Covalent Linkage and Macrocylization Preserve and Enhance Synergistic Interactions in Catalytic Amyloids

The self-assembly of short peptides into catalytic amyloid-like nanomaterials has proven to be a powerful tool in both understanding the evolution of early proteins and identifying new catalysts for practically useful chemical reactions. Here we demonstrate that both parallel and antiparallel arrangements of β-sheets can accommodate metal ions in catalytically productive coordination environments. Moreover, synergistic relationships, identified in catalytic amyloid mixtures, can be captured in macrocyclic and sheet-loop-sheet species, that offer faster rates of assembly and provide more complex asymmetric arrangements of functional groups, thus paving the way for future designs of amyloid-like catalytic proteins. Our findings show how initial catalytic activity in amyloid assemblies can be propagated and improved in more-complex molecules, providing another link in a complex evolutionary chain between short, potentially abiotically produced peptides and modern-day enzymes.     

Late-Stage Hydrocarbon Conjugation and Cyclisation in Synthetic Peptides and Proteins

The conventional S-alkylation of cysteine relies upon using activated electrophiles. Here we demonstrate high-yielding and selective S-alkylation and S-lipidation of cysteines in unprotected synthetic peptides and proteins by using weak electrophiles and a Zn²⁺ promoter. Linear or branched iodoalkanes can S-alkylate cysteine in an unprotected 38-residue Myc peptide fragment and in a 91-residue miniprotein Omomyc, thus highlighting selective late-stage synthetic modifications. Metal-assisted cysteine alkylation is also effective for incorporating dehydroalanine into unprotected peptides and for peptide cyclisation via aliphatic thioether crosslinks, including customising macrocycles to stabilise helical peptides for enhanced uptake and delivery to proteins inside cells. Chemoselective and efficient late-stage Zn²⁺-promoted cysteine alkylation in unprotected peptides and proteins promises many useful applications.     

Effect of Methionine Sulfoxide on the Synthesis and Purification of Aggregation-Prone Peptides

A two-step synthesis for methionine-containing hydrophobic and/or aggregation-prone peptides is presented that takes advantage of the reversibility of methionine oxidation. The use of polar methionine sulfoxide as a building block in solid-phase peptide synthesis improves the synthesis quality and yields the crude peptide, with significantly improved solubility compared to the reduced species. This facilitates the otherwise often laborious peptide purification by high-performance liquid chromatography. The subsequent reduction proceeds quantitatively. This approach has been optimised with the methionine-rich Tar-DNA-binding protein 43 (307–347), but is also more generally applicable, as demonstrated by the syntheses of human calcitonin and two aggregation-prone peptides from the human prion protein.     

Cell-envelope proteinases from lactic acid bacteria: Biochemical features and biotechnological applications

Proteins displayed on the cell surface of lactic acid bacteria (LAB) perform diverse and important biochemical roles. Among these, the cell-envelope proteinases (CEPs) are one of the most widely studied and most exploited for biotechnological applications. CEPs are important players in the proteolytic system of LAB, because they are required by LAB to degrade proteins in the growth media into peptides and/or amino acids required for the nitrogen nutrition of LAB. The most important area of application of CEPs is therefore in protein hydrolysis, especially in dairy products. Also, the physical location of CEPs (i.e., being cell-envelope anchored) allows for relatively easy downstream processing (e.g., extraction) of CEPs. This review describes the biochemical features and organization of CEPs and how this fits them for the purpose of protein hydrolysis. It begins with a focus on the genetic organization and expression of CEPs. The catalytic behavior and cleavage specificities of CEPs from various LAB are also discussed. Following this, the extraction and purification of most CEPs reported to date is described. The industrial applications of CEPs in food technology, health promotion, as well as in the growing area of water purification are discussed. Techniques for improving the production and catalytic efficiency of CEPs are also given an important place in this review.     

Fluorescent Amino Acid Initiated de novo Cyclic Peptides for the Label-Free Assessment of Cell Permeability**

The major obstacle in applying peptides to intracellular targets is their low inherent cell permeability. Standard approaches to attach a fluorophore (e. g. FITC, TAMRA) can change the physicochemical properties of the parent peptide and influence their ability to penetrate and localize in cells. We report a label-free strategy for evaluating the cell permeability of cyclic peptide leads. Fluorescent tryptophan analogues 4-cyanotryptophan (4CNW) and β-(1-azulenyl)-L-alanine (AzAla) were incorporated into in vitro translated macrocyclic peptides by initiator reprogramming. We then demonstrate these efficient blue fluorescent emitters are good tools for monitoring peptide penetration into cells.     

Angiotensin-converting enzyme (ACE) inhibitory peptides from fermented sausages inoculated with Lactobacillus plantarum CD101 and Staphylococcus simulans NJ201

Natural ACE inhibitory peptides derived from food are considered to be an effective supplement for lowering blood pressure. This study investigated the effects of Lactobacillus plantarum CD101 and Staphylococcus simulans NJ201 on proteolysis and the sequence composition of ACE inhibitory peptides in fermented sausages. The ACE inhibitory activity of the inoculated group reached the maximum values on day 35 during the production of fermented sausages. A more positive effect in the hydrolysis of the sarcoplasmic and myofibrillar protein was observed in the inoculated group through sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE). The free amino acid content of the inoculated group was 865.21 ± 12.55 mg/100 g, which was significantly higher (P < 0.05) than that of the control group. The molecular weight distribution showed a significant increase (P < 0.05) in peptides with a molecular weight less than 1 kDa in the inoculated group. A total of thirty-four peptides with high hydrophobicity were identified by liquid chromatograph-mass spectrometer/mass spectrometer (LC–MS/MS). Furthermore, the peptides were isolated and purified using gel filtration chromatography (GFC) and reversed-phased high-performance liquid chromatography (RP-HPLC). Eleven novel peptides were identified by Nano-LC-ESI-MS/MS, in which VALSLSRP with X-Pro structure exhibited the highest ACE inhibition rate (75.36 ± 2.45%). These results indicated that mixed starters are conducive to protein degradation and the formation of peptides with high ACE inhibitory activity, especially peptides less than 1 kDa, and could be used as a functional material for antihypertensive drugs.