Structural systematics of bioactive peptides and their biosynthetic implications.

Structures of natural peptides have been linearized according to their biosynthetic constructions. The structural diversities of these peptides originate from highly complex enzyme systems, that are often found to be integrated to polyenzymes. Polyenzymes are constructed from similar elements or modules, and structural variations can be understood from mutational events and rearrangements at the DNA-level.

Principles of the molecular construction of multienzyme templates for peptide biosynthesis in integrated reaction sequences.

The amino acid sequences of the genes coding for four multienzyme peptide synthetases, operating by the thiotemplate mechanism are compared, to show underlying principles in the biosynthetic mechanism. Alignment with other carboxylic acid activating enzymes shows the sequences. LAY(V/I)I(Y/F)TSGT(T/S)GxPKGV and GELx(L/I)GGxG(V/I) to be involved in MgATP2-binding and adenylate formation, and two other sequences, one containing the element FxLGG(H/D)S(I/L) to be involved in covalent binding of the amino acid. As a general rule, 1000 amino acid building blocks are responsible for the incorporation of one amino acid into the nascent peptide.

Cell-free bioluminescent screening of translation inhibitors.

The possibility of creating new screening methods with a cell-free translation system has been demonstrated with a quantitative determination of diphtheria toxin and some antibiotics (puromycin, kanamycin and tetracycline) as examples. The approach proposed follows from the ability of various substances to inhibit protein synthesis. We used a wheat-germ cell-free translation system stabilized by freeze-drying in the presence of trehalose with the mRNA of the Ca(2+)-activated photoprotein obelin as a reporter template. This freeze-dried cell-free translation system allows prolonged storage of the detecting system before it is required, increases the reproducibility of the results and simplifies the application procedure. The obelin mRNA extends the sensitivity of the method owing to the high sensitivity of detection of the synthesized protein.

Cotranslational formation of active photoprotein obelin in a cell-free translation system: direct ultrahigh sensitive measure of the translation course.

Translation of apoobelin mRNA in a cell-free wheat germ translation system in the presence of coelenterazine and molecular oxygen results in cotranslational formation of active photoprotein. Active obelin formation is recorded by its luminescence, either direct in the translation mixture in the presence of coelenterazine and calcium ions or in aliquots from the translation mixture. In the second case translation is carried out with coelenterazine and EGTA. Registration of the translation course by luminescence of the synthesized product in both cases allows use of apoobelin mRNA at very low concentrations as an internal marker for immediate measure of protein biosynthesis activity of in vitro translation systems. It is shown that the simultaneous translation of any other mRNA does not affect translation of photoprotein mRNAs under standard conditions. Continuous registration of luminescence in a cuvette of a liquid scintillation counter in photon-counting mode varies the time of signal accumulation in a wide temporal range, thus increasing the numerical values of the recorded signals. Registration of photoprotein luminescence during translation can be used to obtain additional information about the translation process, for example codon reading speed, about protein folding, and about the formation of active proteins on ribosomes.