Biomedical subjects

V V Rogov

Publications and source records attributed to V V Rogov.

9 recordsLinked to original sources

PubMed2001

Solution structure and dynamics of the functional domain of Paracoccus denitrificans cytochrome c(552) in both redox states.

A soluble and fully functional 10.5 kDa fragment of the 18.2 kDa membrane-bound cytochrome c(552) from Paracoccus denitrificans has been heterologously expressed and (13)C/(15)N-labeled to study the structural features of this protein in both redox states. Well-resolved solution structures of both the reduced and oxidized states have been determined using high-resolution heteronuclear NMR. The overall protein topology consists of two long terminal helices and three shorter helices surrounding the heme moiety. No significant redox-induced structural differences have been observed. (15)N relaxation rates and heteronuclear NOE values were determined at 500 and 600 MHz. Several residues located around the heme moiety display increased backbone mobility in both oxidation states, while helices I, III, and V as well as the two concatenated beta-turns between Leu30 and Arg36 apparently form a less flexible domain within the protein structure. Major redox-state-dependent differences of the internal backbone mobility on the picosecond-nanosecond time scale were not evident. Hydrogen exchange experiments demonstrated that the slow-exchanging amide proton resonances mainly belong to the helices and beta-turns, corresponding to the regions with high order parameters in the dynamics data. Despite this correlation, the backbone amide protons of the oxidized cytochrome c(552) exchange considerably faster with the solvent compared to the reduced protein. Using both differential scanning calorimetry as well as temperature-dependent NMR spectroscopy, a significant difference in the thermostabilities of the two redox states has been observed, with transition temperatures of 349.9 K (76.8 degrees C) for reduced and 307.5 K (34.4 degrees C) for oxidized cytochrome c(552). These results suggest a clearly distinct backbone stability between the two oxidation states.

Calorimetry, Differential Scanning↗

PubMed1999

Secondary structure and oligomerization behavior of equilibrium unfolding intermediates of the lambda cro repressor.

The thermal unfolding of the wild-type Cro repressor, its disulfide-bridged mutant Cro-V55C (with the Val-55 --> Cys single amino acid substitution), and a CNBr-fragment (13-66)2 of Cro-V55C was studied by Fourier transform infrared spectroscopy and dynamic light scattering. The combined approach reveals that thermal denaturation of Cro-WT and Cro-V55C proceeds in two steps through equilibrium unfolding intermediates. The first thermal transition of the Cro-V55C dimer involves the melting of the alpha-helices and the short beta-strand localized in the N-terminal part of the molecule. This event is accompanied by the formation of tetramers, and also impacts on the hydrogen-bonding interactions of the C-terminal beta-strands. The beta-sheet formed by the C-terminal parts of each polypeptide chain is the major structural feature of the intermediate state of Cro-V55C and unfolds during a second thermal transition, which is accompanied by the dissociation of the tetramers. Cutting of 12 amino acids in the N-terminal region is sufficient to prevent the formation of alpha-helical structure in the CNBr-fragment of Cro-V55C, and to induce tetramerization already at room temperature. The tetramers may persist over a broad temperature range, and start to dissociate only upon thermal unfolding of the beta-sheet structure formed by the C-terminal regions. The wild-type protein is a dimer at room temperature and at protein concentrations of 1.8-5.8 mg/mL. At lower concentrations, the dimers are stable until the onset of thermal unfolding, which is accompanied by the dissociation of the dimers into monomers. At higher protein concentrations, the unfolding is more complex and involves the formation of tetramers at intermediate temperatures. At these intermediate temperatures, the Cro-WT has lost all of its alpha-helical structure and also most of its native beta-sheet structure. Upon further temperature increase, a tendency for an intermolecular association of the beta-strands is observed, which may result in irreversible beta-aggregation at high protein concentrations.

Amino Acid Substitution↗

PubMed1997

Cooperative thermal transitions of bovine and human apo-alpha-lactalbumins: evidence for a new intermediate state.

The thermal denaturation of bovine and human apo-alpha-lactalbumins at neutral pH has been studied by intrinsic protein fluorescence, circular dichroism (CD), and differential scanning microcalorimetry (DSC) methods. Apo-alpha-lactalbumin possesses a thermal transition with a midpoint about 25-30 degrees C under these conditions (pH 8.1, 10 mM borate, 1 mM EGTA), which is reflected in changes in both fluorescence emission maximum and quantum yield. However, the CD showed a decrease in ellipticity at 270 nm with a midpoint at about 10-15 degrees C, while DSC shows the transition within the region of 15-20 degrees C. The non-coincidence of transition monitored by different methods suggests the existence of an intermediate state in the course of the thermal denaturation process. This intermediate state is not the classical molten globule state which occurs at higher temperature (i.e. denatured state at these conditions) [D.A. Dolgikh, R.I. Gilmanshin, E.V. Brazhnikov, V.E. Bychkova, G.V. Semisotnov, S.Y. Venyaminov and O.B. Ptitsyn, FEBS Letters, 136 (1981) 311-315] and has physical properties intermediate between the native and molten globule states.

Animals↗

PubMed1996

Study of tyrosine-containing mutants of ribosomal protein L7/L12 from Escherichia coli.

Three mutant forms of the ribosomal protein L7/L12 with replacements of Ser1, Met14 and Met26 to Tyr were studied by the methods of fluorescence spectroscopy, circular dichroism and microcalorimetry. The amino-acid residue Tyr14 in the protein L7/L12 Tyr14 is located in a region with a more organized structure than Tyr26 in protein L7/L12 Tyr26. The replacements Ser1-->Tyr1 and Met14-->Tyr14 do not affect the secondary structure of protein L7/L12. The replacement Met26-->Tyr26 stabilizes the secondary structure of protein L7/L12. A pH-induced temperature transition was observed in the pH range 5.0-7.3 in protein L7/L12 Tyr14 by tyrosine fluorescence. Analogous transitions were observed for protein L7/L12 Tyr26 by Tyr fluorescence and for the wild type protein L7/L12 by Phe fluorescence. Three pH-dependent states of protein L7/L12 and its mutant forms L7/L12 Tyr1 and L7/L12 Tyr14 were found on the microcalorimetric melting curves. The characteristics of protein L7/L12 Tyr14 are very close to the wild type protein L7/L12 and it is a suitable object for studying the structure of the N-terminal part of molecule by two-dimentional 1H-NMR.

Bacterial Proteins↗

PubMed1996

Reversible association of the equilibrium unfolding intermediate of lambda Cro repressor.

An extended differentiated scanning calorimetry study of the wild-type Cro repressor and of its V55C mutant has revealed a significant concentration dependence of the melting profiles, even though the two polypeptide chains forming the active repressor molecule are covalently bound within the mutant. An analysis of the temperature dependencies of the partial molar heat capacity suggests that in both cases equilibrium unfolding occurs via a highly-populated intermediate state corresponding to polypeptide tetramers. The results of thermodynamic analysis are confirmed by direct glutaraldehyde cross-linking experiments. Judging by heat effects and circular dichroism data, this intermediate state regains about 50% of the ordered structure and melts co-operatively.

Bacteriophage lambda↗

PubMed1996

Circularly permuted dihydrofolate reductase possesses all the properties of the molten globule state, but can resume functional tertiary structure by interaction with its ligands.

It is obvious that functional activity of a protein molecule is closely related to its structure. On the other hand, the understanding of structure-function relationship still remains one of the intriguing problems of molecular biology. There is widespread belief that mutagenesis presents a real way to solve this problem. Following this assumption, we have investigated the effect of circular permutation in dihydrofolate reductase from E. coli on protein structure and functioning. It has been shown that in the absence of ligands two circularly permuted variants of dihydrofolate reductase possess all the properties of the molten globule state. However, after addition of ligands they gain the native-like structural properties and specific activity. This means that the in vitro folding of permuted dihydrofolate reductase is terminated at the stage of the molten globule formation. Interaction of permuted protein with ligands leads to the structural adjustment and formation of active protein molecules.

Animals↗

PubMed1992

Domains in lambda Cro repressor. A calorimetric study.

Thermodynamic properties of a mutant lambda Cro repressor with Cys replacing Val55 were studied calorimetrically. Formation of the S-S cross-link between neighboring Cys55 residues in this dimeric molecule leads to stabilization of a structure formed by the C-terminal parts of the two polypeptide chains, which behave as a single cooperative domain upon protein denaturation by heating. This composite domain is very stable at neutral pH and disrupts at 110 degrees C. The S-S-cross-linked tryptic fragment (residues 22-66), which includes this C-terminal domain, has similar stability. The N-terminal parts of the polypeptide chains do not form any stable structure when isolated, but in S-S-cross-linked dimer, they form a single cooperative block which melts in an all-or-none way 9 degrees C higher than the un-cross-linked protein. The observed cooperation of the distant N-terminal parts in dimer raises questions regarding lambda Cro repressor structure in solution.

Calorimetry↗

PubMed1991

Two-stage thermal unfolding of [Cys55]-substituted Cro repressor of bacteriophage lambda.

It has been shown by scanning calorimetry and 1H NMR spectroscopy that thermal denaturation of mutant lambda phage cro repressor in which Val55 was substituted for Cys, proceeds in 2 stages in contrast to the wild type protein. At neutral pH values, an additional cooperative transition has been observed at about 100 degrees C. Calorimetric measurements on the mutant and its tryptic fragment lead to the conclusion that the two-stage character of thermal unfolding of the mutant is due to a disruption of an additional cooperative domain in the dimer molecule which is stabilized by the S-S crosslink.

Bacteriophage lambda↗

PubMedDate not supplied

[Calorimetric studies of the effect of amino acid replacements 16Gln-Leu and 26Tyr-Asp on the structural organization and stability of the Cro-repressor from phage lambda].

The scanning calorimetry technique has been used to study the influence of amino acid substitutions on thermodynamic parameters of formation and stabilization of a cooperative structure of Cro repressor of bacteriophage lambda molecule. It is shown that substitutions 16Gln-Leu and 26Tyr-Asp enhances the protein molecule stability by 32 degrees C as compared with the wild-type protein. It is also demonstrated that the denaturation enthalpy of the mutated Cro differs slightly from that of the wild-type protein at the same temperature, while the effective enthalpy value is significantly lower. The analysis of excess heat capacity of the mutated protein shows that this complex function determined experimentally is approximated by two functions, which is indicative of the presence of two quasi-independent transitions. Thus, the stabilization and redistribution of intermolecular interactions evoked by the above substitutions leads to disintegration of the single cooperative repressor molecule in two interacting cooperative domains. The plausible mechanism of formation of such a domain structure is discussed on the basis of the available calorimetric data.

Amino Acids↗