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D A Dolgikh

Publications and source records attributed to D A Dolgikh.

At least 37 records · Page 2Linked to original sources
PubMed1984

'Molten-globule' state accumulates in carbonic anhydrase folding.

Kinetics of folding and unfolding of bovine carbonic anhydrase B were monitored by circular dichroism, viscometry and esterase activity. It was shown that kinetic intermediate states accumulating in folding process reveal a native-like compactness and secondary structure but have a symmetrized average environment of aromatic side groups and no esterase activity. These properties allow one to consider these intermediate states as the 'molten-globule' state of a protein molecule previously described by us for several equilibrium forms of bovine and human alpha-lactalbumins and bovine carbonic anhydrase B.

Animals↗
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Protein engineering of de novo protein with predesigned structure and activity.

The de novo protein albebetin has been engineered (J. Mol. Biol. 1992, 225, 927-931) to form a predesigned tertiary fold that has not yet been observed in natural proteins. Analysis of albebetin expressed in a cell-free system and in Escherichia coli revealed its compactness, relative stability, and the secondary structure close to the predesigned one. The blast-transforming biological activity of human interferon was grafted to albebetin by attachment of an eight amino acid interferon fragment to the N-terminus of albebetin next to its first methionine residue. The chimeric protein was expressed in a wheat germ cell-free translation system and tested for its structural properties, receptor binding, and biological activity. According to the tests, albebetin incorporating the active interferon fragment has a compact and relatively stable structure, and binds the murine thymocyte receptor effectively. It activates the blast transformation reaction of thymocyte cells even more efficiently than human interferon at low concentrations.

Amino Acid Sequence↗
PubMedDate not supplied

[An artificial protein, possessing biological activity of HL-60 human promyelocytic leukemia differentiation factor].

The ABB-df artificial protein was prepared by inserting the TGENHR biologically active peptide corresponding to the 41-46 sequence of the differentiation factor for the HL-60 cell line of the human promyelocyte leukemia into the N-terminus of the polypeptide chain of albebetin, an artificial protein with the preset structure. The ABB-df protein was found to induce the differentiation of HL-60 cells and to inhibit their proliferation; its efficiency was almost the same as that of the starting peptide. According to CD spectroscopy, the inclusion of the peptide fragment into albebetin exerts virtually no effect on the regular secondary structure of albebetin.

Amino Acid Sequence↗
PubMedDate not supplied

[Synthetic proteins with antiviral properties of alpha-interferons].

Consensus sequence 30-36 LKDRHDF of human alpha-interferons is inserted into the C- and N-terminal sequences of the artificial protein albeferon using genetic engineering methods in order to obtain artificial proteins with antiviral activity. Albeferon, obtained by Dolgikh et al. (Protein Eng., 1996, vol. 9, pp. 195-201), already contains the IFN-alpha 2 130-137 fragment and possesses antiproliferative activity comparable with that of IFN-alpha 2. According to CD spectroscopy, both proteins have regular secondary structures similar to that of the precursor protein. They exhibit antiviral activities, and the activity of one of them is comparable with that of IFN-alpha 2. At the same time, their cytotoxic properties are displayed only at relatively high concentrations, which substantially exceed the minimal antiviral concentrations.

Amino Acid Sequence↗
PubMedDate not supplied

[Y97V substitution in the horse cytochrome c causes accumulation of the equilibrium intermediate].

Equilibrium unfolding experiments on several mutant forms of horse heart cytochrome c were performed. By means of absorbance spectroscopy, the accumulation of an equilibrium intermediate was revealed upon unfolding of Y97V mutant protein, and its structural properties were characterized. The data obtained allow one to conclude that the equilibrium intermediate corresponds to the earliest kinetic intermediate Ic in cytochrome c folding reaction. A comparative analysis of spectral properties of unfolded states of cytochrome c induced by urea or guanidine hydrochloride is presented.

Amino Acid Substitution↗
PubMedDate not supplied

[The immune response to artificial proteins containing biologically active fragments of human IFN-alpha2 and insulin].

A study was made of the humoral immune response of BALB/c mice to various doses of artificial proteins that contained biologically active fragments of human interferon alpha 2 (IFN-alpha 2) and insulin. The insulin fragment had no effect on the response to any protein construct. The IFN-alpha 2 fragment increased the titer of antibodies against the construct. Mapping of continuous B epitopes with immune sera revealed several antigenic determinants, the C end of the IFN-alpha 2 fragment with the adjacent de novo protein region being immunodominant. More effective binding of serum antibodies with the constructs containing the IFN-alpha 2 fragment was attributed to antibody interaction with the fragment and to a better recognition of the entire protein construct by the immune system.

Animals↗
PubMedDate not supplied

[Introduction of biologically active fragments of interferon-alpha2 and insulin into the artificial protein albebetin affects immunogenicity of the final construct].

The immunogenicity of the artificial protein albebetin and its derivatives with active peptide fragments was investigated. We also studied the influence of the peptides on the immunogenicity of the whole construct and contribution of each component to the immunogenicity. Two of three studied proteins contained active peptides from human IFN-alpha and insulin. Three continuous antigenic sites with different immunogenic potential were recognized in the chimerical proteins. The interferon fragment was the immunodominant site in the albeferon and albeferon-insulin molecules, while the insulin fragment displayed low immunogenic activity. All continuous B-epitopes are located at the boundaries of the secondary structure elements and at the predicted surface-located sites of albebetin molecule. Thus, peptide fragments attached to the artificial protein carrier can influence immunogenicity of the resulting construct.

Amino Acid Sequence↗
PubMedDate not supplied

[De novo proteins with a given spatial structure: new approaches to design and analysis].

Based on the molecular theory of protein structure the de novo protein was designed in order to obtain the tertiary fold which has not yet been observed in natural proteins, namely four-stranded antiparallel beta-sheet covered by two alpha-helixes. The gene coding for this protein (named albebetin) was chemically synthesized, cloned in plasmid with SP6 phage promoter and expressed in mRNA-dependent cell-free translation system. An approach was developed to study albebetin using only nanogram amounts of radio labelled protein without previous purification. The preliminary analysis of its structure by gel-filtration, urea-gradient electrophoresis and limited proteolysis revealed compactness and stability of the de novo protein.

Amino Acid Sequence↗
PubMedDate not supplied

[Expression of neurotoxin II from Naja oxiana cobra venom in Escherichia coli in a hybrid form with thioredoxin].

Neurotoxin II from the venom of cobra Naja oxiana is a short type alpha-neurotoxin, which competitively inhibits nicotinic acetylcholine receptor. The toxin gene was expressed as a construct fused with the thioredoxin gene and the linker encoding the enteropeptidase recognition site and a Met residue between the genes. The fusion protein was mainly cleaved by cyanogen bromide, since enteropeptidase was less effective. The yield of neurotoxin II was 6 mg/l of the bacterial culture. The resulting recombinant protein was identified with native neurotoxin II by its N-terminal analysis, mass spectrometry, and NMR spectroscopy. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 1; see also http://www.maik.ru.

Animals↗
PubMedDate not supplied

[Fluctuating state of the protein globule].

It has been shown that bovine and human alpha-lactalbumins and carbonic anhydrase B can be transformed under different influence into a peculiar state possessing physical characteristics intermediate between those for the native and unfolded states. In this state a protein molecule is compact and has the secondary structure similar to that of the native molecule, but does not melt cooperatively at heating, has an anomalously fast H-D exchange and a more or less symmetrical average environment of aromatic and other side groups. A model of this "intermediate" state of protein molecule is proposed, according to which the intermediate state differs from the native one mainly by the substantial increase of protein structure fluctuations and the sharp decrease of van der Waals and other specific interactions. It has been shown that the transition from the native state to this intermediate state is the phase transition of the first order. The role of the intermediate state in protein folding is discussed.

Animals↗
PubMedDate not supplied

[Protein globule without the unique three-dimensional structure: experimental data for alpha-lactalbumins and general model].

It has been shown that bovine and human alpha-lactalbumins under the influence of acidic pH, high temperatures or low guanidine hydrochloride concentrations are transferred into a state which is quite distinct both from the native and the completely denatured (unfolded) ones. Human alpha-lactalbumin can be transferred into this intermediate state also by removal of the bound to it Ca2+ ion. On the basis of the obtained physical characteristics of this state a model of the "intermediate" state is suggested in which a protein globule is compact, has a secondary structure, but does not possess a unique three-dimensional structure. Phase diagrams of the protein molecule states (native, intermediate and unfolded) are presented and discussed and the role of the intermediate state in protein folding is considered.

Animals↗
PubMedDate not supplied

[Comparison of the structures of different forms of sperm whale myoglobin in solution using wide angle x-ray scattering].

Experimental X-ray diffuse scattering curves in a wide range of angles for oxy-, deoxy-, met- and cyan-forms of sperm whale myoglobin in solution have been obtained. It has been found that scattering indicatrices of these ligand forms of myoglobin are well compatible with each other and differ from the theoretical scattering curve of met-myoglobin, calculated from the structure of this protein in crystal. The conclusions have been drawn: (1) on the similarity of myoglobin structures in solution for all the protein forms studied; (2) on the difference in the structure of the above ligand forms of myoglobin in solution from the structure of the protein in crystal by a slight shift (about 2 A) of the "hairpin" GH.

Animals↗
PubMedDate not supplied

[Artificial proteins with a given spatial structure and biological activity].

Biologically active fragment 131-138 of human interferon alpha 2 carrying blast-transforming activity of the protein was attached to the N-terminus of the de novo protein albebetin with predetermined tertiary structure by means of genetic engineering. The chimeric protein was expressed in a wheat germ cell-free translation system and tested for compactness, stability and biological activity. According to the tests used albebetin with interferon fragment has a compact and relatively stable structure. It binds murine thymocyte receptor with high affinity and activates efficiently thymocyte blast transformation at a concentration of 10(-11) M.

Amino Acid Sequence↗