Native Chemical Ligation Combined with Desulfurization and Deselenization: A General Strategy for Chemical Protein Synthesis

Of the many approaches proposed to generalize the native chemical ligation approach for protein synthesis, the simple procedure of global desulfurization of peptide thiols has become the most widely adopted. In this review, the development of the native ligation–desulfurization strategy is described, focusing on the conversion of Cys to Ala following ligation at N-terminal Cys residues. Subsequent variations on this theme have broadened the scope to other natural amino acids including Phe, Leu, Val, and Lys, and even non-native peptide linkages such as isopeptide bonds on lysine side chains. Using insights from both selenocysteine–peptide side reactions and radical initiated desulfurization procedures, a new method for the selective deselenization of peptides containing both selenocysteine and cysteine residues has been developed. Together, these approaches represent a robust and flexible methodology for the synthesis of complex polypeptides without the use of protecting groups.     

Ultra-stable peptide scaffolds for protein engineering-synthesis and folding of the circular cystine knotted cyclotide cycloviolacin O2

The cyclic cystine knot motif, as defined by the cyclotide peptide family, is an attractive scaffold for protein engineering. To date, however, the utilisation of this scaffold has been limited by the inability to synthesise members of the most diverse and biologically active subfamily, the bracelet cyclotides. This study describes the synthesis and first direct oxidative folding of a bracelet cyclotide-cycloviolacin O2-and thus provides an efficient method for exploring the most potent cyclic cystine knot peptides. The linear chain of cycloviolacin O2 was assembled by solid-phase Fmoc peptide synthesis and cyclised by thioester-mediated native chemical ligation, and the inherent difficulties of folding bracelet cyclotides were successfully overcome in a single-step reaction. The folding pathway was characterised and was found to include predominating fully oxidised intermediates that slowly converted to the native peptide structure.     

Ribosomal Synthesis of Cyclic Peptides with a Fluorogenic Oxidative Coupling Reaction

Ring around the peptides : We demonstrate a new method for the cyclization of peptides that involves the oxidative coupling of 5‐hydroxyindole and benzylamine. After two nonproteinogenic amino acids were incorporated into peptides by reprogramming the genetic code, cyclization took place rapidly upon the addition of K₃Fe(CN)₆ and generated a conjugated, fluorescent, heterocyclic structure.

     

Expanding the Scope of Sortase‐Mediated Ligations by Using Sortase Homologues

Sortase‐catalyzed transacylation reactions are widely used for the construction of non‐natural protein derivatives. However, the most commonly used enzyme for these strategies (sortase A from Staphylococcus aureus) is limited by its narrow substrate scope. To expand the range of substrates compatible with sortase‐mediated reactions, we characterized the in vitro substrate preferences of eight sortase A homologues. From these studies, we identified sortase A enzymes that recognize multiple substrates that are unreactive toward sortase A from S. aureus. We further exploited the ability of sortase A from Streptococcus pneumoniae to recognize an LPATS substrate to perform a site‐specific modification of the N‐terminal serine residue in the naturally occurring antimicrobial peptide DCD‐1L. Finally, we unexpectedly observed that certain substrates (LPATXG, X=Nle, Leu, Phe, Tyr) were susceptible to transacylation at alternative sites within the substrate motif, and sortase A from S. pneumoniae was capable of forming oligomers. Overall, this work provides a foundation for the further development of sortase enzymes for use in protein modification.     

Effects of Arginine Deimination and Citrulline Side-Chain Length on Peptide Secondary Structure Formation

Post-translational modifications expand the chemical functionality of peptides and proteins beyond that originating from the encoded amino acids, but studies on the structural effects of these modifications have been limited. Arginine undergoes deimination to give citrulline (Cit), converting the positively charged guanidinium moiety into a neutral urea group. Herein, we report the effect of Arg deimination on secondary structure formation. To understand the reason for the number of methylene units in Cit, the effect of Cit side-chain length on secondary structure formation was also studied. Ala-based peptides and β-hairpin peptides were used to study α-helix and β-sheet formation, respectively. Peptides containing Cit analogues were prepared by an orthogonal protecting group strategy coupled with solid-phase carbamylation. The CD data for the Ala-based peptides were analyzed by using modified Lifson–Roig theory, showing that the helix propensity of Arg decreased upon deimination and that either shortening or lengthening Cit also decreased the helix propensity. The β-hairpin peptides were analyzed by NMR methods, showing minimal change in strand formation energetics upon Arg deimination. Altering the Cit side-chain length did not affect strand formation energetics either. These results should be useful for the preparation of urea-bearing systems and the design of peptides incorporating urea-bearing residues with varying side-chain length.     

The Conserved Glu in the Cyclotide Cycloviolacin O2 Has a Key Structural Role

Cyclotides are a large family of plant peptides that are characterised by a head‐to‐tail circular backbone and three disulfide bonds that are arranged in a cystine knot. This unique structural feature, which is referred to as a cyclic cystine knot, gives the cyclotides remarkable stability against chemical and biological degradation. In addition to their natural function as insecticides for plant defence, the cyclotides have a range of bioactivities with pharmaceutical relevance, including cytotoxicity against cancer cell lines. A glutamic acid residue, aside from the invariable disulfide array, is the most conserved feature throughout the cyclotide family, and it has recently been shown to be crucial for biological activity. Here we have used solution‐state NMR spectroscopy to determine the three‐dimensional structures of the potent cytotoxic cyclotide cycloviolacin O2, and an inactive analogue in which this conserved glutamic acid has been methylated. The structures of the peptides show that the glutamic acid has a key structural role in coordinating a set of hydrogen bonds in native cycloviolacin O2; this interaction is disrupted in the methylated analogue. The proposed mechanism of action of cyclotides is membrane disruption and these results suggest that the glutamic acid is linked to cyclotide function by stabilising the structure to allow efficient aggregation in membranes, rather than in a direct interaction with a target receptor.     

Specificity of Lipoprotein Chaperones for the Characteristic Lipidated Structural Motifs of their Cognate Lipoproteins

Lipoprotein‐binding chaperones mediate intracellular transport of lipidated proteins and determine their proper localisation and functioning. Understanding of the exact structural parameters that determine recognition and transport by different chaperones is of major interest. We have synthesised several lipid‐modified peptides, representative of different lipoprotein classes, and have investigated their binding to the relevant chaperones PDEδ, UNC119a, UNC119b, and galectins‐1 and ‐3. Our results demonstrate that PDEδ recognises S‐isoprenylated C‐terminal peptidic structures but not N‐myristoylated peptides. In contrast, UNC119 proteins bind only mono‐N‐myristoylated, but do not recognise doubly lipidated and S‐isoprenylated peptides at the C terminus. For galectins‐1 and ‐3, neither binding to N‐acylated, nor to C‐terminally prenylated peptides could be determined. These results shed light on the specificity of the chaperone‐mediated cellular lipoprotein transport systems.     

Kinetic Analysis of PRMT1 Reveals Multifactorial Processivity and a Sequential Ordered Mechanism

Arginine methylation is a prevalent post‐translational modification in eukaryotic cells. Two significant debates exist within the field: do these enzymes dimethylate their substrates in a processive or distributive manner, and do these enzymes operate using a random or sequential method of bisubstrate binding? We revealed that human protein arginine N‐methyltransferase 1 (PRMT1) enzyme kinetics are dependent on substrate sequence. Further, peptides containing an Nη‐hydroxyarginine generally demonstrated substrate inhibition and had improved K_M values, which evoked a possible role in inhibitor design. We also revealed that the perceived degree of enzyme processivity is a function of both cofactor and enzyme concentration, suggesting that previous conclusions about PRMT sequential methyl transfer mechanisms require reassessment. Finally, we demonstrated a sequential ordered Bi–Bi kinetic mechanism for PRMT1, based on steady‐state kinetic analysis. Together, our data indicate a PRMT1 mechanism of action and processivity that might also extend to other functionally and structurally conserved PRMTs.     

Design of photocontrolled RNA-binding peptidomimetics

Positively constrained: the first examples of photocontrolled RNA binding peptides are described. The large number of positively charged sides chains in the Rev response element (RRE) of an HIV type I targeting α-helix imposes constraints on the choice of azobenzene-derived crosslinker.     

The Outcomes of Decorated Prolines in the Discovery of Antimicrobial Peptides from Temporin-L

Previously, we identified a potent antimicrobial analogue of temporin L (TL), [Pro³]TL, in which glutamine at position 3 was substituted with proline. In this study, a series of analogues in which position 3 is substituted with non-natural proline derivatives, was investigated for correlations between the conformational properties of the compounds and their antibacterial, cytotoxic, and hemolytic activities. Non-natural proline analogues with substituents at position 4 of the pyrrolidine ring were considered. Structure–activity relationship (SAR) studies of these analogues were performed by means of antimicrobial and cytotoxicity assays along with circular dichroism (CD) and NMR spectroscopic analyses for selected compounds. The most promising peptides were additionally evaluated for their activity against some representative veterinary microbial strains to compare with those from human strains. We identified novel analogues with interesting properties that make them attractive lead compounds.     

Hydrocracking Brazilian Marlim vacuum residue with natural limonite. Part 1: catalytic activity of natural limonite

An experimental study examined the catalytic effects of natural Australian (AL) and Brazilian (BL) limonites used in hydrocracking Brazilian Marlim vacuum residue (ML-VR). The catalytic behavior of the limonites was compared with a conventional NiO-MoO₃-Al₂O₃ (NiMo) catalyst. Diphenylmethane (DPM) and 1-methylnaphthalene (1-MN) were used as standards. The order in which coke and gas formation were suppressed during hydrocracking of ML-VR was NiMo>BL>AL, which is the same order as for the hydrogenation activity observed with the standard compounds. By contrast, the limonite catalysts exhibited relatively higher conversions and distillate yields in ML-VR hydrocracking than did the NiMo catalyst with the order of conversion and distillate yield (yield of the fraction with boiling point of 540 °C) being AL>BL>NiMo, which is the same order obtained for catalytic cracking of the two standards. Coke formation was effectively suppressed at high hydrogen pressures. The limonite catalysts showed lower activities for nitrogen and sulfur removal than did NiMo, but both proved to have a larger activity for nickel removal.     

Isolation, amino acid sequence and biological activities of novel long-chain polyamine-associated peptide toxins from the sponge Axinyssa aculeata

A novel family of functionalized peptide toxins, aculeines (ACUs), was isolated from the marine sponge Axinyssa aculeate. ACUs are polypeptides with N-terminal residues that are modified by the addition of long-chain polyamines (LCPA). Aculeines were present in the sponge extract as a complex mixture with differing polyamine chain lengths and peptide structures. ACU-A and B, which were purified in this study, share a common polypeptide chain but differ in their N-terminal residue modifications. The amino acid sequence of the polypeptide portion of ACU-A and B was deduced from 3' and 5' RACE, and supported by Edman degradation and mass spectral analysis of peptide fragments. ACU induced convulsions upon intracerebroventricular (i.c.v.) injection in mice, and disrupted neuronal membrane integrity in electrophysiological assays. ACU also lysed erythrocytes with a potency that differed between animal species. Here we describe the isolation, amino acid sequence, and biological activity of this new group of cytotoxic sponge peptides.