the natural glycosyl groups can be completely removed from the folded D-proteins to afford the desired chirally inverted D-protein targets using naturally occurring O-GlcNAcase. This approach enabled the efficient chemical syntheses of several important but difficult-to-fold D-proteins incorporating disulfide bonds including the mirror-image tumor necrosis factor alpha (D-TNFα) homotrimer and the mirror-image receptor-binding domain of the Omicron spike protein (D-RBD). Our work establishes the use of O-GlcNAc to facilitate D-protein synthesis and folding and proves that D-proteins bearing O-GlcNAc can be good substrates for naturally occurring O-GlcNAcase.
Assuntos
Acetilglucosaminidase , Proteínas , Peptídeos , Polissacarídeos , GlucosaminaRESUMO
Mirror-image proteins (D-proteins) are useful in biomedical research for purposes such as mirror-image screening for D-peptide drug discovery, but the chemical synthesis of many D-proteins is often low yielding due to the poor solubility or aggregation of their constituent peptide segments. Here, we report a Lys-C protease-cleavable solubilizing tag and its use to synthesize difficult-to-obtain D-proteins. Our tag is easily installed onto multiple amino acids such as DLys, DSer, DThr, and/or the N-terminal amino acid of hydrophobic D-peptides, is impervious to various reaction conditions, such as peptide synthesis, ligation, desulfurization, and transition metal-mediated deprotection, and yet can be completely removed by Lys-C protease under denaturing conditions to give the desired D-protein. The efficacy and practicality of the new method were exemplified in the synthesis of two challenging D-proteins: D-enantiomers of programmed cell death protein 1 IgV domain and SARS-CoV-2 envelope protein, in high yield. This work demonstrates that the enzymatic cleavage of solubilizing tags under denaturing conditions is feasible, thus paving the way for the production of more D-proteins.
Assuntos
Peptídeos , Proteínas , Proteínas/química , Peptídeos/química , Aminoácidos/química , Técnicas de Química Sintética/métodos , Peptídeo Hidrolases , EndopeptidasesRESUMO
Membrane-associated D-proteins are an important class of synthetic molecules needed for D-peptide drug discovery, but their chemical synthesis using canonical ligation methods such as native chemical ligation is often hampered by the poor solubility of their constituent peptide segments. Here, we describe a Backbone-Installed Split Intein-Assisted Ligation (BISIAL) method for the synthesis of these proteins, wherein the native L-forms of the N- and C-intein fragments of the unique consensus-fast (Cfa) (i.e. L-CfaN and L-CfaC ) are separately installed onto the two D-peptide segments to be ligated via a removable backbone modification. The ligation proceeds smoothly at micromolar (µM) concentrations under strongly chaotropic conditions (8.0â M urea), and the subsequent removal of the backbone modification groups affords the desired D-proteins without leaving any "ligation scar" on the products. The effectiveness and practicality of the BISIAL method are exemplified by the synthesis of the D-enantiomers of the extracellular domains of T cell immunoglobulin and ITIM domain (TIGIT) and tropomyosin receptor kinase C (TrkC). The BISIAL method further expands the chemical protein synthesis ligation toolkit and provides practical access to challenging D-protein targets.
Assuntos
Inteínas , Proteínas , Peptídeos/química , Processamento de ProteínaRESUMO
Disulfide-rich proteins are useful as drugs or tool molecules in biomedical studies, but their synthesis is complicated by the difficulties associated with their folding. Here, we describe a removable glycosylation modification (RGM) strategy that expedites the chemical synthesis of correctly folded proteins with multiple or even interchain disulfide bonds. Our strategy comprises the introduction of simple O-linked ß-N-acetylglucosamine (O-GlcNAc) groups at the Ser/Thr sites that effectively improve the folding of disulfide-rich proteins by stabilization of their folding intermediates. After folding, the O-GlcNAc groups can be efficiently removed using O-GlcNAcase (OGA) to afford the correctly folded proteins. Using this strategy, we completed the synthesis of correctly folded hepcidin, an iron-regulating hormone bearing four pairs of disulfide-bonds, and the first total synthesis of correctly folded interleukin-5 (IL-5), a 26 kDa homodimer cytokine responsible for eosinophil growth and differentiation.
Assuntos
AcetilglucosaminaRESUMO
The ß2-adrenergic receptor (ß2AR) is a G-protein-coupled receptor (GPCR) that responds to the hormone adrenaline and is an important drug target in the context of respiratory diseases, including asthma. ß2AR function can be regulated by post-translational modifications such as phosphorylation and ubiquitination at the C-terminus, but access to the full-length ß2AR with well-defined and homogeneous modification patterns critical for biochemical and biophysical studies remains challenging. Here, we report a practical synthesis of differentially modified, full-length ß2AR based on a combined native chemical ligation (NCL) and sortase ligation strategy. An array of homogeneous samples of full-length ß2ARs with distinct modification patterns, including a full-length ß2AR bearing both monoubiquitination and octaphosphorylation modifications, were successfully prepared for the first time. Using these homogeneously modified full-length ß2AR receptors, we found that different phosphorylation patterns mediate different interactions with ß-arrestin1 as reflected in different agonist binding affinities. Our experiments also indicated that ubiquitination can further modulate interactions between ß2AR and ß-arrestin1. Access to full-length ß2AR with well-defined and homogeneous modification patterns at the C-terminus opens a door to further in-depth mechanistic studies into the structure and dynamics of ß2AR complexes with downstream transducer proteins, including G proteins, arrestins, and GPCR kinases.
Assuntos
Processamento de Proteína Pós-Traducional , Receptores Adrenérgicos beta 2/química , Regulação Alostérica , Aminoaciltransferases/química , Proteínas de Bactérias/química , Cisteína Endopeptidases/química , Humanos , Fosforilação , Receptores Adrenérgicos beta 2/metabolismo , Staphylococcus aureus/enzimologia , Ubiquitinação , beta-Arrestina 1/metabolismoRESUMO
The combination of distinct peptide ligation techniques to facilitate chemical protein synthesis represents one of the long-standing goals in the field. A new combination ligation method of N-to-C sequential native chemical ligation and Ser/Thr ligation (NCL-STL) is described for the first time. This method relies on the peptide salicylaldehyde S,S-propanedithioacetal (SALPDT)-ester prepared by a new 1,3-propanedithiol-mediated reaction. The peptide SALPDT-ester, which is compatible with NCL, can be fully activated by N-chlorosuccinimide (NCS)/AgNO3 in aqueous solution to afford peptide SAL-ester for use in the subsequent STL. The practicality of the combined NCL-STL method is illustrated by the synthesis of S-palmitoylated matrix-2 (S-palm M2) ion channel from Influenza A virus and S-palmitoylated interferon-induced transmembrane protein 3 (S-palm IFITM3). This approach expands the multiple-segments peptide ligation toolkit for producing important and complex custom-made protein samples by chemical protein synthesis.
Assuntos
Aldeídos/química , Ésteres/química , Proteínas de Membrana/síntese química , Propano/química , Proteínas de Ligação a RNA/síntese química , Serina/química , Compostos de Sulfidrila/química , Treonina/química , Proteínas da Matriz Viral/síntese química , Humanos , Proteínas de Membrana/química , Estrutura Molecular , Proteínas de Ligação a RNA/química , Proteínas da Matriz Viral/químicaRESUMO
The preparation of native S-palmitoylated (S-palm) membrane proteins is one of the unsolved challenges in chemical protein synthesis. Herein, we report the first chemical synthesis of S-palm membrane proteins by removable-backbone-modification-assisted Ser/Thr ligation (RBMGABA -assisted STL). This method involves two critical steps: 1)â synthesis of S-palm peptides by a new γ-aminobutyric acid based RBM (RBMGABA ) strategy, and 2)â ligation of the S-palm RBM-modified peptides to give the desired S-palm product by the STL method. The utility of the RBMGABA -assisted STL method was demonstrated by the synthesis of rabbit S-palm sarcolipin (SLN) and S-palm matrix-2 (M2) ion channel. The synthesis of S-palm membrane proteins highlights the importance of developing non-NCL methods for chemical protein synthesis.
Assuntos
Proteínas de Membrana/química , Palmitatos/química , Peptídeos/síntese química , Serina/química , Treonina/química , Sequência de Aminoácidos , Aminobutiratos/química , Animais , Canais Iônicos/síntese química , Proteínas Musculares/síntese química , Proteolipídeos/síntese química , Coelhos , Técnicas de Síntese em Fase Sólida , SolubilidadeRESUMO
The use of synthetic bridges as surrogates for disulfide bonds has emerged as a practical strategy to obviate the poor stability of some disulfide-containing peptides. However, peptides incorporating large-span synthetic bridges are still beyond the reach of existing methods. Herein, we report a native chemical ligation (NCL)-assisted diaminodiacid (DADA) strategy that enables the robust generation of disulfide surrogate peptides incorporating surrogate bridges up to 50â amino acids in length. This strategy provides access to some highly desirable but otherwise impossible-to-obtain disulfide surrogates of bioactive peptide. The bioactivities and structures of the synthetic disulfide surrogates were verified by voltage clamp assays, NMR, and X-ray crystallography; and stability studies established that the disulfide replacements effectively overcame the problems of disulfide reduction and scrambling that often plague these pharmacologically important peptides.
RESUMO
With the growing requirement for otherwise-difficult-to-obtain proteins, it is necessary to develop more efficient chemical protein synthesis methods for rapid access to designed protein samples. In particular, a one-pot multi-segment condensation method, with only one purification step to obtain the final product, is expected to demonstrate unique benefits in chemical protein synthesis, such as the requirement of fewer handling procedures and the higher efficiency in obtaining aimed protein samples. The utilization of the one-pot multi-segment condensation strategy is demonstrated via the synthesis of a series of post-translational modification (PTM) or disease-associated peptides or proteins for basic and advanced scientific research. This review summarizes the recent one-pot multi-segment condensation methods utilized in chemical protein synthesis, in which two aspects of drive-strategies will be mainly included: a kinetically controlled strategy and a protecting group-removal strategy, respectively. On one hand, the activities of peptides in N-terminal thiol amino acids or C-terminal acyl donors can be largely different based on the differences in properties, such as steric hindrance, migration rates, electrophilicity, and introduction of active elements such as selenium, etc. Using the different activities, regio-selective peptide ligation can be performed in a kinetically controlled manner. On the other hand, the protecting group-removal strategy involves various moieties, which can block the activity of functional groups arising from N-terminal thiol amino acids or C-terminal acyl donors, and they can be removed by using additives, and pH- or photo-stimulation conditions with further achievement of chemical protein synthesis by the one-pot strategy.
Assuntos
Proteínas/síntese química , Estrutura Molecular , Peptídeos/química , Processamento de Proteína Pós-Traducional , Proteínas/química , Proteínas/metabolismoRESUMO
During the total chemical synthesis of the water-soluble globular Haemophilus Influenzae DNA ligase (Hin-Lig), we observed the surprising phenomenon of a soluble peptide segment that failed to undergo native chemical ligation. Based on dynamic light scattering and transmission electron microscopy experiments, we determined that the peptide formed soluble colloidal particles in a homogeneous solution containing 6 m guanidine hydrochloride. Conventional peptide performance-improving strategies, such as installation of a terminal/side-chain Arg tag or O-acyl isopeptide, failed to enable the reaction, presumably because of their inability to disrupt the formation of soluble colloidal particles. However, a removable backbone modification strategy recently developed for the synthesis of membrane proteins did disrupt the formation of the colloids, and the desired ligation of this soluble but unreactive system was eventually accomplished. This work demonstrates that an appropriate solution dispersion state, in addition to good peptide solubility, is a prerequisite for successful peptide ligation.
Assuntos
Proteínas de Bactérias/metabolismo , DNA Ligases/metabolismo , Haemophilus influenzae/enzimologia , Peptídeos/síntese química , Técnicas de Síntese em Fase Sólida/métodos , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cromatografia Líquida de Alta Pressão , Coloides/química , DNA Ligases/química , DNA Ligases/genética , Guanidina/química , Histidina/genética , Histidina/metabolismo , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Peptídeos/análise , Peptídeos/química , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Espectrometria de Massas em TandemRESUMO
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.
Assuntos
Proteínas de Membrana/síntese química , Técnicas de Síntese em Fase Sólida/métodos , Antiporters/síntese química , Antiporters/química , Detergentes/química , Proteínas de Escherichia coli/síntese química , Proteínas de Escherichia coli/química , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/química , Espectroscopia de Ressonância Magnética , Proteínas de Membrana/química , Isótopos de Nitrogênio , Canais de Potássio Corretores do Fluxo de Internalização/síntese química , Canais de Potássio Corretores do Fluxo de Internalização/química , Redobramento de Proteína , Solubilidade , Solventes/química , Proteínas da Matriz Viral/síntese química , Proteínas da Matriz Viral/química , Proteínas Virais/síntese química , Proteínas Virais/química , Canal Kir5.1RESUMO
Post-translational modifications (e.g., ubiquitylation) of histones play important roles in dynamic regulation of chromatin. Histone ubiquitylation has been speculated to directly influence the structure and dynamics of nucleosomes. However, structural information for ubiquitylated nucleosomes is still lacking. Here we report an alternative strategy for total chemical synthesis of homogenous histone H2B-K34-ubiquitylation (H2B-K34Ub) by using acid-cleavable auxiliary-mediated ligation of peptide hydrazides for site-specific ubiquitylation. Synthetic H2B-K34Ub was efficiently incorporated into nucleosomes and further used for single-particle cryo-electron microscopy (cryo-EM) imaging. The cryo-EM structure of the nucleosome containing H2B-K34Ub suggests that two flexible ubiquitin domains protrude between the DNA chains of the nucleosomes. The DNA chains around the H2B-K34 sites shift and provide more space for ubiquitin to protrude. These analyses indicated local and slight structural influences on the nucleosome with ubiquitylation at the H2B-K34 site.
Assuntos
Histonas/síntese química , Nucleossomos/química , Microscopia Crioeletrônica , Histonas/química , Nucleossomos/metabolismo , Estrutura Terciária de Proteína , UbiquitinaçãoRESUMO
Biochemical studies of cellular processes involving polyubiquitin have gained increasing attention. More tools are needed to identify ubiquitin (Ub)-binding proteins. We report diazirine-based photoaffinity probes that can capture Ub-binding proteins in cell lysates, and show that diazirines are preferable to aryl azides as the photo-crosslinking group, since they decrease non-selective capture. Photoaffinity probes containing at least two Ub units were required to effectively capture Ub-binding proteins. Different capture selectivity was observed for probes containing diubiquitin moieties with different types of linkages, thus indicating the potential to develop linkage-dependent probes for selectively profiling Ub-binding proteins under various cellular conditions.
Assuntos
Diazometano/química , Marcadores de Fotoafinidade/síntese química , Ubiquitinas/isolamento & purificação , Humanos , Modelos Moleculares , Estrutura Molecular , Marcadores de Fotoafinidade/química , Ubiquitinas/químicaRESUMO
Chemical ubiquitination is an effective approach for accessing structurally defined, atypical ubiquitin (Ub) chains that are difficult to prepare by other techniques. Herein, we describe a strategy that uses a readily accessible premade isopeptide-linked 76-mer (isoUb), which has an N-terminal Cys and a C-terminal hydrazide, as the key building block to assemble atypical Ub chains in a modular fashion. This method avoids the use of auxiliary-modified Lys and instead employs the canonical and therefore more robust Cys-based native chemical ligation technique. The efficiency and capacity of this isoUb-based strategy is exemplified by the cost-effective synthesis of several linkage- and length-defined atypical Ub chains, including K27-linked tetra-Ub and K11/K48-branched tri-, tetra-, penta-, and hexa-Ubs.
RESUMO
Chemical protein synthesis can provide access to proteins with post-translational modifications or site-specific labelings. Although this technology is finding increasing applications in the studies of water-soluble globular proteins, chemical synthesis of membrane proteins remains elusive. In this report, a general and robust removable backbone modification (RBM) method is developed for the chemical synthesis of membrane proteins. This method uses an activated O-to-N acyl transfer auxiliary to install in the Fmoc solid-phase peptide synthesis process a RBM group with switchable reactivity toward trifluoroacetic acid. The method can be applied to versatile membrane proteins because the RBM group can be placed at any primary amino acid. With RBM, the membrane proteins and their segments behave almost as if they were water-soluble peptides and can be easily handled in the process of ligation, purification, and mass characterizations. After the full-length protein is assembled, the RBM group can be readily removed by trifluoroacetic acid. The efficiency and usefulness of the new method has been demonstrated by the successful synthesis of a two-transmembrane-domain protein (HCV p7 ion channel) with site-specific isotopic labeling and a four-transmembrane-domain protein (multidrug resistance transporter EmrE). This method enables practical synthesis of small- to medium-sized membrane proteins or membrane protein domains for biochemical and biophysical studies.
Assuntos
Proteínas de Membrana/síntese química , Técnicas de Síntese em Fase Sólida/métodos , Proteínas de Membrana/química , Modelos Moleculares , Peptídeos/químicaRESUMO
Longer amyloid-beta (Aß) peptides (43 to 49 amino acids) play essential roles in the pathology of Alzheimer's disease (AD). The difficulty in the preparation of longer Aß peptides is still an obstacle to elucidate their roles in AD. Herein we report a robust and efficient strategy for the chemical synthesis of longer Aß peptides (Aß48 and Aß49). A key feature of this method is the installation of removable Arg4-tagged backbone modification groups into the hydrophobic region of Aß. This modification can improve the handling properties of the purification, ligation and mass characterization of longer Aß peptides. The practicability of the new method has been demonstrated by the successful synthesis of Aß48 and Aß49 peptides.
Assuntos
Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/síntese química , Sequência de Aminoácidos , Arginina/química , Técnicas de Química Sintética , Interações Hidrofóbicas e HidrofílicasRESUMO
A new thiol protecting group Hmb(off/on) is described, which has a switchable activity that may be useful in the chemical synthesis of proteins. When placed on the side chain of Cys, Cys(Hmb(off)) is stable to trifluoroacetic acid (TFA) in the process of solid-phase peptide synthesis. When Cys(Hmb(off)) is treated with neutral aqueous buffers, it is cleanly converted to acid-labile Cys(Hmb(on)), which can later be fully deprotected by TFA to generate free Cys. The utility of Cys(Hmb(off/on)) is demonstrated by the chemical synthesis of an erythropoietin segment, EPO[Cys(98)-Arg(166)]-OH through native chemical ligation.
Assuntos
Peptídeos/química , Peptídeos/síntese química , Técnicas de Síntese em Fase Sólida , Compostos de Sulfidrila/químicaRESUMO
Mambalgins are a class of 57-residue polypeptide toxins isolated from the venom of the African mamba. They exhibit potent analgesic effects by inhibiting the acid-sensing ion channels. Classified as members of the family of three-finger toxins, mambalgins contain four pairs of disulfide bridges that help to stabilize the three-finger scaffold. Here, we report the chemical synthesis of functional mambalgin-1/2/3 by using one-step two-segment hydrazide-based native chemical ligation. The two-segment ligation approach reported here may enable efficient production of mambalgin toxins. These synthetic mambalgins are useful compounds for development of diagnostic or therapeutic reagents. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.
Assuntos
Venenos Elapídicos/síntese química , Peptídeos/síntese química , Azidas/química , Dissulfetos/química , Venenos Elapídicos/química , Modelos Moleculares , Estrutura Molecular , Peptídeos/químicaRESUMO
Chemical synthesis or semi-synthesis of membrane proteins can provide unique molecular tools, such as site-specific isotope labeling or post-translationally modified membrane proteins to gain insight into their biophysical and functional characteristics. However, during preparation, purification, and ligation of transmembrane peptides, tremendous challenges are encountered owing to their hydrophobic nature. This review focuses on the recent advances in chemical synthesis strategies of membrane proteins. These strategies help to solubilize the hydrophobic transmembrane peptide sequences under standard purification and chemical ligation conditions to improve their handling properties. Biophysical and functional studies of synthetic membrane proteins are reviewed as well.