Kirpichnikov Mp

De novo design, synthesis and study of albebetin, a polypeptide with a predetermined three-dimensional structure. Probing the structure at the nanogram level.

The de novo polypeptide named albebetin was designed to form the tertiary fold that has not yet been observed in natural proteins. The design was based on the molecular theory of protein structures. The gene coding for this polypeptide was chemically synthesized. For the initial characterization of a protein structure, a new approach has been developed that uses only nanogram amounts of a polypeptide without its previous purification. This approach includes the biosynthesis of radiolabeled protein in a cell-free translation system with subsequent analysis of its compactness and structure by size-exclusion chromatography, urea-gradient electrophoresis and limited proteolysis. According to all tests used, albebetin has a compact stable structure.

Comparative analysis of proapoptotic activity of cytochrome c mutants in living cells

A non-traumatic electroporation procedure was developed to load exogenous cytochrome c into the cytoplasm and to study the apoptotic effect of cytochrome c, its K72-substitued mutants and “yeast → horse” hybrid cytochrome c in living WEHI-3 cells. The minimum apoptosis-activating intracellular concentration of horse heart cytochrome c was estimated to be 2.7 ± 0.5 μM (47 ± 9 fg/cell). The equieffective concentrations of the K72A-, K72E- and K72L-substituted mutants of cytochrome c were five-, 15- and 70-fold higher. The “yeast → horse” hybrid created by introducing S2D, K4E, A7K, T8K, and K11V substitutions (horse protein numbering) and deleting five N-terminal residues in yeast cytochrome c did not evoke apoptotic activity in mammalian cells. The apoptotic function of cytochrome c was abolished by the K72W substitution. The K72W-substituted cytochrome c possesses reduced affinity to the apoptotic protease activating factor-1 (Apaf-1) and forms an inactive complex. This mutant is competent as a respiratory-chain electron carrier and well suited for knock-in studies of cytochrome c-mediated apoptosis.

Biochemical characterization of human enteropeptidase light chain

The synthetic gene encoding human enteropeptidase light chain (L-HEP) was cloned into plasmid pET-32a downstream from the gene of fusion partner thioredoxin immediately after the DNA sequence encoding the enteropeptidase recognition site. The fusion protein thioredoxin (Trx)/L-HEP was expressed in Escherichia coli BL21(DE3). Autocatalytic cleavage of the fusion protein and activation of recombinant L-HEP were achieved by solubilization of inclusion bodies and refolding of Trx/L-HEP fusion protein. The kinetic parameters of human and bovine enteropeptidases in the presence of different concentrations of Ca2+ and Na+ for cleavage of the specific substrate GD4K-na and nonspecific substrates such as small ester Z-Lys-SBzl and chromogenic substrates Z-Ala-X-Arg-pNA have been comparatively analyzed. It is demonstrated that positively charged ions increased the Michaelis constant (Km) for cleavage of specific substrate GD4K-na, while the catalytic constant (kcat) remained practically unchanged. L-HEP demonstrated secondary specificity to the chromogenic substrate Z-Ala-Phe-Arg-pNA with kcat/Km 260 mM−1·sec−1. Enzymatic activity of L-HEP was suppressed by inhibitors of trypsin-like and cysteine (E-64), but not metallo-, amino-, or chymotrypsin-like proteinases. L-HEP was active over a broad range of pH (6–9) with optimum activity at pH 7.5, and it demonstrated high stability to different denaturing agents.

Immunotoxins containing A‐chain of mistletoe lectin I are more active than immunotoxins with ricin A‐chain

Conjugates of anti‐CD25 monoclonal antibodies against cell surface IL‐2 receptor with MLIA and RTA were prepared and investigated. Both of the immunotoxins had high specific cytotoxic activity on target cells. The IC50 value of the anti‐CD25/MLIA immunotoxin was 15‐fold greater than that of the anti‐CD25/RTA. Previous studies of the anti‐CD5 immunotoxins with MLIA and RTA showed that the anti‐CD5/MLIA IT was 80‐fold more active than anti‐CD5/RTA IT [Tonevitsky et al. (1991) Int. J. Immunopharmacol. 13, 1037–1041]. The surface hydrophobicity of the MLI A‐chain was 4‐fold higher than that of the ricin A‐chain as estimated by binding with ANS. In model experiments with small unilamellar DMPC liposomes, MLIA but not RTA increased the turbidity of liposome suspensions at pH 4.5. Our results indicate that the greater cytotoxic activity of the MLI A‐chain immunotoxin probably provided a higher surface hydrophobicity of the protein and the ability to interact with phospholipid membranes.

Cytotoxicity of albebetin oligomers depends on cross-β-sheet formation

Prefibrillar cytotoxicity was suggested as a common amyloid characteristic. We showed two types of albebetin prefibrillar oligomers are formed during incubation at pH 7.3. Initial round‐shaped oligomers consist of 10–15 molecules determined by atomic force microscopy, do not bind thioflavin‐T and do not affect viability of granular neurons and SH‐SY5Y cells. They are converted into ca. 30–40‐mers possessing cross‐β‐sheet and reducing viability of neuronal cells. Neither monomers nor fibrils possess cytotoxicity. We suggest that oligomeric size is important for stabilising cross‐β‐sheet core critical for cytotoxicity. As albebetin was used as a carrier‐protein for drug delivery, examination of amyloidogenicity is required prior polypeptide biomedical applications.

The role of structural domains in RIP II toxin model membrane binding

The interaction of plant toxin ricin and MLI binding subunits to liposomes containing monosialoganglioside (GM1), bearing a terminal galactose residue, has been examined as a possible receptor model. For the first time we demonstrate that ricin B‐chain but not ricin provokes liposome aggregation at 10 M% GM1 concentration, whereas in the presence of either ricin A‐chain or galactose the aggregation is inhibited. The B‐subunit of plant toxin MLI from Viscum album has similar lectin specificity and activity but cannot aggregate GM1 liposomes. The ability of the B‐chain to aggregate liposomes adds a new crucial step in the toxin transmembrane penetration mechanism. We demonstrate here possible ricin B‐chain interactions with membranes proceeding via two sites, namely (a) a galactose‐binding domain and (b) a hydrophobic interchain domain. In close contact with two phospholipid bilayers, ricin B‐chain may determine the geometry of the fusion site. These events can provoke A‐chain translocation which follows membrane fusion.

Key role of barstar Cys-40 residue in the mechanism of heat denaturation of bacterial ribonuclease complexes with barstar

The mechanism by which barnase and binase are stabilized in their complexes with barstar and the role of the Cys‐40 residue of barstar in that stabilization have been investigated by scanning microcalorimetry. Melting of ribonuclease complexes with barstar and its Cys‐82‐Ala mutant is described by two 2‐state transitions. The lower‐temperature one corresponds to barstar denaturation and the higher‐temperature transition to ribonuclease melting. The barstar mutation Cys‐40‐Ala, which is within the principal barnase‐binding region of barstar, simplifies the melting to a single 2‐state transition. The presence of residue Cys‐40 in barstar results in additional stabilization of ribonuclease in the complex.

Interactions between cro repressor and the model specific binding site

Binding of λ phage cro repressor to the synthetic half of OR3, the most conservative half of the specific binding sites, was investigated by proton nuclear magnetic resonance spectroscopy. It was found that the α‐helical segment (27–36) of the protein was involved in specific interactions with the model binding site. The 3‐dimensional structure of cro repressor does not change noticeably upon complex formation. Intercalation can be excluded as a possible means of interaction.

cI and lexA repressors consist of three cro-like domains.

The switching of phages X and 434 from the lysogenic state to the lytic growth or vice versa depends mainly on 2 repressors: cro and cI. This switching can be induced by external agents such as UV light. On the other hand, various DNAdamaging agents (including UV light) induce the ‘SOS-system’ of Escherichia coli [l] which is normally inhibited by IexA repressor. Among the stages of the ‘SOS-system’ work one includes recA protein activation which cleaves proteolytically IexA repressor [2,3]; the same protein cleaves also c1 repressor 1451. binding of c1 to ~2 (which is cooperative with binding to 0~1) turns on (and, moreover, stimulates) the transcription of the c1 gene from PRM promoter. At the same time cro repressor does not bind cooperatively with the sites 0~1, 0~2, OR3 and its binding to OR3 turns off the transcription of c1 gene from mM promoter [lO,l 11. IexA protein binds with 2 DNA regions (each with an approximate 2-fold internal symmetry) before the 1exA gene and with one DNA region before the recA gene turning off the transcription of those genes [ 14,151.

Three-dimensional structure of binase in solution

We present the spatial structure of binase, a small extracellular ribonuclease, derived from 1H‐NMR* data in aqueous solution. The total of 20 structures were obtained via torsion angle dynamics using DYANA program with experimental NOE and hydrogen bond distance constraints and φ and χ1 dihedral angle constraints. The final structures were energy minimised with ECEPP/2 potential in FANTOM program. Binase consists of three α‐helices in N‐terminal part (residues 6–16, 26–32 and 41–44), five‐stranded antiparallel β‐sheet in C‐terminal part (residues 51–55, 70–75, 86–90, 94–99 and 104–108) and two‐stranded parallel β‐sheet (residues 22–24 and 49–51). Three loops (residues 36–39, 56–67 and 76–83), which play significant role in biological functioning of binase, are flexible in solution. The differences between binase and barnase spatial structures in solution explain the differences in thermostability of binase, barnase and their hybrids.