Carrier Protein Interaction with Competing Adenylation and Epimerization Domains in a Nonribosomal Peptide Synthetase Analyzed by FRET.

In multi-domain nonribosomal peptide synthetases (NRPSs) the order of domains and their catalytic specificities dictate the structure of the peptide product. Peptidyl-carrier proteins (PCPs) bind activated amino acids and channel elongating peptidyl intermediates along the protein template. To this end, fine-tuned interactions with the catalytic domains and large-scale PCP translocations are necessary. Despite crystal structure snapshots of several PCP-domain interactions, the conformational dynamics under catalytic conditions in solution remain poorly understood. We report a FRET reporter of gramicidin S synthetase 1 (GrsA; with A-PCP-E domains) to study for the first time the interaction between PCP and adenylation (A) domain in the presence of an epimerization (E) domain, a competing downstream partner for the PCP. Bulk FRET measurements showed that upon PCP aminoacylation a conformational shift towards PCP binding to the A domain occurs, indicating the E domain acts on its PCP substrate out of a disfavored conformational equilibrium. Furthermore, the A domain was found to preferably bind the D-Phe-S-Ppant-PCP stereoisomer, suggesting it helps in establishing the stereoisomeric mixture in favor of the D-aminoacyl moiety. These observations surprisingly show that the conformational logic can deviate from the order of domains and thus reveal new principles in the multi-domain interplay of NRPSs.

Non-Destructive Evaluation of the Cutting Surface of Hardwood Finger Joints.

In this study, the surface parameters wettability, roughness, and adhesive penetration, which are important for wood bonding, were investigated and evaluated utilizing non-destructive methods after different mechanical processing. For this purpose, beech and birch finger joints were prepared with different cutting combinations (three cutters with different sharpness levels and two feed rates) in an industrial process. Effects and interactions on the surface parameters resulting from the different cutting combinations were evaluated using three Full Factorial Designs. The various cutting parameters had a predominantly significant influence on the surface parameters. The effects and identified interactions highlight the complexity of the cutting surface and the importance of wood bonding. In this respect, a new finding is that with sharper cutters, higher contact angles of the adhesives occur. The methods (contact angle measurement, laser scanning microscopy, and brightfield microscopy) used were well suited to make effects visible and quantifiable, which can be of interest for the quality control of the wood processing industry. The results can help to better understand and evaluate the design of wood surfaces via machining and the bonding of hardwoods. Possibly the results can contribute to further standardizing the production of load-bearing hardwood finger joints and making them more efficient.