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N A Rodionova

Publications and source records attributed to N A Rodionova.

At least 19 recordsLinked to original sources
PubMed2001

A novel peptide recognition mode revealed by the X-ray structure of a core U2AF35/U2AF65 heterodimer.

U2 auxiliary factor (U2AF) is an essential splicing factor that recognizes the 3' splice site and recruits the U2 snRNP to the branch point. The X-ray structure of the human core U2AF heterodimer, consisting of the U2AF35 central domain and a proline-rich region of U2AF65, has been determined at 2.2 A resolution. The structure reveals a novel protein-protein recognition strategy, in which an atypical RNA recognition motif (RRM) of U2AF35 and the U2AF65 polyproline segment interact via reciprocal "tongue-in-groove" tryptophan residues. Complementary biochemical experiments demonstrate that the core U2AF heterodimer binds RNA, and that the interacting tryptophan side chains are essential for U2AF dimerization. Atypical RRMs in other splicing factors may serve as protein-protein interaction motifs elsewhere during spliceosome assembly.

Amino Acid Sequence↗
PubMed2001

Biophysical analysis of the endoplasmic reticulum-resident chaperone/heat shock protein gp96/GRP94 and its complex with peptide antigen.

Animals vaccinated with heat shock protein (HSP)--peptide complexes develop specific protective immunity against cancers from which the HSPs were originally isolated. This autologous specific immunity has been demonstrated using a number of HSP--peptide antigen complexes. A prototypical HSP-based cancer vaccine is the gp96--peptide antigen complex, which is currently undergoing human clinical trials. Here, we analyzed the structure of a recombinant wild-type and a mutant gp96 protein and their peptide complexes using a number of biophysical techniques. Gel filtration chromatography, dynamic light scattering, and equilibrium analytical ultracentrifugation demonstrated that both a wild-type gp96 and a gp96 mutant lacking a dimerization domain formed higher order structures. More detailed analysis using scanning transmission electron microscopy indicated that both the wild-type and dimerization deletion mutant gp96 protein were organized, unexpectedly, into large aggregates. Size distributions ranged from dimers to octamers and higher. Circular dichroism and intrinsic Trp fluorescence suggested that the gp96 dimerization domain deletion mutant protein was more compact than the wild-type gp96. A fluorescent peptide antigen was synthesized, and the peptide-binding properties of wild-type and the dimerization domain deletion mutant gp96 were studied. Fluorescence lifetime and anisotropy decay showed that the bound antigenic peptide was located in a hydrophobic pocket, with considerable free space for the rotation of the probe. Deletion of the dimerization domain affected the peptide-binding microenvironment, although peptide-binding affinity was reduced by only a small extent. Peptide--gp96 complexes were extremely stable, persisting for many days in the cold. The extraordinary stability of peptide--gp96 complexes and the plasticity of the peptide-binding pocket support the proposed relay of diverse peptides to MHC and/or other molecules via molecular recognition.

Animals↗
PubMed1997

[Denatured transitions of the molecular chaperone GroEL from Escherichia coli].

Conformational changes of oligomeric particle of GroEL chaperone from E. coli in solution were studied, which proceed during its denaturation upon the action of elevated urea concentration, temperature, and extremal pH values by the methods of CD, light scattering, scanning microcalorimetry, hydrophobic probe binding, and ATPase activity measurements. The ranges of changing the external conditions; within which GroEL retains its structure and functions, were determined. Denaturation transitions were found to be cooperative, pronounced, and irreversible. In the pH range from 6.0 to 9.6, the three-step change of the ATPase activity of GroEL was shown to occur with half-transition pH1/2 of 6.3, 8.5, and 9.3. It does not result in any essential structural changes and is probably associated with a protonation/deprotonation of amino acid residues important for the GroEL ATPase activity.

Adenosine Triphosphatases↗
PubMed1995

Characterization of two membrane-bound forms of OmpA.

The insertion of the outer membrane protein A (OmpA) into lipid bilayers was studied by limited proteolysis, polarized Fourier transform infrared (FTIR) spectroscopy, and fluorescence spectroscopy. In the native state, OmpA is thought to form a barrel of eight antiparallel beta-strands. For the present study, it was isolated in an unfolded form, purified, and exposed to performed vesicles of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), and three phospholipids that were brominated in different positions of their sn-2 chains (4,5-BrPC, 9,10-BrPC, and 11,12-BrPC). Limited proteolysis revealed two membrane-bound forms of OmpA, namely an "adsorbed" (35 kDa) and an "inserted" (30 kDa) form [Surrey, T., & Jähnig, F. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 7457-7461]. Which form was found after membrane binding and refolding depended on the lipids used and on the temperature. Polarized attenuated total reflection (ATR)-FTIR spectra were recorded with OmpA bound to germanium-supported bilayers in both forms. The position of the amide I' band indicated quite large fractions of beta-structure of OmpA in both membrane-bound forms (35-45% in the adsorbed form and 45-55% in the inserted form). Measurements of the linear dichroism of the amide I' bands in the inserted form are consistent with an antiparallel beta-barrel in which the strands are inclined at about 36 degrees from the membrane normal. The average angle of the beta-strands to the bilayer normal is likely larger in the 35 kDa form than in the inserted form.(ABSTRACT TRUNCATED AT 250 WORDS)

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1991

Study of the "molten globule" intermediate state in protein folding by a hydrophobic fluorescent probe.

Binding of the hydrophobic fluorescent probe, 1-anilino-naphthalene-8-sulfonate (ANS), to synthetic polypeptides and proteins with a different structural organization has been studied. It has been shown that ANS has a much stronger affinity to the protein "molten globule" state, with a pronounced secondary structure and compactness, but without a tightly packed tertiary structure as compared with its affinity to the native and coil-like proteins, or to coil-like, alpha-helical, or beta-structural hydrophilic homopolypeptides. The possibility of using ANS for the study of equilibrium and kinetic molten globule intermediates is demonstrated, with carbonic anhydrase, beta-lactamase, and alpha-lactalbumin as examples.

Anilino Naphthalenesulfonates↗
PubMed1990

Two slow stages in refolding of bovine carbonic anhydrase B are due to proline isomerization.

Kinetics of refolding of bovine carbonic anhydrase B have been studied by the "double-jump" technique (i.e. the dependence of protein refolding on delay time in the unfolded state after fast unfolding). It is shown that two stages (the slow with a relaxation time of t1/2 approximately equal to 120 s and the superslow with t1/2 approximately equal to 600 s) observed during refolding of bovine carbonic anhydrase B are due to trans-cis isomerization of proline residues. The dependences of rate constants of these processes on temperature and on the final denaturant concentration were measured. Activation energies of both processes are the same, Ea = 18(+/- 2) kcal/mol. The rate constants of protein refolding do not depend on the final concentration of urea under native conditions. In addition, the rate of isomerization of essential proline residues in the "molten globule" intermediate state of bovine carbonic anhydrase was measured and found to be equal to that for unstructural polypeptides. The effect of several proline residues on carbonic anhydrase refolding is discussed.

Animals↗
PubMed1987

Sequential mechanism of refolding of carbonic anhydrase B.

The kinetics of refolding of bovine carbonic anhydrase B was studied by a variety of methods over a wide range of times (from milliseconds to hours). It has been shown that protein refolding proceeds through three stages. At the first stage (t1/2 approximately equal to 0.03 s) hydrophobic clusters and a compact state of the chain are formed. At the second stage (t1/2 approximately equal to 140 s) hydrophobic clusters are desolvated and the rigid native-like hydrophobic core is formed. At the third stage (t1/2 approximately equal to 600 s) the native active protein is formed.

Animals↗
PubMed1987

beta-Glucosidases from cellulolytic fungi Aspergillus terreus, Geotrichum candidum, and Trichoderma longibrachiatum as typical glycosidases.

By ethanol precipitation (v/v) and chromatography on Sephadex SP, DEAE (or DEAE-cellulose), and G-200 beta-glucosidases (EC 3.2.1.21) from the culture filtrates of cellulolytic fungi Aspergillus terreus, Geotrichum candidum, and Trichoderma longibrachiatum grown on the medium with cellulose containing materials were isolated. The enzymes were homogenous as shown by different techniques. The substrate specificities of the obtained enzymes were studied. beta-Glucosidases had higher affinity for p-nitrophenyl-beta-D-glucopyranoside than for cellobiose (Km 1.25, 0.34, 0.20 and 5.4, 2.0, 1.2 mM, respectively) and were able to hydrolyze both laminaribiose and gentiobiose; but they were unable to cleave cotton fiber, carboxymethylcellulose, and other glycans to reducing sugars. They showed transglycosylase activity. Ki values for arylglucosidase activity of beta-glucosidases from A. terreus, G. candidum, and T. longibrachiatum in the presence of either glucose or glucono-1,5-lactone were 12.2, 6.0, 2.1 and 0.20, 0.19, 0.07 mM, respectively. The Mr's were estimated by gel filtration and by sedimentation equilibrium centrifugation to 200,000, 200,000, 350,000, respectively. The isoelectric points of beta-glucosidases were 4.8, 5.9, and 4.2, respectively. The optimum temperatures and pH's were 60, 50, and 50 degrees C and at pH 4.5, 4.5, and 4.8-5.7, respectively. These properties appear to relate beta-glucosidases obtained in the present study to typical glycosidases.

Aspergillus↗
PubMed1982

[Properties of beta-glucosidase from the cellulolytic fungus Geotrichum candidum 3c].

beta-Glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) splitting p-nitrophenyl-beta-D-glucosidase was isolated from cellular preparation of the fungus Geotrichum candidum 3c "cellocandine G10x" and purified 38-fold. The enzyme was homogeneous during ultracentrifugation, gel-filtration, isoelectrofocusing and disc-electrophoresis in polyacrylamide gel and had a pI of 4.2, sedimentation coefficient of 2.6S and molecular weight of 120 000. The enzyme had maximal activity at pH 5.6, 45 degrees and retained up to 45% of its activity under optimal conditions (pH, t degrees) after 48 hr incubation. beta-Glucosidase did not split disaccharides, e. g. lactose cellobiose, laminaribiose, gentibiose as well as o-nitrophenyl-beta-D-galactoside, methyl-beta-D-xyloside. The enzyme hydrolyzed p-nitrophenyl-beta-D-glucopyranoside (Km = 0.18 mM) and some plant beta-glucosides, such as phenylethyl-beta-D-glucopyranoside from rose petals and diosgenine tetrasaccharide from deltoid dioscorea (Km = 0.26 mM).

Cellulase↗
PubMed1977

[beta-Glucosidases from fungus Geotrichum candidum].

beta-Glucosidases from Geotrichum candidum 3C cellulase preparation were separated from C1 enzymes and beta-1,4-glucanases by means of DEAE-Sephadex A-50 chromatography, gel filtration through P-150 Biogel and chromatography on CM-cellulose, and then were fractionated by isoelectric focusing using carrier ampholites with pH ranges 3-6 and 4-6. beta-Glucosidases with pI 3.8, 4.2, 4.6, 5.1, 5.6 and 6.2 were found in cellulase preparation from G. candidum 3C. Molecular weight of beta-glucosidases with pI 3.8, 4.2, 4.6 and 6.2, isolated under isoelectric focusing, were estimated by means of gel filtration through Sephadex G-200 to be 35000, 123000, 188000 and 223000 respectively. beta-Glucosidases with pI 3.8, 4.6, 5.6 and 6.2 hydrolyzed cellobiose and did not attack p-nitrophenyl-beta-D-glucopyranoside; those with pI 4.2 and 5.6 hydrolyzed p-nitrophenyl-beta-D-glucopyranoside and plant glucoside, protodioscin, and did not split cellobiose. All the beta-glucosidases studied did not hydrolyze laminaribose, beta-D-methylsylopyranoside, alder O-methylglucuronoxylane, o-nitrophenyl-beta-D-galactopyranoside and p-nitrophenyl-alpha-D-glucopyranoside. beta-Cellobiase with pI 6.2 hydrolzed lactoses, cellobioses with pI 3.8 and pI 5.6 splited gentiobiose. beta-Glucosidase with pI 4.6 did not attack any substrate studied, except cellobiose.

Cellulase↗
PubMed1977

[Fractionation and purification of endo-1,4-beta-xylanases and exo-1,4-beta-xylosidases of Aspergillus niger].

Two endo-1,4-beta-zylanases (m. w. 24,000 and 41,000) and six exo-1,4-beta-xylosidases, differing in their molecular weights and isoelectric points, were found in a xylanase preparation from Aspergillus niger, using different methods of fractionation. An electrophoretically homogeneous exo-1,4-beta-xylosidase (m. w. 30,000) purified 120-fold, with pI 4.6, having optimal effect on methyl-beta-D-xyloside at pH 3.0 was obtained. Exo-1,4-beta-xylosidase splits off xylose from the ends of the xylan chains at xylotriose, xylobiose and methyl-beta-D-xyloside and is characterized by a high transglycosilase activity. An electrophoretically homogeneous endo-1,4-beta-xylanase (m. w. 24,000) purified 250-fold, with pI 4.2 and optimal effect on carboxymethylxylan at pH 4.2 was isolated. Endo-1,4-beta-xylanase splits arabinoglucuronoxylan to form xylooligosaccharides; however, it does not hydrolyze xylobiose.

Aspergillus↗