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1.
Thermal denaturation and aggregation of apoform of glycogen phosphorylase b. Effect of crowding agents and chaperones
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Tatyana B. Eronina Natalia A. Chebotareva Svetlana G. Roman Sergey Yu. Kleymenov Valentina F. Makeeva Nikolay B. Poliansky Konstantin O. Muranov Boris I. Kurganov 《Biopolymers》2014,101(5):504-516
The effect of protein and chemical chaperones and crowders on thermal stability and aggregation of apoform of rabbit muscle glycogen phosphorylase b (apoPhb) has been studied at 37°C. Proline suppressed heat‐induced loss in ability of apoPhb to reconstitution at 37°C, whereas α‐crystallin did not reveal a protective action. To compare the antiaggregation activity of intact and crosslinked α‐crystallins, an adsorption capacity (AC) of a protein chaperone with respect to a target protein was estimated. This parameter is a measure of the antiaggregation activity. Crosslinking of α‐crystallin results in 11‐fold decrease in the initial AC. The nonlinear character of the relative initial rate of apoPhb aggregation versus the [intact α‐crystallin]/[apoPhb] ratio plot is indicative of the decrease in the AC of α‐crystallin with increasing the [α‐crystallin]/[apoPhb] ratio and can be interpreted as an evidence for dynamic chaperone structure and polydispersity of α‐crystallin–target protein complexes. As for chemical chaperones, a semisaturation concentration of the latter was used as a characteristic of the antiaggregation activity. A decrease in the semisaturation concentration for proline was observed in the presence of the crowders (polyethylene glycol and Ficoll‐70). © 2013 Wiley Periodicals, Inc. Biopolymers 101: 504–516, 2014. 相似文献
2.
Replication slippage may cause parallel evolution in the secondary structures of mitochondrial transfer RNAs 总被引:9,自引:4,他引:5
Presence of the dihydrouridine (D) stem in the mitochondrial cysteine tRNA
is unusually variable among lepidosaurian reptiles. Phylogenetic and
comparative analyses of cysteine tRNA gene sequences identify eight
parallel losses of the D-stem, resulting in D-arm replacement loops.
Sampling within the monophyletic Acrodonta provides no evidence for
reversal. Slipped-strand mispairing of noncontiguous repeated sequences
during replication or direct replication slippage can explain repeats
observed within cysteine tRNAs that contain a D-arm replacement loop. These
two mechanisms involving replication slippage can account for the loss of
the cysteine tRNA D-stem in several lepidosaurian lineages, and may
represent general mechanisms by which the secondary structures of
mitochondrial tRNAs are altered.
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3.
The complete cDNA sequence and protein reading frame of a developmentally
regulated hemocyanin subunit in the Dungeness crab (Cancer magister) is
presented. The protein sequence is aligned with 18 potentially homologous
hemocyanin-type proteins displaying apparent sequence similarities.
Functional domains are identified, and a comparison of predicted
hydrophilicities, surface probabilities, and regional backbone
flexibilities provides evidence for a remarkable degree of structural
conservation among the proteins surveyed. Parsimony analysis of the protein
sequence alignment identifies four monophyletic groups on the arthropodan
branch of the hemocyanin gene tree: crustacean hemocyanins, insect
hexamerins, chelicerate hemocyanins, and arthropodan prophenoloxidases.
They form a monophyletic group relative to molluscan hemocyanins and
nonarthropodan tyrosinases. Arthropodan prophenoloxidases, although
functionally similar to tyrosinases, appear to belong to the arthropodan
hexamer- type hemolymph proteins as opposed to molluscan hemocyanins and
tyrosinases.
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4.
Vera A. Borzova Kira A. Markossian Natalia A. Chebotareva Sergey Yu. Kleymenov Nikolay B. Poliansky Konstantin O. Muranov Vita A. Stein-Margolina Vladimir V. Shubin Denis I. Markov Boris I. Kurganov 《PloS one》2016,11(4)
Thermal aggregation of bovine serum albumin (BSA) has been studied using dynamic light scattering, asymmetric flow field-flow fractionation and analytical ultracentrifugation. The studies were carried out at fixed temperatures (60°C, 65°C, 70°C and 80°C) in 0.1 M phosphate buffer, pH 7.0, at BSA concentration of 1 mg/ml. Thermal denaturation of the protein was studied by differential scanning calorimetry. Analysis of the experimental data shows that at 65°C the stage of protein unfolding and individual stages of protein aggregation are markedly separated in time. This circumstance allowed us to propose the following mechanism of thermal aggregation of BSA. Protein unfolding results in the formation of two forms of the non-native protein with different propensity to aggregation. One of the forms (highly reactive unfolded form, Uhr) is characterized by a high rate of aggregation. Aggregation of Uhr leads to the formation of primary aggregates with the hydrodynamic radius (Rh,1) of 10.3 nm. The second form (low reactive unfolded form, Ulr) participates in the aggregation process by its attachment to the primary aggregates produced by the Uhr form and possesses ability for self-aggregation with formation of stable small-sized aggregates (Ast). At complete exhaustion of Ulr, secondary aggregates with the hydrodynamic radius (Rh,2) of 12.8 nm are formed. At 60°C the rates of unfolding and aggregation are commensurate, at 70°C the rates of formation of the primary and secondary aggregates are commensurate, at 80°C the registration of the initial stages of aggregation is complicated by formation of large-sized aggregates. 相似文献
5.
Krivandin AV Murugova TN Kuklin AI Muranov KO Poliansky NB Aksenov VL Ostrovsky MA 《Biochemistry. Biokhimii?a》2010,75(11):1324-1330
The structure of the oligomeric protein α-crystallin from bovine eye lens was investigated by small-angle neutron scattering
(SANS) with contrast variation. Based on the SANS curves, the match point for α-crystallin (43% D2O) and its average scattering length density at this point (2.4•1010 cm-2) were evaluated. The radius of gyration and the distance distri- bution functions for α-crystallin were calculated. On the
basis of these calculations, it was concluded that α-crystallin is characterized by homogeneous distribution of scattering
density in the domains inaccessible for water penetration, and all polypeptide subunits in α-crystallin oligomers undergo
equal deuteration. The latter indicates that all α-crystallin subunits are equally accessible for water and presumably for
some other low molecular weight substances. These conclusions on the α-crystallin structure (homogeneous distribution of scattering
density and equal accessibility of all subunits for low molecular weight substances) should be taken into account when elaborating
a-crystallin quaternary structure models. 相似文献
6.
K. O. Muranov N. B. Poliansky V. C. Kurova A. M. Riabokon N. L. Sheremet A. A. Fedorov K. I. Bannik A. N. Abrosimova M. A. Ostrovsky 《Biophysics》2010,55(5):870-877
Four randomized groups of male mice Fl (C57Black/CBA) were investigated: (a) UV-irraidated (UV-A, 15 min daily over 10 months,
51 ± 7 W/m2); (b) γ-ray irradiated (2 Gy), single; (c) influence of combination of UV- and γ-ray treatment; (d) aging. The lens opacities
were measured at the seventh and tenth month. An expert method based on a six-point scale was used for cataract measurement.
At the seventh month, the median of lens opacities were Aging group = 0; UV-irradiated group = 2.5; γ-irradiated group = 4.75;
γ- and UV-irradiated group = 6.0. The difference between all groups was significant (p < 0.004, Kruskall-Wallis ANOVA test). The Conover post hock test has shown a significant difference for all comparison pairs
(p < 0.002) with the exception of the γ- and UV-irradiated group. At the tenth month, the lens opacities strongly increased:
Aging group = 2.5; UV-irradiated group = 5.0; γ-irradiated group = 6.5; γ- and UV-irradiated group = 7.5 (median). The difference
between groups was significant (p < 0.0001, Kruskall-Willis ANOVA test). The Conover post hock test has shown a significant difference for all comparison pairs
(p < 0.003) with the exception of the UV-irradiated group and γ-irradiated group. No formation of specific lens opacities for
any group was found. Morphology and protein composition were investigated at the tenth month. The results of a study of morphological
changes show destructive and degenerative impairments of the capsule, epithelium cages, and lens fibers. However, no specific
changes related to certain particular actions have been found. In addition, there were no specific changes in the protein
composition of either water-soluble and water-insoluble fractions estimated by the differential gel electrophoresis technique.
The data mean that aging, UV treatment, and γ-radiation causes similar lens changes. It was supposed that UV treatment and/or
γ-radiation act as an aging factor on the lens. 相似文献
7.
Roman SG Chebotareva NA Eronina TB Kleymenov SY Makeeva VF Poliansky NB Muranov KO Kurganov BI 《Biochemistry》2011,50(49):10607-10623
The effect of crowding on the chaperone-like activity of α-crystallin has been studied using aggregation of UV-irradiated glycogen phosphorylase b (Phb) from rabbit skeletal muscle as an aggregation test system. The merit of this test system is the possibility of testing agents that directly affect the stage of aggregation of the protein molecules. It was shown that the solution of Phb denatured by UV contained aggregates with a hydrodynamic radius of 10.4 nm. These aggregates are relatively stable at 20 °C; however, they reveal a tendency to stick further in the presence of crowding agents. The study of the effect of α-crystallin on the aggregation of UV-irradiated Phb in the presence of the crowding agents by dynamic light scattering at 37 °C showed that under crowding conditions the antiaggregation ability of α-crystallin was weakened. On the basis of the analytical ultracentrifugation, size-exclusion chromatography, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis data, the scheme of interaction of UV-irradiated Phb and α-crystallin has been proposed. It is assumed that chaperone-target protein complexes of two types are formed, namely, the complexes of dissociated forms of α-crystallin with a protein substrate and high-mass α-crystallin-denatured protein complexes. The complexes of the first type reveal a weak propensity to aggregate even under crowding conditions. The complexes of the second type are characterized by the lower rate of aggregation in comparison with that of original UV-irradiated Phb. However, crowding stimulates the rate of aggregation of these complexes, resulting in the above-mentioned decrease in the chaperone-like activity of α-crystallin. 相似文献
8.
Mechanism of thermal aggregation of yeast alcohol dehydrogenase I: role of intramolecular chaperone 总被引:1,自引:0,他引:1
Markossian KA Golub NV Khanova HA Levitsky DI Poliansky NB Muranov KO Kurganov BI 《Biochimica et biophysica acta》2008,1784(9):1286-1293
Kinetics of thermal aggregation of yeast alcohol dehydrogenase I (yADH) have been studied using dynamic light scattering at a fixed temperature (56 degrees C) and under the conditions where the temperature was elevated at a constant rate (1 K/min). The initial parts of the dependences of the hydrodynamic radius on time (or temperature) follow the exponential law. At rather high values of time splitting of the population of aggregates into two components occurs. It is assumed that such peculiarities of the kinetics of thermal aggregation of yADH are due to the presence of a sequence -YSGVCHTDLHAWHGDWPLPVK- in the polypeptide chain possessing chaperone-like activity. Thermodynamic parameters for thermal denaturation of yADH have been calculated from the differential scanning calorimetry data. 相似文献
9.
Microsatellite allele frequencies in humans and chimpanzees, with implications for constraints on allele size 总被引:24,自引:6,他引:18
The distributions of allele sizes at eight simple-sequence repeat (SSR) or
microsatellite loci in chimpanzees are found and compared with the
distributions previously obtained from several human populations. At
several loci, the differences in average allele size between chimpanzees
and humans are sufficiently small that there might be a constraint on the
evolution of average allele size. Furthermore, a model that allows for a
bias in the mutation process shows that for some loci a weak bias can
account for the observations. Several alleles at one of the loci (Mfd 59)
were sequenced. Differences between alleles of different lengths were found
to be more complex than previously assumed. An 8-base-pair deletion was
present in the nonvariable region of the chimpanzee locus. This locus
contains a previously unrecognized repeated region, which is imperfect in
humans and perfect in chimpanzees. The apparently greater opportunity for
mutation conferred by the two perfect repeat regions in chimpanzees is
reflected in the higher variance in repeat number at Mfd 59 in chimpanzees
than in humans. These data indicate that interspecific differences in
allele length are not always attributable to simple changes in the number
of repeats.
相似文献
10.
Monoclonal antibodies against chicken type V collagen: production, specificity, and use for immunocytochemical localization in embryonic cornea and other organs 总被引:23,自引:17,他引:6
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TF Linsenmayer JM Fitch TM Schmid Zak NB E Gibney RD Sanderson R Mayne 《The Journal of cell biology》1983,96(1):124-132
Two monoclonal antibodies have been produced against chick type V collagen and shown to be highly specific for separate, conformational dependent determinants within this molecule. When used for immunocytochemical tissue localization, these antibodies show that a major site for the in situ deposition of type V is within the extracellular matrices of many dense connective tissues. In these, however, it is largely in a form unavailable to the antibodies, thus requiring a specific “unmasking” treatment to obtain successful immunocytochemical staining. The specificity of these two IgG antibodies was determined by inhibition ELISA, in which only type V and no other known collagen shows inhibition. In ELISA, mixtures of the two antibodies give an additive binding reaction to the collagen, suggesting that each is against a different antigenic determinant. That both antigenic determinants are conformational dependent, being either in, or closely associated with, the collagen helix is demonstrated by the loss of antibody binding to molecules that have been thermally denatured. The temperature at which this occurs, as assayed by inhibition ELISA, is very similar to that at which the collagen helix melts, as determined by optical rotation. This gives strong additional evidence that the antibodies are directed against the collagen. The antibodies were used for indirect immunofluorescence analyses of cryostat sections of corneas and other organs from 17 to 18-day-old chick embryos. Of all tissues examined only Bowman’s membrane gave a strong staining reaction with cryostat sections of unfixed material. Staining in other areas of the cornea and in other tissues was very light or nonexistent. When, however, sections were pretreated with pepsin dissolved in dilute HAc or, surprisingly, with the dilute HAc itself dramatic new staining by the antibodies was observed in most tissues examined. The staining, which was specific for the anti-type V collagen antibodies, was largely confined to extracellular matrices of dense connective tissues. Experiments using protease inhibitors suggested that the “unmasking” did not involve proteolysis. We do not yet know the mechanism of this unmasking; however, one possibility is that the dilute acid causes swelling or conformational changes in a type-V collagen-containing supramolecular structure. Further studies should allow us to determine whether this is the case. 相似文献