Total chemical synthesis of crambin

Crambin is a small (46 amino acids) protein isolated from the seeds of the plant Crambe abyssinica. Crambin has been extensively used as a model protein for the development of advanced crystallography and NMR techniques and for computational folding studies. We set out to establish synthetic access to crambin. Initially, we synthesized the 46 amino acid polypeptide by native chemical ligation of two distinct sets of peptide segments (15 + 31 and 31 + 15 residues). The synthetic polypeptide chain folded in good yield to give native crambin containing three disulfide bonds. The chemically synthesized crambin was characterized by LC-MS and by 2D-NMR. However, the 31-residue peptide segments were difficult to purify, and this caused an overall low yield for the synthesis. To overcome this problem, we synthesized crambin by the native chemical ligation of three segments (15 + 16 + 15 residues). Total synthesis using the ligation of three segments gave more than a 10-fold increase in yield and a protein product of exceptionally high purity. This work demonstrates the efficacy of chemical protein synthesis by the native chemical ligation of three segments and establishes efficient synthetic access to the important model protein crambin for experimental studies of protein folding and stability.     

Structural investigation of the HIV-1 envelope glycoprotein gp160 cleavage site

The selective proteolytic activation of the HIV-1 envelope glycoprotein gp160 by furin and other precursor convertases (PCs) occurs at the carboxyl side of the sequence Arg508-Glu-Lys-Arg511 (site 1), in spite of the presence of another consensus sequence: Lys500-Ala-Lys-Arg503 (site 2). We report on the solution structural analysis of a 19-residue synthetic peptide, p498, which spans the two gp160-processing sites 1 and 2, and is properly digested by furin at site 1. A molecular model is obtained for p498, by means of molecular dynamics simulations, from NMR data collected in trifluoroethanol/water. The peptide N-terminal side presents a 9-residue helical segment, enclosing the processing site 2; the C-terminal segment can be described as a loop exposing the processing site 1. A hypothesis for the docking of p498 onto the catalytic domain of human furin, modeled by homology and fitting previous site-directed mutagenesis studies, is also presented. p498 site 1 is shown to have easy access to the furin catalytic site, unlike the nonphysiological site 2. Finally, on the basis of available data, we suggest a possible structural motif required for the gp160-PCs recognition.     

Semisynthesis and folding of the potassium channel KcsA

In this contribution we describe the semisynthesis of the potassium channel, KcsA. A truncated form of KcsA, comprising the first 125 amino acids of the 160-amino acid protein, was synthesized using expressed protein ligation. This truncated form corresponds to the entire membrane-spanning region of the protein and is similar to the construct previously used in crystallographic studies on the KcsA protein. The ligation reaction was carried out using an N-terminal recombinant peptide alpha-thioester, corresponding to residues 1-73 of KcsA, and a synthetic C-terminal peptide corresponding to residues 74-125. Chemical synthesis of the C-peptide was accomplished by optimized Boc-SPPS techniques. A dual fusion strategy, involving glutathione-S-transferase (GST) and the GyrA intein, was developed for recombinant expression of the N-peptide alpha-thioester. The fusion protein, expressed in the insoluble form as inclusion bodies, was refolded and then cleaved successively to remove the GST tag and the intein, thereby releasing the N-peptide alpha-thioester. Following chemical ligation, the KcsA polypeptide was folded into the tetrameric state by incorporation into lipid vesicles. The correctness of the folded state was verified by the ability of the KcsA tetramer to bind to agitoxin-2. To our knowledge, this work represents the first reported semisynthesis of a polytopic membrane protein and highlights the potential application of native chemical ligation and expressed protein ligation for the (semi)synthesis of integral membrane proteins.     

Ionization state of the catalytic dyad Asp25/25' in the HIV-1 protease: NMR studies of site-specifically 13C labelled HIV-1 protease prepared by total chemical synthesis

Total chemical synthesis was used to site-specifically (13)C-label active site Asp25 and Asp25' residues in HIV-1 protease and in several chemically synthesized analogues of the enzyme molecule. (13)C NMR measurements were consistent with a monoprotonated state for the catalytic dyad formed by the interacting Asp25, Asp25' side chain carboxyls.     

半胱氨酸肽片段连接法合成ω-芋螺毒素MVIIA

ω-芋螺毒素MVIIA是已上市的镇痛药Ziconotide的有效成分.采用标准Fmoc保护策略在聚苯乙烯树脂上合成ω-MVIIA比较困难,是固相合成中的"困难肽".本研究将ω-MVIIA分为N-端15肽硫酯和C-端10肽两个片段采用标准Fmoc保护策略分别合成,再通过半胱氨酸肽片段连接得到全长的ω-芋螺毒素MVIIA肽链.该方法提高了合成ω-芋螺毒素MVIIA产率.该研究为"困难肽"的合成提供了较好的参考方法.     

Mirror image forms of snow flea antifreeze protein prepared by total chemical synthesis have identical antifreeze activities

The recently discovered glycine-rich snow flea antifreeze protein (sfAFP) has no sequence homology with any known proteins. No experimental structure has been reported for this interesting protein molecule. Here we report the total chemical synthesis of the mirror image forms of sfAFP (i.e., L-sfAFP, the native protein, and D-sfAFP, the native protein's enantiomer). The predicted 81 amino acid residue polypeptide chain of sfAFP contains Cys residues at positions 1, 13, 28, and 43 and was prepared from four synthetic peptide segments by sequential native chemical ligation. After purification, the full-length synthetic polypeptide was folded at 4 degrees C to form the sfAFP protein containing two disulfides. Chemically synthesized sfAFP had the expected antifreeze activity in an ice recrystallization inhibition assay. Mirror image D-sfAFP protein was prepared by the same synthetic strategy, using peptide segments made from d-amino acids, and had an identical but opposite-sign CD spectrum. As expected, D-sfAFP displays the same antifreeze properties as L-sfAFP, because ice presents an achiral surface for sfAFP binding. Facile synthetic access to sfAFP will enable determination of its molecular structure and systematic elucidation of the molecular basis of the antifreeze properties of this unique protein.     

Chemical synthesis of homogeneous human glycosyl-interferon-β that exhibits potent antitumor activity in vivo

Chemical synthesis of homogeneous human glycoproteins exhibiting bioactivity in vivo has been a challenging task. In an effort to overcome this long-standing problem, we selected interferon-β and examined its synthesis. The 166 residue polypeptide chain of interferon-β was prepared by covalent condensation of two synthetic peptide segments and a glycosylated synthetic peptide bearing a complex-type glycan of biological origin. The peptides were covalently condensed by native chemical ligation. Selective desulfurization followed by deprotection of the two Cys(Acm) residues gave the target full-length polypeptide chain of interferon-β bearing either a complex-type sialyl biantennary oligosaccharide or its asialo form. Subsequent folding with concomitant formation of the native disulfide bond afforded correctly folded homogeneous glycosyl-interferon-β. The chemically synthesized sialyl interferon-β exhibited potent antitumor activity in vivo.     

Studies on the insolubility of a transmembrane peptide from signal peptide peptidase

We have undertaken fundamental studies on the solubility properties of a peptide derived from the fourth transmembrane (TM) domain of signal peptide peptidase, a 7-TM intramembrane-cleaving protease. We have found that by disfavoring secondary structure formation we are able to greatly improve the solubility, handling, and purification properties of this peptide. Our findings suggest that preventing secondary structure formation by reversible modification of the polypeptide backbone of hydrophobic transmembrane peptides may be a useful strategy for the total chemical protein synthesis of integral membrane proteins.     

Chemical synthesis of Ub-AMC via ligation of peptide hydrazides

The C-terminal conjugate of ubiquitin with 7-amino-4-methylcoumarin (Ub-AMC) is an important probe for fluorescencebased analysis of deubiquitinating enzyme (DUB) activity. It is important to develop more efficient methods for the preparation of Ub-AMC because the currently available technology is still expensive for scaled-up production. In the present work we report an efficient strategy for total chemical synthesis of Ub-AMC through ligation of peptide hydrazides. Three peptide segments are assembled via N-to-C sequential ligation and the resulting product is converted to Ub-AMC via TCEP-mediated desulfurization. The synthetic Ub-AMC is shown to have expected biological functions throug     

The Effects of Side-Chain Configurations of a Retro–Inverso-Type Inhibitor on the Human T-Cell Leukemia Virus (HTLV)-1 Protease

In this study, the effects of side-chain configurations of D-Ile residues of a retro–inverso (RI)-type inhibitor on the human T-cell leukemia virus type 1 (HTLV-1) protease containing a hydroxyethylamine dipeptide isostere were clarified. Prior to evaluation using the RI-type inhibitor, the effects of side-chain configurations of Ile residues of the substrate peptide on the HTLV-1 protease were examined to estimate the influence of side-chain configurations on enzyme activity. Based on the estimation of the influence of side-chain configurations on protease affinity, the RI-type inhibitors containing a D-allo-Ile residue in the corresponding substrate sequence, instead of a D-Ile residue, were synthesized via 9-fluorenylmethoxycarbonyl-based solid-phase peptide synthesis. Refolded recombinant HTLV-1 protease (1-116, L40I) was used for the simple and short evaluation of the inhibitory activities of the synthesized RI-type inhibitors. The results clearly indicated that mimicking the whole topology, comprising both the main- and side-chain structures of the parent inhibitor, is effective for the design of potent RI-modified protease inhibitors.     

Functional characterization and NMR spectroscopy on full-length Vpu from HIV-1 prepared by total chemical synthesis

Vpu is an 81-residue integral membrane protein encoded in the HIV-1 genome that is of considerable interest because it plays important roles in the release of virus particles from infected cells and in the degradation of the cellular receptor. We report here the total chemical synthesis of full-length Vpu(1-81) as well as a site-specifically (15)N-labeled analogue, Vpu(2-81), using native chemical ligation methodologies and also report a structural and functional comparison of these constructs with recombinant protein obtained via bacterial expression. The structures of the synthetic and expressed polypeptides were similar in lipid micelles using solution NMR spectroscopy. Solid-state NMR spectra of the polypeptides in aligned hydrated lipid bilayers indicated that their overall topologies were also very comparable. Further, the channel activity of the synthetic protein was found to be analogous to that previously characterized for the recombinant protein. We have thus demonstrated that using solid phase peptide synthesis and chemical ligation it is feasible to obtain large quantities of a purified and homogeneous membrane protein in a structurally and functionally relevant form for future structural and characterization studies.     

Design and construction of an open multistranded beta-sheet polypeptide stabilized by a disulfide bridge

The design and characterization of an open eight-stranded beta-sheet in a synthetic, 2-fold symmetric 70-residue peptide is described. The design strategy involves the generation of a 35-residue four-stranded beta-sheet peptide in which successive hairpins are nucleated by appropriately positioned (D)Pro-Xxx sequences. Oxidative dimerization using a single Cys residue positioned at the center of the C-terminal strand results in a disulfide-bridged eight-stranded structure. Nuclear Overhauser effects firmly establish an eight-stranded beta-sheet in methanol. In water, the outer strands are frayed, but a well-defined four-stranded beta-sheet stabilized by a disulfide bridge and a hydrophobic cluster is determined from NMR data. Comparison of the precursor peptide with the disulfide-bridged dimer reveals considerable enhancement of beta-sheet content in the latter, suggesting that the disulfide cross-link is an effective strategy for the stabilization of beta-sheets.     

Engineered Histidine-Rich Peptides Enhance Endosomal Escape for Antibody-Targeted Intracellular Delivery of Functional Proteins

Currently, the clinical application of protein/peptide therapeutics is mainly limited to the modulation of diseases in extracellular spaces. Intracellular targets are hardly accessed, owing largely to the endosomal entrapment of internalized proteins/peptides. Here, we report a strategy to design and construct peptides that enable endosome-to-cytosol delivery based on an extension of the “histidine switch” principle. By substituting the Arg/Lys residues in cationic cell-penetrating peptides (CPPs) with histidine, we obtained peptides with pH-dependent membrane-perturbation activity. These peptides do not randomly penetrate cells like CPPs, but imitate the endosomal escape of CPPs following cellular uptake. Working with one such 16-residue peptide (hsLMWP) with high endosomal escape capacity, we engineered modular fusion proteins and achieved antibody-targeted delivery of diverse protein cargoes—including the pro-apoptotic protein BID (BH3-interacting domain death agonist) and Cre recombinase—into the cytosol of multiple cancer cell types. After extensive in vitro testing, an in vivo analysis with xenograft mice ultimately demonstrated that a trastuzumab-hsLMWP-BID fusion conferred strong anti-tumor efficacy without apparent side effects. Notably, our fusion protein features a modular design, allowing flexible applications for any antibody/cargo combination of choice. Therefore, the potential applications extend throughout life science and biomedicine, including gene editing, cancer treatment, and immunotherapy.     

Solid phase synthesis and opioid receptor binding properties of presumable dermorphin precursor derivatives.

Presumable dermorphin precursor peptide derivatives comprised of 35 amino acids and their fragments, which are based on the amino acid sequence determined by recombinant deoxyribonucleic acid (DNA) techniques, were synthesized by the solid phase method. A 35-residue peptide amide containing L-Ala2-dermorphin sequence at the N-terminus (1) as well as its D-Ala2 isomer (2) and the C-terminal 20-residue peptide amide were found to be unexpectedly stable against aminopeptidase M digestion and in rat brain membrane fractions mixture, suggesting that the C-terminal Glu-rich moiety of 1 and 2 serves to protect from enzymatic breakdown. In the opioid receptor binding assay, 2 showed 40 and 25-fold higher affinities than 1 for mu and delta-receptors, respectively. The N-terminal 15-residue peptide fragment of 2 showed greatly increased affinities for both receptors, being one half of those of dermorphin, whereas that of 1 showed low affinities. Opioid receptor binding properties of these synthetic peptides may be useful in investigation of the processing to dermorphin.     

Solid-phase synthesis of peptide and glycopeptide thioesters through side-chain-anchoring strategies

An efficient new strategy for the synthesis of peptide and glycopeptide thioesters is described. The method relies on the side-chain immobilization of a variety of Fmoc-amino acids, protected at their C-termini, on solid supports. Once anchored, peptides were constructed using solid-phase peptide synthesis according to the Fmoc protocol. After unmasking the C-terminal carboxylate, either thiols or amino acid thioesters were coupled to afford, after cleavage, peptide and glycopeptide thioesters in high yields. Using this method a significant proportion of the proteinogenic amino acids could be incorporated as C-terminal amino acid residues, therefore providing access to a large number of potential targets that can serve as acyl donors in subsequent ligation reactions. The utility of this methodology was exemplified in the synthesis of a 28 amino acid glycopeptide thioester, which was further elaborated to an N-terminal fragment of the glycoprotein erythropoietin (EPO) by native chemical ligation.     

多肽酰肼连接法合成C端香豆素修饰的泛素

C端用香豆素修饰的泛素分子（Ub-AMC）是研究蛋白质泛素化过程的重要探针.该探针分子的制备目前主要依靠生物表达结合化学修饰的方法,合成效率较低.本文使用多肽酰肼连接反应,发展出化学全合成Ub-AMC分子的新路线.该方法通过N到C顺序两次连接实现了目标分子的组装,再通过自由基脱硫反应得到天然结构的Ub-AMC分子,有望实现较大量的合成.通过酶学活性实验,证实了通过新方法合成的Ub-AMC具有预期的生物活性.     

Chemoenzymatic synthesis of HIV-1 V3 glycopeptides carrying two N-glycans and effects of glycosylation on the peptide domain

[structure: see text] A highly efficient chemoenzymatic synthesis of HIV-1 V3 domain glycopeptides carrying two N-linked core tri- and pentasaccharides was achieved. The synthesis consisted of two key steps: a solid-phase synthesis of the cyclic, 47-mer V3 domain peptide containing two GlcNAc residues and a novel endoglycosidase-catalyzed transglycosylation that simultaneously added two N-glycan moieties to the peptide precursor from the oligosaccharide oxazoline donor substrates. The availability of the synthetic glycopeptides allowed the probing of the effects of glycosylation on the HIV-1 V3 domain. It was demonstrated that glycosylation influenced the global conformations of the V3 domain and provided protection of the V3 domain against protease digestion.     

Evolutionarily conserved functional mechanics across pepsin-like and retroviral aspartic proteases

The biological function of the aspartic protease from HIV-1 has recently been related to the conformational flexibility of its structural scaffold. Here, we use a multistep strategy to investigate whether the same mechanism affects the functionality in the pepsin-like fold. (i) We identify the set of conserved residues by using sequence-alignment techniques. These residues cluster in three distinct regions: near the cleavage-site cavity, in the four beta-sheets cross-linking the two lobes, and in a solvent-exposed region below the long beta-hairpin in the N-terminal lobe. (ii) We elucidate the role played by the conserved residues for the enzymatic functionality of one representative member of the fold family, the human beta-secretase, by means of classical molecular dynamics (MD). The conserved regions exhibit little overall mobility and yet are involved into the most important modes of structural fluctuations. These modes influence the substrate-catalytic aspartates distance through a relative rotation of the N- and C-terminal lobes. (iii) We investigate the effects of this modulation by estimating the reaction free energy at different representative substrate/enzyme conformations. The activation free energy is strongly affected by large-scale protein motions, similarly to what has been observed in the HIV-1 enzyme. (iv) We extend our findings to all other members of the two eukaryotic and retroviral fold families by recurring to a simple, topology-based, energy functional. This analysis reveals a sophisticated mechanism of enzymatic activity modulation common to all aspartic proteases. We suggest that aspartic proteases have been evolutionarily selected to possess similar functional motions despite the observed fold variations.     

Enzymatic coupling of specific peptides at nonspecific ligation sites: effect of Asp189Glu mutation in trypsin on substrate mimetic-mediated reactions

Two main drawbacks seriously restrict the synthetic value of proteases as reagents in peptide fragment coupling: (i) native proteolytic activity and, thus, risk of undesired peptide cleavage; (ii) limited enzyme specificities restricting the amino acid residues between which a peptide bond can be formed. While the latter can be overcome by the use of substrate mimetics achieving peptide bond formation at nonspecific ligation sites, the risk of proteolytic cleavage still remains and hinders the wide acceptance of this powerful strategy for peptide coupling. This paper reports on the effect of the trypsin point mutant Asp189Glu on substrate mimetic-mediated reactions. The effect of this mutation on the steady-state hydrolysis of substrate mimetics of the 4-guanidinophenyl ester type and on trypsin-specific Lys- and Arg-containing peptides was investigated. The results were confirmed by enzymatic coupling reactions using substrate mimetics as the acyl donor and specific amino acid-containing peptides as the acyl acceptor. The competition assay verifies the predicted shift in substrate preference from Lys and Arg to the substrate mimetics and, thus, from cleavage to synthesis of peptide bonds. The combination of results obtained qualifies the trypsin mutant D189E as the first substrate mimetic-specific peptide ligase.