Protein and Peptide Letters - Volume 12, Issue 8, 2005

C-terminal peptide α-thioesters are valuable intermediates in the synthesis/semisynthesis of proteins by native chemical ligation. They are prepared either by solid-phase peptide synthesis (SPPS) or biosynthetically by protein splicing techniques. The present paper reviews the different methods available for the chemical synthesis of peptide α-thioesters using Fmoc-based SPPS.

An overview of the applications of Nα-(1-phenyl-2-mercaptoethyl) auxiliary is presented. We describe the on resin preparation (C α-carboxy and thioester) of Nα-auxiliary derivatives of glycine and the synthesis and incorporation of preformed Nα-auxiliary derivatives of glycine and alanine with the protection schemes, including the thiazolidine strategy for SPPS. Such approaches allowed the synthesis of the protein cytochro Read More

Chemoselective ligation strategies have previously provided synthetic access to water-soluble proteins with novel properties, and more recently these strategies have been used to prepare ion channels. Examples of ion channels prepared by total chemical synthesis include bacterial mechanosensitive channels, and viral ion channels. Chemical protein synthesis allows for the generation of ion channel proteins with bot Read More

Here we report a bi-directional and interchangeable three-segment peptide ligation of N, M, and C-segments, mimicking the reverse process of protein splicing to form, in tandem, a tripartite NMC-peptide using a synthetic intein, a role served by the M-segment with an N-terminal Ser or Thr and a C-terminal thioester.

Intein-mediated protein splicing is facilitated by four separate but coordinated nucleophilic displacement reactions that result in the excision of the intein and the ligation of the flanking polypeptides, called the exteins. These reactions are catalyzed by the intein plus the first downstream extein amino acid without the assistance of cofactors or auxiliary enzymes. Non-canonical inteins missing conserved nucleophilic residues at th Read More

Expressed protein ligation has become a frequently used technique to insert non-standard amino acids into proteins. The technique has been adapted to insert selenocysteine residues in place of cysteine residue in proteins, taking advantage of the similarity in the chemistries of sulfur and selenium. This replacement can confer unique structural and catalytic properties to enzymes and proteins. The development of this tech Read More

This article reviews the methodology and recent applications of expressed protein ligation for NMR structural studies of proteins and protein complexes.

We review intein-mediated approaches for the site-specific modifications of proteins and highlight their applications in (1) the site-specific in vitro and in vivo biotinylation of proteins for protein arrays and (2) the site-specific in vivo labeling of proteins in living cells.

Specific enzyme immobilization has moved into the focus for many applications in biochemical research fields. Expressed Protein Ligation (EPL) has been proven to be ideal to selectively label proteins at single positions. Applying this technique to enzymes of the aldo/keto reductase superfamily provides a new approach to generate native or modified redox enzymes for direct and indirect immobilization.

Interactions between G proteins and GPCRs are fundamental for transmitting signals for a multitude of physiologic responses. Little is known regarding the protein-protein interface between the G protein and the receptor, much less the mechanisms for receptor activation of G proteins. Here, we will describe how expressed protein ligation will aid in the study of protein-protein interactions between semi-synthetic G alpha Read More

The present paper reviews the use of expressed protein ligation for the biosynthesis of backbone cyclized polypeptides. This general method allows the in vivo and in vitro biosynthesis of cyclic polypeptides using recombinant DNA expression techniques. Biosynthetic access to backbone cyclic peptides opens the possibility to generate cell-based combinatorial libraries that can be screened inside living cells for their ability to att Read More

High throughput screening of SICLOPPS libraries afforded six distinct cyclic peptides that inhibit Escherichia coli growth both in liquid and solid media. One of these peptides (LN05) reduced both bacterial growth rate and caused cell aggregation in liquid media. Mutant bacteria immune to LN05 action were obtained at a frequency of 10-7. Overexpression of an E. coli genomic library in the presence of LN05 production resul Read More

A 3:1 combination of sucrose and glycine provides significantly greater protection against pressure-induced inactivation of yeast alcohol dehydrogenase than either solute alone. Trehalose, alone, gives much greater protection than sucrose alone, but not so in combination with glycine. These are striking new findings that cannot be accounted for by current theories of protein stabilization.

In this paper we propose constructing an improved two-level neural network to predict protein secondary structure. Firstly, we code the whole protein composition information as the inputs to the first-level network besides the evolutionary information. Secondly, we calculate the reliability score for each residue position based on the output of the first-level network, and the role of the second-level network is to take full adv Read More

The Nelore bull (Bos taurus indicus) seminal plasma proteome was analyzed by MALDI-TOF MS and twodimensional gel electrophoresis. A total of 260 spots were visualized in the 2-DE gel (pI range 3-10) and 13 spots could be identified by peptide mass fingerprinting corresponding to 11 different polypeptides. The results allowed the creation of the first proteomic map of Bos taurus indicus seminal plasma. The roles of the Read More

BnSP-7 and BnSP-6, two Lys49-phospholipase A2 isolated from Bothrops neuwiedi pauloensis snake venom, were co-crystallized with α-tocopherol and X-ray diffraction data were collected for both complexes (2.2 and 2.6 Å). A new "alternative" quaternary conformation for these two complexes compared with all other dimeric Lys49-PLA2 has been observed.

CutC is a novel copper homeostasis protein containing 248 amino acids. Here we report the cloning, expression, purification, crystallization and preliminary X-ray crystallographic studies of CutC from Shigella flexneri 2a. Purification of CutC and its selenomethionine (SeMet) derivate were done using immobilized metal ion affinity chromatography, size-exclusion and ion-exchange chromatography. The purified proteins were Read More