Objective. To study the antibacterial activity of lysostaphin against staphylococci various species, as well as its effect on the viability of Vero cells. Materials and Methods. Lysostaphin was obtained by genetic engineering. Purification of the protein was carried out on SP-sepharose, the purity was determined by electrophoresis in PAGE by Lamley. The susceptibility to lysostaphin of 9 species 175 strains of staphylococci was studied. Identification was performed by MALDI-TOF MS, antibiotic susceptibility by EUCAST (v. 11.0). The MIC of lysostaphin was studied by the method of serial dilutions with concentrations between 0.015 and 512 mg/l. For 72 hours, the viability of Vero cells with lysostaphin at concentrations of 0.5-32.0 mg/l was determined by the MTT method with counting of living cells according to their growth curve. The results were analyzed by ANOVA followed by Dunnett’s test. Results. A kinetic study of S. aureus growth in the presence of revealed an inhibitory effect of endopeptidase (MIC 0.06 mg/l). Lysostaphin was characterized by pronounced activity against clinical methicillinsensitive S. aureus. The MIC ranged between 0.03 and 0.5 mg/l and the MIC50/90 was 0.125⁄0.5 mg/l. For methicillin-resistant S. aureus MIC50/90 0.25⁄0.5 mg/l. The MIC50 for MRSE was 2 times higher than for MSSE – 1 mg/l. The maximum MIC value was determined against isolates of S. warneri and S. hominis – 64 mg/l, the lowest for S. saprophyticus – 0.5 mg/l. MIC50 of lysostaphin against MRSA was 4 times lower than that of vancomycin, MIC90 was 3 times lower. Differences in viable cells depending on the concentration of lysostaphin were not found. Conclusions. Significant activity of lysostaphin against staphylococci was revealed, which is several times higher than vancomycin against MRSA. Lysostaphin was also effective against methicillin-resistant S. aureus. The high anti-staphylococcal activity and cytocompatibility of lysostaphin are promising for its further study in the prevention and treatment of staphylococcal orthopedic infections.

Ribonuclease from Bacillus intermedius (binase) is a small basic protein with antitumour activity. The three-dimensional structure of the binase mutant form Glu43Ala/Phe81Ala was determined at 1.98 Å resolution and its functional properties, such as the kinetic parameters characterizing the hydrolysis of polyinosinic acid and cytotoxicity towards Kasumi-1 cells, were investigated. In all crystal structures of binase studied previously the characteristic dimer is present, with the active site of one subunit being blocked owing to interactions within the dimer. In contrast to this, the new mutant form is not dimeric in the crystal. The catalytic efficiency of the mutant form is increased 1.7-fold and its cytotoxic properties are enhanced compared with the wild-type enzyme.

ErbB is a family of epidermal growth factor receptors representing an important class of receptor tyrosine kinases that play a leading role in cellular growth, development, and differentiation. Transmembrane domains of these receptors transduce biochemical signals across the plasma membrane via lateral homo- and heterodimerization. The relatively small size of ErbB transmembrane domain complexes with detergents or lipids makes it possible to study their detailed spatial structure using three-dimensional heteronuclear high-resolution NMR spectroscopy. Here, we describe an efficient expression system and a purification procedure for preparative-scale production of transmembrane peptides from all four ErbB proteins—ErbB1, ErbB2, ErbB3, and ErbB4—for the purpose of structural studies. The recombinant peptides were produced in Escherichia coli BL21(DE3)pLysS cells as N-terminal extensions of thioredoxin A. The fusion proteins were cleaved with the light chain of human enterokinase. Several (10–30) milligrams of purified isotope-labeled transmembrane peptides were isolated using a simple and convenient procedure, which consists of consecutive steps of immobilized metal affinity chromatography and cation-exchange chromatography. The purified peptides were reconstituted in a lipid/detergent environment (micelles or bicelles) and characterized using dynamic light scattering and CD and NMR spectroscopy. The data obtained indicate that purified ErbB transmembrane peptides are suitable for structural and dynamic studies of their homo- and heterodimer complexes using high resolution NMR spectroscopy.

Specific interactions between transmembrane α-helices, to a large extent, determine the biological function of integral membrane proteins upon normal development and in pathological states of an organism. Various membrane-like media, partially those mimicking the conditions of multicomponent biological membranes, are used to study the structural and thermodynamic features that define the character of oligomerization of transmembrane helical segments. The choice of the composition of the membrane-mimicking medium is conducted in an effort to obtain a biologically relevant conformation of the protein complex and a sample that would be stable enough to allow to perform a series of long-term experiments with its use. In the present work, heteronuclear NMR spectroscopy and molecular dynamics simulations were used to demonstrate that the two most widely used media (detergent DPC micelles and lipid DMPC/DHPC bicelles) enable to perform structural studies of the specific interactions between transmembrane α-helices by the example of dimerizing the transmembrane domain of the bitopic protein glycophorin A. However, a number of peculiarities place lipid bicelles closer to natural lipid bilayers in terms of their physical properties.

The structures of two crystal modifications of the W34F mutant ribonuclease from the bacterium Bacillus intermedius (binase) were solved and refined at 1.7 and 1.1 Å resolution. The kinetic parameters of the hydrolysis of substrates of different length (GpU, GpUp, and poly(I)) by binase and its W34F mutant were investigated and compared. The catalytic activity of the enzymes was shown to increase with increasing length of the substrate. The substitution of tryptophan for phenylalanine does not lead to a change in the activity of the enzyme but results in a decrease of the binding constants for substrates containing more than one phosphate groups. A comparison of the structure of the mutant enzyme with the previously established structures of binase and its complexes with sulfate ions and guanosine monophosphate showed that the difference in their kinetic parameters is related to the fact that the mutant ribonuclease cannot bind the second phosphate group. Both crystal modifications of the mutant binase contain dimers, like in the crystal structure of binase studied previously. In these dimers, only one enzyme molecule can bind the substrate molecule. Since the dimers were found in the crystals grown under four different conditions, it can be suggested that the enzyme can exist as dimers in solution as well. Mutants of binase, which could exclude the formation of dimers, are suggested.