Vol 52, No 1 (2018)

A new series of heat-stable (st) mutants of bacteriophage T5, which contains deletions in the tRNA gene region, has been isolated. An accurate mapping of the deletion boundaries for more than 30 mutants of phage T5 has been carried out. As a result of the analysis of nucleotide sequences flanking the deleted regions in wild-type phage DNA, it has been shown that they all contain short, direct repeats of different lengths (2–35 nucleotide residues), and that only one repetition is retained in the mutant phage DNA. On the basis of the obtained results, it was suggested that deletion mutants of the phage T5 are formed as a result of illegal recombination occurring with the participation of short repeats in DNA (SHDIR). Based on the example of two mutants, it has been shown that the resistance to thermal inactivation depends on the size of the deleted region.

A full analysis has been conducted of the sequences and secondary structures of viral type-I or related IRESs identified in all of the elements that correspond to the previously described minimal fragment of the enterovirus C IRES, which mimics the glycine tRNA anticodon hairpin in the IRES structure and is necessary for the specific binding of glycyl—tRNA synthetase. Experiments on human glycyl—tRNA synthetase binding with the mRNA fragments of several taxonomically distant viruses showed that the binding constants of these complexes are similar. These results indicate that the regulation of translation initiation via glycyl—tRNA synthetase must be a universal mechanism for these viruses and the corresponding parts of their mRNAs must have similar spatial structures. Furthermore, at least one additional mRNA hairpin with the glycyl anticodon loop has been found in all analyzed viral type-I IRESs. It seems plausible that this extra hairpin is associated with the second RNA-binding site of the glycyl—tRNA synthetase dimer and stabilizes its complex with the viral mRNA.

The formation of ribosomal 48S initiation complexes at the start codon of the mRNA leader sequence that encodes obelin has been studied using the method of primer extension inhibition (toeprinting). Experiments have been performed in a system composed of purified individual components required to initiate translation. The influence of the dominant negative mutant of factor eIF4A (R362Q) on translation initiation has been studied. It has been shown that the presence of the mutant in reaction mixture has no effect on efficiency of formation of the 48S complexes at start codon of the template studied.

Laccase belongs to the family of copper-containing oxidases. A study was made of the mechanism that sustains the incorporation of copper ions into the T2/T3 centers of recombinant two-domain laccase Streptomyces griseoflavus Ac-993. The occupancy of the T3 center by copper ions was found to increase with an increasing copper content in the culture medium and after dialysis of the protein preparation against a copper sulfate-containing buffer. The T2 center was filled only when overproducer strain cells were grown at a higher copper concentration in the medium. Two-domain laccases were assumed to possess a channel that serves to deliver copper ions to the T3 center during the formation of the three-dimensional laccase conformation and dialysis of the protein preparation. A narrower channel leads to the T2 center in two-domain laccases compared with three-domain ones, rendering the center less accessible for copper atoms. The incorporation of copper ions into the T2 center of two-domain laccases is likely to occur in the course of their biosynthesis or the formation of a functional trimer.

Possible combinations of βαβ-units and Π-like modules in proteins in both right- and left-handed forms have been analyzed in detail. The correlation between the mutual arrangement of the structural elements in the polypeptide chain and their handedness has been shown. In the βαβΠ combinations, which is encountered most frequently in proteins, the Π-module follows the βαβ unit along the chain and both elements are right-handed. In the Πβαβ combinations, where the Π-module is located at the N end and the βαβ-unit follows it, the former is left-handed and the latter is right-handed. In relatively rare combinations of the left-handed βαβ-units and right-handed Π-modules, the βαβ-unit follows Π-module in the chain. The combinations of left-handed Π-modules and the left-handed βαβ-units are unobservable in proteins. It has also been shown that the Π-modules with a β-strand—α-helix—arch—β-strand structure are observed in proteins only in a right-handed form and half of them (51%) contains cis-prolines in their arches. These arches of nonhomologous proteins, as well as the positions of cis-prolines, nearly coincide when superimposed. The superimposed Π-modules also demonstrate that their overall folds are very similar. Structural alignment of their amino acid sequences has shown that the Π-modules have very similar sequence patterns of the key hydrophobic, hydrophilic, glycine, and cis-proline residues.

Studies on the process of spontaneous protein folding into a unique native state are an important issue of molecular biology. Apomyoglobin from the sperm whale is a convenient model for these studies in vitro. Here, we present the results of equilibrium and kinetic experiments carried out in a study on the folding and unfolding of eight mutant apomyoglobin forms of with hydrophobic amino acid substitutions on the protein surface. Calculated values of apparent constants of folding/unfolding rates, as well as the data on equilibrium conformational transitions in the urea concentration range of 0–6 M at 11°C are given. Based on the obtained information on the kinetic properties of the studied proteins, a Φ-value analysis of the transition state has been performed and values of urea concentrations corresponding to the midpoint of the transition from the native to intermediate state have been determined for the given forms of mutant apomyoglobin. It has been found that a significant increase in the stability of the native state can be achieved by a small number of amino acid substitutions on the protein surface. It has been shown that the substitution of only one amino acid residue exclusively affects the height of the energy barrier that separates different states of apomyoglobin.

Studying the effect of cysteine bridges on different energy levels of multistage folding proteins will enable a better understanding of the process of folding and functioning of globular proteins. In particular, it will create prospects for directed change in the stability and rate of protein folding. In this work, using the method of differential scanning microcalorimetry, we have studied the effect of three cysteine bridges introduced in different structural elements of the green fluorescent protein on the denaturation enthalpies, activation energies, and heat-capacity increments when this protein passes from native to intermediate and transition states. The studies have allowed us to confirm that, with this protein denaturation, the process hardly damages the structure initially, but then changes occur in the protein structure in the region of 4–6 beta sheets. The cysteine bridge introduced in this region decreases the hydration of the second transition state and increases the hydration of the second intermediate state during the thermal denaturation of the green fluorescent protein.

The L1 protuberance of the ribosome includes two domain ribosomal protein L1 and three helices of 23S rRNA (H76, H77, and H78) with interconnecting loops A and B. Helix 78 consists of two parts, i.e., H78a and H78b. A comparison of the available structural data of L1-RNA complexes with the obtained kinetic data made it possible to determine the influence of the nonconserved regions of Thermus thermophilus L1-protuberance on the mutual affinity of the L1 protein and 23S rRNA. It has been shown that the N-terminal helix of the protein and 78b helix of 23S rRNA are essential for the formation of an additional intermolecular contact, which is separated in the protein from the main site of L1-rRNA interaction by a flexible connection. This results in a rise in the TthL1-rRNA affinity. At the same time, the elongation of the 76 helix has no effect on rRNA-protein binding.

The products of the reassembly reaction of tetradecameric two-ring quaternary structure of GroEL chaperonin under the pressure of its heptameric co-chaperonin GroES have been visualized by electron microscopy. It has been shown that one-ring heptameric oligomers of GroEL have been formed at the beginning (after ~5 min) of the reaction, while at the final stage of the reaction (after ~70 min), both onering heptamers in complex with one GroES and two-rings tetradecamers in complexes with one (asymmetrical complex) or two (symmetrical complex) GroES heptamers are present. The relationship between the data of light scattering, native electrophoresis, and electron microscopy obtained earlier has been discussed.

The scientific interest to the structural and functional properties of actin is determined by its abundance in cells. Being an important component of the cytoskeleton, actin is involved in many protein-protein interactions. Using crystal structures and molecular models, we have mapped the amino acid residues that are involved in these interactions and form the ATP-binding site of the actin monomer. Moreover, using mass spectrometry and high-performance liquid chromatography methods, we have discovered the regions of the amino acid sequence of actin that form the core of the actin fibril. According to the bioinformatic analysis, these regions are amyloidogenic and are located in the C-terminal region and in the hinge between the first and third subdomains. The data obtained are applicable to chordate actin, because multiple alignment revealed highly conserved amino acid sequences. In turn, the comparison of the chordate actin with the bacterial homologs showed the presence of numerous amino acid substitutions and insertions.