Incorporation of non-natural amino acids into proteins

Chemical and biological diversity of protein structures and functions can be widely expanded by position-specific incorporation of non-natural amino acids carrying a variety of specialty side groups. After the pioneering works of Schultz's group and Chamberlin's group in 1989, noticeable progress has been made in expanding types of amino acids, in finding novel methods of tRNA aminoacylation and in extending genetic codes for directing the positions. Aminoacylation of tRNA with non-natural amino acids has been achieved by directed evolution of aminoacyl-tRNA synthetases or some ribozymes. Codons have been extended to include four-base codons or non-natural base pairs. Multiple incorporation of different non-natural amino acids has been achieved by the use of a different four-base codon for each tRNA. The combination of these novel techniques has opened the possibility of synthesising non-natural mutant proteins in living cells.     

Incorporation of shikimate and other precursors into aromatic amino acids and prenylquinones of isolated spinach chloroplasts

Under conditions of photosynthesis, shikimate-[1,6-¹⁴C] and D,L-tyrosine-[β-¹⁴C] were incorporated into the aromatic amino acids Phe, Tyr and Trp, and the prenylquinone and α-tocopherol by intact spinach chloroplasts. This might indicate the presence of enzymes of shikimate pathway in chloroplasts.     

Tunicamycin-induced growth and inhibition of glucosamine incorporation into cell walls of rice coleoptiles

Tunicamycin, 0.25 to 2.5 μ M , promotes elongation of rice coleoptile sections after a 2 h lag. Tunicamycin decreased the minimum stress-relaxation time of the cell wall, T₀; and the wall loosening is recognized as the cause of this growth promotion. Bacitracin did not have significant effects on growth or T₀ except for inhibition of elongation at high concentration. Coleoptile sections were incubated with [¹⁴C]-glucosamine, and the synthetic pathway of the hexosamine-containing cell wall component was examined by a pulse-chase experiment. This component seems to be synthesized in the particulate fraction and secreted mainly into the hemicellulose I fraction. Tunicamycin strongly inhibited glucosamine incorporation into the particulate fraction and stopped the labeling of the cell wall. At 2.5 μ M , tunicamycin had no effect on incorporation of mannose, leucine or proline. These results suggest that the hexosamine-containing wall component is a kind of asparagine-linked glycoprotein, and that this component plays a principal role in formation of the cell wall network and in growth regulation.     

Simulated hypogravity and proline incorporation into salt-extractable macromolecules from cell walls

Proline [U-¹⁴C] was fed to shoots of intact Tagetes patula grown normally, on horizontal clinostats, or on vertical clinostats rotating at 15 rev/hr. After various periods the incorporation of ¹⁴C into salt-extractable material from the cell walls of stems, petioles, leaves and flowers was determined. The cell walls of the gravity-compensated plants (grown on horizontal clinostats) has the highest amount of salt-extractable radioactivity. A 2- to 9-fold increase was observed in comparison to either the normal or vertical clinostat plant controls. Some physico-chemical properties of the salt-extractable fraction suggest that it consists of highly charged, low MW entities, possibly short chain peptides. On acid hydrolysis this material yields radioactive aspartic acid, glutamic acid and proline. The presence of labelled hydroxyproline is suggested. After acid hydrolysis of the cell walls of leaves, it was found that ca 4 times the amount of ¹⁴C was incorporated in the hypogravity-grown plant compared to the controls. It appears likely that extensibility changes in tissues under simulated hypogravity required additional cell wall protein.     

Incorporation of 19F-substituted aromatic amino acids into membrane proteins from chromatophores of Rhodospirillum rubrum G9+

Chromatophore membranes were isolated from cells of the carotenoidless mutant Rhodospirillum rubrum G9⁺ grown in the presence of several fluorinated aromatic amino acids, solubilized using SDS and the extent of incorporation analyzed using high-resolution ¹⁹F-NMR spectroscopy. 3- and 4-¹⁹F-phenylalanine, 6-¹⁹F-tryptophan and 3-¹⁹F-tyrosine were biosynthetically incorporated into membrane proteins whereas 5-¹⁹F-tryptophan and 2-¹⁹F-phenylalanine were inhibitors of cell growth. The polypeptide chains of the major chromatophore membrane protein the light-harvesting complex, were isolated and shown by high-resolution ¹⁹F-NMR to contain 3-¹⁹F-phenylalanine, which is known to be situated principally within the membrane hydrocarbon layer. Broad-band ¹⁹F-NMR spectra of 3-¹⁹F-phenylalanine-labelled chromatophores showed the phenyl ring to be immobilized within the membrane.     

Hemicellulosic polymers of cell walls of zea coleoptiles

Hemicellulosic polymers comprised about 43% of the primary walls of Zea mays L. cv WF9 × Bear 38 coleoptiles; these polymers were separated by an alkali-gradient into three major fractions. Fraction 1 (GAX I) was solubilized from walls with 0.01 to 0.045 n KOH and consisted of novel glucuronoarabino(galacto)xylans. Nearly six of every seven residues of these xylans were substituted predominantly with single arabinosyl sidegroups. Fraction 2 (GAX II), material released by 0.45 to 0.8 n KOH, was also enriched with glucuronoarabinoxylan, but only two of every three xylose residues was substituted. This xylan was similar to those found in Zea and other Graminaceous species. Both of these xylan fractions contained uronic acid, terminal- and 4-linked galactosyl, and small amounts of 2-, 3-, 5-, and 3,5-linked arabinosyl units. Fraction 3 (MG-GAX) was released by 2.0 to 3.0 n KOH and consisted of about 60% mixed-linked glucan and about 40% glucuronoarabinoxylan. This fraction represented about half of the total hemicellulosic material of the primary walls of these coleoptiles.     

Incorporation of tellurium into amino acids and proteins in a tellurium-tolerant fungi

Aspergillus fumigatus, Aspergillus terreus, and Penicillium chrysogenum, a tellurium tolerant fungi, are able to grow on sulfur free medium amended with 0.2% (w/v) tellurite. Tellurium was incorporated into several types of low and high molecular weight proteins. The newly detected telluro-proteins contained an extraordinary high level of tellurium, as well as telluro-cysteine, telluro-cystine, telluro-methionine, and serine.