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Biomedical subjects

D A Dolgikh

Publications and source records attributed to D A Dolgikh.

At least 19 recordsLinked to original sources
PubMed2006

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 (k(cat)) remained practically unchanged. L-HEP demonstrated secondary specificity to the chromogenic substrate Z-Ala-Phe-Arg-pNA with k(cat)/Km 260 mM(-1) x 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.

Animals↗
PubMed2006

Amyloidogenic properties of the artificial protein albebetin and its biologically active derivatives. The role of electrostatic interactions in fibril formation.

The artificial protein albebetin (ABB) and its derivatives containing biologically active fragments of natural proteins form fibrils at physiological pH. The amyloid nature of the fibrils was confirmed by far UV circular dichroism spectra indicating for rich beta-structure, thioflavin T binding assays, and examination of the obtained polymers by atomic force microscopy. Fusing of short peptides--octapeptide of human alpha(2)-interferon (130-137) or hexapeptide HLDF-6 (41-46) of human leukemia differentiation factor--with the N-terminus of ABB led to increased amyloidogenicity of the protein: the rate of fibril formation increased and the morphology of fibrils became more complex. The presence of free hexapeptide HLDF-6 in the ABB solution had the same effect. Increasing ionic strength also activated the process of amyloid formation, but to less extent than did the peptides fused with ABB or added to the ABB solution. We suggest an important role of electrostatic interactions in formation of ABB fibrils. The foregoing ways (addition of salt or peptides) allow decrease in electrostatic repulsion between ABB molecules carrying large negative charge (-12) at neutral pH, thus promoting fibril formation.

Amyloid↗
PubMed2005

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 microM (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.

Animals↗
PubMed2001

Folding of circular permutants with decreased contact order: general trend balanced by protein stability.

To examine the influence of contact order and stability on the refolding rate constant for two-state proteins, we have analysed the folding kinetics of the small beta-alpha-beta protein S6 and two of its circular permutants with relative contact orders of 0.19, 0.15 and 0.12. Data reveal a small but significant increase of the refolding rate constant (log k(f)) with decreasing contact order. At the same time, the decreased contact order is correlated to losses in global stability and alterations of the folding nucleus. When the differences in stability are accounted for by addition of Na2SO4 or by comparison of the folding kinetics at the transition mid-point, the dependence between log k(f) and contact order becomes stronger and follows the general correlation for two-state proteins. The observation emphasizes the combined action of topology and stability in controlling the rate constant of protein folding.

Binding Sites↗
PubMed2000

Zn(2+)-mediated structure formation and compaction of the "natively unfolded" human prothymosin alpha.

Human recombinant prothymosin alpha (ProTalpha) is known to have coil-like conformation at neutral pH; i.e., it belongs to the class of "natively unfolded" proteins. By means of circular dichroism, SAXS, and ANS fluorescence, we have investigated the effect of several divalent cations on the structure of this protein. Results of these studies are consistent with the conclusion that ProTalpha conformation is unaffected by large excess of Ca(2+), Mg(2+), Mn(2+), Cu(2+), and Ni(2+). However, Zn(2+) induces compaction and considerable rearrangement of the protein structure. This means that ProTalpha can specifically interact with Zn(2+) (K(D) approximately 10(-3) M), and such interactions induce folding of the natively unfolded protein into a compact partially folded (premolten globule-like) conformation. It is possible that these structural changes may be important for the function of this protein.

Amino Acid Sequence↗
PubMed1999

Natively unfolded human prothymosin alpha adopts partially folded collapsed conformation at acidic pH.

Prothymosin alpha has previously been shown to be unfolded at neutral pH, thus belonging to a growing family of "natively unfolded" proteins. The structural properties and conformational stability of recombinant human prothymosin alpha were characterized at neutral and acidic pH by gel filtration, SAXS, circular dichroism, ANS fluorescence, (1)H NMR, and resistance to urea-induced unfolding. Interestingly, prothymosin alpha underwent a cooperative transition from the unfolded state into a partially folded conformation on lowering the pH. This conformation of prothymosin alpha is a compact denatured state, with structural properties different from those of the molten globule. The formation of alpha-helical structure by the glutamic acid-rich elements of the protein accompanied by the partial hydrophobic collapse is expected at lower pH due to the neutralization of the negatively charged residues. It is possible that such conformational changes may be associated with the protein function.

Circular Dichroism↗
PubMed1999

Structure and stability of recombinant protein depend on the extra N-terminal methionine residue: S6 permutein from direct and fusion expression systems.

Two permuted variants of S6 ribosomal protein were obtained in direct and fusion expression systems, respectively. The product of direct expression contained the extra N-terminal methionine residue. The structural properties and conformational stability of these permuteins were compared using 1-D (1)H-NMR, circular dichroism, intrinsic fluorescence, differential scanning calorimetry and resistance to urea-induced unfolding. A pronounced difference in all the parameters studied has been demonstrated. This means that the structure of recombinant protein can be sensitive to peculiarities of the expression and purification procedures, leading particularly to the presence or absence of the Met at the first position in the target protein sequence.

Calorimetry, Differential Scanning↗
PubMed1998

Can grafting of an octapeptide improve the structure of a de novo protein?

Structural properties and conformational stability of de novo proteins -- albebetin and albeferon (albebetin with a grafted interferon fragment) -- were studied by means of CD spectroscopy, gel filtration and urea-induced unfolding. The results allow us to conclude that albebetin possesses the properties of the molten globule state. Grafting of the octapeptide to the N-terminus of this de novo protein affects its structure. We show here that albeferon maintains a secondary structure content of albebetin; it becomes more compact and much more stable toward urea-induced unfolding as compared to albebetin and even possesses some weak tertiary structure (at least around Tyr7). This means that the structure of the artificial protein albebetin can be improved by a simple procedure of octapeptide grafting to its N-terminus.

Chromatography, Gel↗
PubMed1998

[Expression of mutant horse cytochrome c genes in Escherichia coli].

Here we describe genetically engineered constructs for the expression in Escherichia coli of genes for horse cytochrome c mutants. These constructs allow the expression of the cytochrome c genes together with hemeligase, an enzyme which covalently links heme to cytochrome. Careful selection of producer strains and the adjustment of the conditions of expression provided for expression levels of 10-15 mg of protein per liter of culture. This is by an order of magnitude greater than the expression previously achieved in yeast. A series of horse cytochrome c mutants were obtained in this way.

Animals↗
PubMed1997

S6 permutein shows that the unusual target topology is not responsible for the absence of rigid tertiary structure in de novo protein albebetin.

Ribosomal protein S6 from Thermus thermophilus was modified to form the unusual unique topology designed earlier for a de novo protein albebetin. The S6 gene was cloned, sequenced and circularly permutated by means of genetic engineering methods. The permutated gene was expressed in Escherichia coli and the permutein was isolated and investigated by means of circular dichroism, fluorescence spectroscopy and scanning microcalorimetry. The permutated protein revealed a pronounced secondary structure close to that of the wild type S6 protein and a rigid tertiary structure possessing cooperative temperature melting. It means that the unusual new topology of albebetin is compatible with a rigid tertiary structure, it may be realized in natural proteins and it is not responsible for the absence of rigid structure in albebetin.

Circular Dichroism↗
PubMed1996

The de novo protein with grafted biological function: transferring of interferon blast-transforming activity to albebetin.

The de novo protein albebetin has been designed recently to form a predetermined tertiary fold that has not yet been observed in natural proteins. An eight amino acid fragment (131-138) of human interferon alpha(2) carrying the blast-transforming activity of the protein was attached to the N-terminus of albebetin next to its initiatory methionine residue. The gene of chimeric protein was expressed in a wheat germ cell-free translation system and synthesized protein was tested for its compactness and stability. Its ability for receptor binding was also studied. We have shown that albebetin with attached octapeptide is practically as compact as natural proteins of corresponding molecular weight and possesses high stability toward the urea-induced unfolding. It binds murine thymocyte receptor at a high affinity and activates the thymocyte blast transformation efficiently at a concentration of 10(-11) M.

Amino Acid Sequence↗
PubMed1994

A new approach to artificial and modified proteins: theory-based design, synthesis in a cell-free system and fast testing of structural properties by radiolabels.

A novel approach to the creation of artificial and modified proteins has been elaborated. The approach includes a sequence design based on the molecular theory of protein secondary structure and folding patterns, gene expression in a cell-free system and testing of structural properties of the synthesized polypeptides at a nanogram level using radiolabelled chains. The approach has been applied to a new synthetic protein albebetin which has been designed to form a 3-D fold which does not contradict any structural rule but has been never observed up to now in natural proteins. Using size-exclusion chromatography, urea-gradient electrophoresis and limited proteolysis of a radiolabelled chain, it has been shown that the artificial protein is nearly as compact as natural proteins, cooperatively unfolds at high urea concentrations and has some structural features of a definite structure consistent with the designed one. As albebetin has been designed as consisting of two structural repeats, a 'half-albebetin' (one of these repeats) has also been synthesized and studied. It was shown that 'half-albebetin' is also compact.

Amino Acid Sequence↗
PubMed1992

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.

Amino Acid Sequence↗
PubMed1991

Expression of the envelope antigen F1 of Yersinia pestis is mediated by the product of caf1M gene having homology with the chaperone protein PapD of Escherichia coli.

The effective synthesis of the envelope antigen F1 of Y. pestis in E. coli HB101 is mediated by the expression of the caf1M gene. This gene was sequenced, and the protein encoded was found to have a significant homology with the chaperone protein PapD of uropathogenic E. coli. The data presented allow one to suppose Caf1M and PapD proteins perform similar functions in the biogenesis of the Y. pestis capsule and E. coli P-pili, respectively.

Adhesins, Escherichia coli↗
PubMed1985

Compact state of a protein molecule with pronounced small-scale mobility: bovine alpha-lactalbumin.

We describe a novel physical state of a protein molecule which is nearly as compact as the native state and has pronounced secondary structure, but differs from the native state by the large increase of thermal fluctuations (in particular, by the large mobility of side groups). This state has been characterized in detail for the acid form of bovine alpha-lactalbumin as a result of the study of physical properties of this state by a large variety of different methods (hydrodynamics, diffuse X-ray scattering, circular dichroism and infrared spectra, polarization of the luminescence, proton magnetic resonance, deuterium exchange and microcalorimetry). It has been shown that bovine alpha-lactalbumin can be transformed into a similar state by thermal denaturation. This process is thermodynamically two state (i.e. all-or-none transition), which means that this state differs from the native one by a phase transition of the first order.

Animals↗