Calibrating catalytic DNA nanostructures for site-selective protein modification

Many biomedical fields rely on proteins that are selectively modified with novel chemical entities. These are often attached using reactive or catalytic moieties, but the position where these moieties are attached is often poorly controlled. In this work, we assess how catalyst position affects the efficiency and selectivity of protein modification. For this, we anchored a template DNA strand to the active site of three different proteins, which were subsequently hybridized to DNA strands that contained catalysts at different positions. We found that catalysts operating via a covalently bound reactant intermediate show a strong correlation between their distance to the protein surface and their efficiency and that the site-selectivity of the modification is affected by the position of the catalyst. Our results are rationalized using computational simulations, showing that one-point anchoring of the DNA construct leads to notable differences in the site of modification

Tuning Activity of Antimicrobial Peptides by Lipidation

Antimicrobial peptides (AMPs) are amino acid-based bioactive molecules that specifically target microbes. As such, they are a potent class of antibiotics, especially against bacterial infections. Naturally occurring AMPs are usually too long to be considered for therapeutic applications. To solve this, short sequences that mimic the activity of AMPs are designed. However, such endeavors are often accompanied with a reduction in antibacterial activity. To counter this, lipophilic molecules can be attached that function as a lipid anchor and target the short sequence to the bacterial membrane. For a range of short AMPs, this strategy has proven to lead to more active constructs. Although these lipidated short AMPs often work as complex target specific surfactants, more delicate modes of action that do not deviate too much from the nonlipidated counterparts are also known. This is readily observed by the large differences in activities that are detected when alterations in the lipid chain length and chirality of the amino acids residues are implemented. It is not uncommon to see that inactive or poorly active short AMPs can be turned into potent antibacterial agents. Importantly, selectivity of the short lipidated AMPs (lipoAMPs) for the bacterial membrane can be enhanced by alteration of the amino acid chirality. This strategy has led to lipoAMPs with submicromolar activities; in fact, activities that rival that of vancomycin have been observed for several short AMPs. Future research needs to determine (i) the effect of lipidation on the formation of lipid rafts in the bacterial membrane, (ii) if structural complications like branched lipids or chiral substituents on the lipid chain can be used to further increase the activity and selectivity of the conjugates, and (iii) if additional functionalities other than a membrane-anchoring ability can be bestowed on the lipid chain, e.g., redox activity or scavenger for small molecular components that traverse the lipid membrane. The interplay between degree of lipophilicity and the chirality of the amino acids of the AMP also needs further exploration, especially to see if more potent and selective (lipo)AMPs can be obtained that can be applied systemically. It may also be advisable to measure the most potent lipoAMPs in a centralized facility in order to obtain objective and comparable antibacterial activities

Biosynthesis of 2-aminooctanoic acid and its use to terminally modify a lactoferricin B peptide derivative for improved antimicrobial activity

Terminal modification of peptides is frequently used to improve their hydrophobicity. While N-terminal modification with fatty acids (lipidation) has been reported previously, C-terminal lipidation is limited as it requires the use of linkers. Here we report the use of a biocatalyst for the production of an unnatural fatty amino acid, (S)-2-aminooctanoic acid (2-AOA) with enantiomeric excess gt 98% ee and the subsequent use of 2-AOA to modify and improve the activity of an antimicrobial peptide. A transaminase originating from Chromobacterium violaceum was employed with a conversion efficiency 52-80% depending on the ratio of amino group donor to acceptor. 2-AOA is a fatty acid with amino functionality, which allowed direct C- and N-terminal conjugation respectively to an antimicrobial peptide (AMP) derived from lactoferricin B. The antibacterial activity of the modified peptides was improved by up to 16-fold. Furthermore, minimal inhibitory concentrations (MIC) of C-terminally modified peptide were always lower than N-terminally conjugated peptides. The C-terminally modified peptide exhibited MIC values of 25 mu g/ml for Escherichia coli, 50 mu g/ml for Bacillus subtilis, 100 mu g/ml for Salmonella typhimurium, 200 mu g/ml for Pseudomonas aeruginosa and 400 mu g/ml for Staphylococcus aureus. The C-terminally modified peptide was the only peptide tested that showed complete inhibition of growth of S. aureus