Vol 45, No 3 (2019)

The discovery of fluorescent proteins (FP) in 1962 and the following design of genetically encoded biomarkers and biosensors revolutionized the study of living systems. At present, researchers have access to FPs of a wide range of colors with a wide variety of specialized properties for visualization of biological processes in vivo using high-resolution spectroscopy techniques. GFP-like fluorescent proteins are widely used in cell biology as markers of various biological targets in a living cell. With the development of methods for visualizing processes in living organisms, the scientists have directed their efforts to designing new bright and photostable biomarkers of different colors with improved photophysical characteristics. The X-ray study plays an important role in design of the new biomarkers with improved properties. It allows us to determine the structure–function relations as a guide for directed change of the FPs properties meeting the requirements of modern research methods. The review presents the basic structures of the chromophores of the GFP-like fluorescent proteins determined by the X-ray method.

Currently, soybean occupies the first place as a global protein source for replacing fish meal in animal and aquaculture feeds. Since at the stage of fish postembryonic development, before the switch to active feeding, the efficacy of proteolysis in larvae is not high enough, soybean proteins in starter feeds should be hydrolyzed. The hydrolysate composition and behavior of the resulting protein fragments were shown to differ when soybean proteins were hydrolyzed by different enzyme preparations: the enzyme complex from the hepatopancreas of the Kamchatka crab (EC HPKC), protosubtilin, and the enzyme complex from pyloric appendage of cod (EC PAC). The most active enzyme preparation among them was EC HPKC, which demonstrated a high proteolytic activity at room temperature. Upon hydrolysis by EC HPKC, the yield of soluble hydrolysis products was 92% per weight of the initial protein material. Depending on the incubation time, the hydrolysates contained up to 60% of free amino acids (per weight of the hydrolyzed protein mixture) and short peptides less than 3 kDa. The use of protosubtilin or EC PAC at room temperature resulted in the intensive gelation and coagulation of the formed protein fragments resistant to further degradation. In order to achieve the yield of soluble hydrolysis products comparable with that for EC HPKC, it was necessary to increase the temperature. The yield of soluble products upon the EC PAC-induced hydrolysis of soybean proteins at 37°C achieved 82–88% of the initial protein material. The greatest part of the hydrolysate was represented by low-molecular-weight peptides with a molecular weight lower than 10 kDa and free amino acids (20.16% of the weight of the hydrolyzed protein mixture). Although the optimal temperature for the protosubtilin activity is 40–60°C according to the manufacturer’s data we did not perform hydrolysis in the presence of protosubtilin at this temperature because of the hazard to sulfur-containing amino acids. The content of free amino acids and the size of protein fragments in the soybean protein hydrolysates obtained upon the EC HPKC-induced hydrolysis at room temperature and with EC PAC at 37°C met the requirements for the fish starter feeds. Manipulations with such parameters as the hydrolysis time and the enzyme complex/protein ratio for the used enzyme preparations allow to prepare soybean protein hydrolysates differing in their hydrolysis degree.

A novel derivative of green fluorescent protein chromophore (Z)-5-((E)-3-(4-hydroxyphenyl)allylidene)-2,3-dimethyl-3,5-dihydro-4H-imidazol-4-one was synthesised. The desired compound was prepared by the synthetic approach based on the use of carboxyimidate. Comparison of optical properties of the obtained compound and of the classical GFP chromophore analogue (Z)-5-(4-hydroxybenzylidene)-2,3-dimethyl-3,5-dihydro-4H-imidazol-4-one showed the shift of absorption and emission maxima to the long-wavelength region induced by the increase of the conjugated π-system in the benzylidene fragment.

The synthesis of deoxyuridine triphosphate derivatives with alkyl groups of various chain length attached to the pyrimidine base by the trans-alkene linker was performed. The efficiency of the incorporation of modified nucleotides during the synthesis of DNA with a high degree of modification by the primer extension reaction with Taq DNA polymerase was studied.