[Limited Trypsinolysis of GroES: The Effect on the Interaction with GroEL and Assembly In Vitro].

GroES is a heptameric partner of tetradecameric molecular chaperone GroEL, which ensures the correct folding and assembly of numerous cellular proteins both in vitro and in vivo. This work demonstrates the results of a study of structural aspects of GroES that affect its interaction with GroEL and reassembly. The effect of limited trypsinolysis of GroES on these processes has been studied. It has been shown that limited trypsinolysis of GroES is only strongly pronounced outside the complex with GroEL and results in the cleavage of the peptide bond between Lys20 and Ser21. The N-terminal fragment (~2 kDa) is retained in the GroES particle, which maintains its heptaoligomeric structure but loses the ability to interact with GroEL and dissociates upon a change in the pH from 7 to 8. Trypsin-nicked GroES cannot reassemble after urea-induced unfolding, while the urea-induced unfolding of intact GroES is fully reversible. The reported results indicate the important role of the N-terminal part of GroES subunit in the assembly of its heptameric structure and the interaction with GroEL.

[Influence of sociohygienic factors on the shaping of the nutritional status in children and teenagers].

There was revealed the structure of deteriorations in the nutritional status of schoolchildren in the city: the most of students has normal nutritional status, but there was noted the high prevalence of excessive body weight and obesity among children and teenagers. Risk factors for development of deteriorations of the nutrition state were detected as follows: irrational food regimen, qualitative compartment offood, factors of educational environment, lifestyle. The main role in system of control of the nutritional status in children is referred to the correction of socio-hygienic factors which prove to be the priority ones in the shaping of the nutritional status in students. As the main condition determining the nutrition state of the up-to-date schoolchildren and the quality of their life in the whole the social cultural level of children and adolescents must be regarded as a result of the hygienic education and training in fundamentals of healthy lifestyle. Priority protective factors of the gain in the part of schoolchildren with normal nutritional status (optimalfood regimen, optimal dietary habits, sufficient level of physical activity) laidfrom the child age in conditions of the family, sufficient level of the physical activity and the implementation of the other element of hygienically expedient day regimen served as the base for the elaboration of the system of the control of nutritional status. Algorithm of the control of the nutritional status in the students of educational institutions includes the creation of healthcare educational environment, optimization of nutrition and physical activity, the shaping of the culture of healthy lifestyle, health-improving measures for children with disorders of nutritional status and their psychological pedagogical supports at the stage of the correction of the nutritional status, improvement of the medical service for the early detection of deviations of nutritional status with the estimation of the efficiency of the system ofpreventive and health-improving measures.

Phylogenetic analysis of variants of the Puumala virus (Hantaviridae: Orthohantavirus) circulating in the Saratov region.

The objective is to determine the complete nucleotide sequence and conduct a phylogenetic analysis of genome variants of the Puumala virus isolated in the Saratov region. MATERIALS AND METHODS: The samples for the study were field material collected in the Gagarinsky (formerly Saratovsky), Engelssky, Novoburassky and Khvalynsky districts of the Saratov region in the period from 2019 to 2022. To specifically enrich the Puumala virus genome in the samples, were used PCR and developed a specific primer panel. Next, the resulting PCR products were sequenced and the fragments were assembled into one sequence for each segment of the virus genome. To construct phylogenetic trees, the maximum parsimony algorithm was used. RESULTS: Genetic variants of the Puumala virus isolated in the Saratov region have a high degree of genome similarity to each other, which indicates their unity of origin. According to phylogenetic analysis, they all form a separate branch in the cluster formed by hantaviruses from other subjects of the Volga Federal District. The virus variants from the Republics of Udmurtia and Tatarstan, as well as from the Samara and Ulyanovsk regions, are closest to the samples from the Saratov region. CONCLUSION: The data obtained show the presence of a pronounced territorial confinement of strains to certain regions or areas that are the natural biotopes of their carriers. This makes it possible to fairly accurately determine the territory of possible infection of patients and/or the circulation of carriers of these virus variants based on the sequence of individual segments of their genome.

Molecular chaperone GroEL/ES: unfolding and refolding processes.

Molecular chaperones are a special class of heat shock proteins (Hsp) that assist the folding and formation of the quaternary structure of other proteins both in vivo and in vitro. However, some chaperones are complex oligomeric proteins, and one of the intriguing questions is how the chaperones fold. The representatives of the Escherichia coli chaperone system GroEL (Hsp60) and GroES (Hsp10) have been studied most intensively. GroEL consists of 14 identical subunits combined into two interacting ring-like structures of seven subunits each, while the co-chaperone GroES interacting with GroEL consists of seven identical subunits combined into a dome-like oligomeric structure. In spite of their complex quaternary structure, GroEL and GroES fold well both in vivo and in vitro. However, the specific oligomerization of GroEL subunits is dependent on ligands and external conditions. This review analyzes the literature and our own data on the study of unfolding (denaturation) and refolding (renaturation) processes of these molecular chaperones and the effect of ligands and solvent composition. Such analysis seems to be useful for understanding the folding mechanism not only of the GroEL/GroES complex, but also of other oligomeric protein complexes.

[Kinetic and equilibrium parameters of erythromycin sorption on various sorbents].

Optimal conditions for erythromycin sorption on supercoupled isoporous Purolight sorbents (styrosorbs) S-106, S-150 and S-160 and on macroporous sulfocation exchanger KU-23 were determined. High selectivity of erythromycin sorption on the supercoupled isoporous cation exchanger S-106 was shown, that was evident from the high distribution coefficient. The effective diffusion coefficient on the cation exchanger S-106 was higher by a factor of 10(2) (1.1 x 10(-11) m2/s) that was in favour of a comparatively high diffusion rate.

Multy-state protein: Determination of carbonic anhydrase free-energy landscape.

Studies of the folding pathway of large proteins whose kinetics is complicated due to the formation of several intermediate states are most frequently impeded or totally impossible because of rapid folding phase occurring during instrument dead time. In this paper the obtaining of energy characteristics of one of such proteins-carbonic anhydrase B-is reported. Tryptophan fluorescence and absorption methods have been used to measure the folding and unfolding kinetics of carbonic anhydrase B at different urea concentrations. In spite of the fact that the formation of the initial intermediate state of this protein takes place during the instrument dead time, the population of this state has been estimated in a wide range of urea concentrations. The use of the population of the rapidly formed intermediate state and the effective rates of slow phases of the protein folding/unfolding permitted us to calculate free energies of all the protein states and the height of energy barriers between them. It has been shown that folding of carbonic anhydrase B can be described by a consecutive reaction scheme. The possibility to obtain energy characteristics of carbonic anhydrase would allow studying structural characteristics of both intermediate and transition states via site-directed mutations.

Dataset concerning GroEL chaperonin interaction with proteins.

GroEL chaperonin is well-known to interact with a wide variety of polypeptide chains. Here we show the data related to our previous work (http://dx.doi.org/10.1016/j.pep.2015.11.020[1]), and concerning the interaction of GroEL with native (lysozyme, α-lactalbumin) and denatured (lysozyme, α-lactalbumin and pepsin) proteins in solution. The use of affinity chromatography on the base of denatured pepsin for GroEL purification from fluorescent impurities is represented as well.

Protein globularization during folding. A study by synchrotron small-angle X-ray scattering.

Various conformational states of polypeptide chains were investigated by synchrotron small-angle X-ray scattering (SAXS). SAXS patterns of proteins and model polypeptides in globular states (native and "molten globule") and in non-globular states (unfolded protein as well as randomly coiled, partially alpha-helical and partially beta-structural synthetic polypeptides) were analyzed in terms of Guinier and Kratky plots. Large differences in the SAXS pattern have been found between globular and non-globular conformations of the polypeptide chains, and they have been interpreted in terms of differences in the shape and size of the globular and non-globular scatterers with the same molecular mass. The equilibrium and time-resolved unfolding curves of bovine carbonic anhydrase and yeast phosphoglycerate kinase were monitored by integrated SAXS intensity, and were found to be coincident with the curves measured by other physicochemical techniques, such as tryptophan fluorescence and peptide circular dichroism spectra. The intermolecular association of the protein "molten globule"-like intermediates accumulated during the guanidine hydrochloride-induced unfolding of bovine carbonic anhydrase has been investigated by various SAXS parameters. It has been shown that the integrated SAXS intensity is much less sensitive to the protein intermolecular association than the zero angle intensity and the radius of gyration. We propose the integrated SAXS intensity as a global parameter which is particularly appropriate for fast kinetic studies of protein coil to globule transitions. Time-resolved refolding curves of the above proteins were monitored by the integrated SAXS intensity to investigate the globularization process in protein folding. Two fast kinetic processes for bovine carbonic anhydrase and two fast (each within two seconds) as well as two slow (within 500 seconds) kinetic processes for yeast phosphoglycerate kinase have been recorded. The kinetic processes reflect both protein intramolecular globularization and its intermolecular association.

Ligands regulate GroEL thermostability.

Escherichia coli heat-shock proteins GroEL and GroES stimulate (in an ATP-dependent manner) the folding of various proteins. In this study scanning microcalorimetry was applied to investigate GroEL thermostability in the presence of its ligands. Mg2+ and K+ ions stabilize while ADP destabilizes the GroEL molecule against the action of temperature. Furthermore, ADP essentially increases the number of binding sites for the hydrophobic probe (ANS) and the number of GroEL SH-groups accessible to Ellman's reagent as well as the accessibility of the protein to the action of trypsin. The interaction of GroEL with GroES in the presence of Mg2+-ADP eliminates the destabilizing effect of ADP on the GroEL molecule against the action of temperature and Ellman's reagent but does not change its hydrophobicity and accessibility to trypsin.

[Monomeric form of the molecular chaperone GroEL: structure, stability, and oligomerization]. / Monomernaia forma molekuliarnogo shaperona GroEL: struktura, stabil'nost' i oligomerizatsiia.

The structure and stability in solution of the monomeric form of GroEL were studied by the methods of circular dichroism, binding of a hydrophobic probe, limited proteolysis, modification of thiol groups, sedimentation, and size-exclusion chromatography. The monomeric GroEL at 23 degrees C was shown to be a globular protein with a pronounced secondary and a rigid tertiary structure. It exhibited no marked tendency to oligomerization in the absence of adenine nucleotides. However, the free monomeric GroEL was substantially less stable to urea and heat than the corresponding subunit in the composition of native oligomeric particles. The monomeric form also bound the hydrophobic probe, 8-anilino-1-naphthalenesulfonic acid, by an order of magnitude better than the subunit in the oligomeric particles. The ATP-induced oligomerization process of both folded and unfolded GroEL monomers was studied. The oligomerization rate was found to be the same for both monomers, and, therefore, should be limited by the ATP-dependent "arrangement" of the sites in the folded monomers responsible for the oligomerization rather than by the spontaneous refolding of monomers.

[Denatured transitions of the molecular chaperone GroEL from Escherichia coli]. / Denaturatsionnye perekhody molekulianogo shaperona GroEL iz Escherichiacoli.

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.

[Biosynthesis and conformational state of 17-kDa and 27-kDa N-terminal fragments of elongation factor EF-2 in solution]. / Biosintez i konformationnoe sostoianie v rastvore 17-kDa- i 27-KDa- N-kontsevykh fragmentov faktora élongatsii EF-2.

N-Terminal fragments of the rat liver elongation factor EF-2 containing 162 (17 kDa) and 244 (27 kDa) amino acid residues of 857 (95 kDa) residues of the native protein were synthesized in E. coli cells and in a wheat germ cell-free translation system, and their conformations were studied. Both fragments were synthesized as inclusion bodies (nonspecific molecular aggregates). The conformations of the fragments in a solution were studied at neutral pH values by CD, fluorescence spectroscopy, scanning microcalorimetry, viscosimetry, gel-filtration, limited proteolysis, and interaction with monospecific anti-EF-2 antibodies and GroEL/ES molecular chaperone. Under nondenaturing conditions, both fragments existed in a solution as associates within a broad range of molecular masses, contained a considerable amount of elements of the intramolecular secondary structure, and represented globules without rigid tertiary structure (molten globules). A rigid tertiary structure was not formed even after the interaction of the fragments with the GroEL/ES molecular chaperone, thus indicating that the C-terminal fragment is essential for the formation of the rigid tertiary structure. Both fragments contained conformational antigenic determinants similar to those in the whole protein; i.e., despite the absence of the rigid tertiary structure, the fragments contained elements whose structure was similar to that of the corresponding regions in the whole protein.

[The interaction of the GroEL chaperone with early kinetic intermediates of renaturing proteins inhibits the formation of their native structure]. / Vzaimodeistvie shaperona GroEL s rannimi kineticheskimi promezhutochnymi sostoianiiami renaturiruiushchikh belkov ingibiruet formirovanie ikh nativnoi struktury.

The main function of the chaperone GroEL is to prevent nonspecific association of nonnative protein chains and provide their correct folding. In the present work, the renaturation kinetics of three globular proteins (human alpha-lactalbumin, bovine carbonic anhydrase, and yeast phosphoglycerate kinase) in the presence of different molar excess of GroEL (up to 10-fold) was studied. It was shown that the formation of the native structure during the refolding of these proteins is retarded with an increase in GroEL molar excess due to the interaction of kinetic protein intermediates with the chaperone. Mg(2+)-ATP and Mg(2+)-ADP weaken this interaction and decrease the retarding effect of GroEL on the protein refolding kinetics. The theoretical modeling of protein folding in the presence of GroEL showed that the experimentally observed linear increase in the protein refolding half-time with increasing molar excess of GroEL must occur only when the protein adopts its native structure outside of GroEL (i.e. in the free state), while the refolding of the protein in the complex with GroEL is inhibited. The dissociation constants of GroEL complexed with the kinetic intermediates of the proteins studied were evaluated, and a simple mechanism of the functioning of GroEL as a molecular chaperone was proposed.

[The dynamic physical development of schoolchildren in Nizhni Novgorod]. / Dinamika fizicheskogo razvitiia shkol'nikov Nizhnego Novgoroda.

In the past 55 years, the trends in the physical development of school children of a large industrial city have been in agreement with the global ones in the growth and development of children and adolescents. In the past decade, there have been unfavourable tendencies in the physical development of schoolchildren. The physical development of children is an indicator of the social and economic status, living conditions, and the environment. Follow-ups should be an obligatory element of monitoring the population's health.

Affinity chromatography of GroEL chaperonin based on denatured proteins: role of electrostatic interactions in regulation of GroEL affinity for protein substrates.

The chaperonin GroEL of the heat shock protein family from Escherichia coli cells can bind various polypeptides lacking rigid tertiary structure and thus prevent their nonspecific association and provide for acquisition of native conformation. In the present work we studied the interaction of GroEL with six denatured proteins (alpha-lactalbumin, ribonuclease A, egg lysozyme in the presence of dithiothreitol, pepsin, beta-casein, and apocytochrome c) possessing negative or positive total charge at neutral pH values and different in hydrophobicity (affinity for a hydrophobic probe ANS). To prevent the influence of nonspecific association of non-native proteins on their interaction with GroEL and make easier the recording of the complexing, the proteins were covalently attached to BrCN-activated Sepharose. At low ionic strength (lower than 60 mM), tight binding of the negatively charged denatured proteins with GroEL (which is also negatively charged) needed relatively low concentrations (approximately 10 mM) of bivalent cations Mg2+ or Ca2+. At the high ionic strength (approximately 600 mM), a tight complex was produced also in the absence of bivalent cations. In contrast, positively charged denatured proteins tightly interacted with GroEL irrespectively of the presence of bivalent cations and ionic strength of the solution (from 20 to 600 mM). These features of GroEL interaction with positively and negatively charged denatured proteins were confirmed by polarized fluorescence (fluorescence anisotropy). The findings suggest that the affinity of GroEL for denatured proteins can be determined by the balance of hydrophobic and electrostatic interactions.

Reactivation of 3-phosphoglycerate kinase from its unfolded proteolytic fragments.

Limited trypsinolysis of pig muscle 3-phosphoglycerate kinase yielded a nicked enzyme without loss of catalytic activity [Jiang, S. X. & Vas, M. (1988) FEBS Lett. 231, 151-154]. The reactivation rate of the nicked enzyme after denaturation does not differ substantially from the reactivation rate of the denatured intact enzyme: t 1/2 varies between 70-110 s at 25 degrees C, pH 7.0 in both cases. Thus, the absence of a covalent linkage between the two proteolytic fragments of the enzyme molecule apparently does not affect the refolding. The two proteolytic fragments can be separated by FPLC under denaturing conditions. Fluorescence spectra of the isolated fragments may indicate that the tryptic cleavage site is within the N-terminal domain. Thus, the larger fragment (molecular mass about 30 kDa) probably contains the whole nucleotide-binding C-terminal domain plus a small part of the N-terminal domain. The inactive isolated fragments were used in renaturation experiments to study the reassembly of active 3-phosphoglycerate kinase. Kinetic measurements revealed the presence of a bimolecular rate-limiting step of reactivation. Separate preincubation of the fragments under renaturing conditions did not cause substantial acceleration of reactivation. This implies that assembly of the separate structural units (possibly domains) may limit the reactivation of the intact enzyme.

Incomplete refolding of a fragment of the N-terminal domain of pig muscle 3-phosphoglycerate kinase that lacks a subdomain. Comparison with refolding of the complementary C-terminal fragment.

Refolding of pig muscle 3-phosphoglycerate kinase (PGK) from a mixture of its complementary proteolytic fragments that did not correspond to the individual domains resulted in a high degree of reactivation [Vas, M., Sinev, M.A., Kotova, N. & Semisotnov, G.V. (1990) Eur. J. Biochem. 189, 575--579]. An independent refolding of the 27.7 kDa C-terminal proteolytic fragment (which encompasses the whole C domain) has been noted, but the refolding ability of the 16.8-kDa N-terminal proteolytic fragment, which lacks a single subdomain from the N domain, remained to be seen. Here the refolding processes of the isolated fragments are compared. Within the first few seconds of initiation of refolding, pulse-proteolysis experiments show the formation of a structure with moderate protease resistance for both fragments. This structure, however, remains unchanged upon further incubation of the N-terminal fragment, whereas refolding of the C-terminal fragment continues as detected by a further increase in proteolytic resistance. The non-native character of the folding intermediate of the N fragment is indicated by the elevated fluorescence intensity of the bound hydrophobic probe 8-anilino-1-naphtalene sulphonate. Its CD spectrum shows the formation of secondary structure distinct from the native one. The noncooperative phase-transition observed in microcalorimetry indicates the absence of a rigid tertiary structure, in contrast with the refolded C-terminal fragment for which a cooperative transition is seen. Size-exclusion chromatography supported the globular character of the intermediate, and showed its propensity to form dimers. No binding of the substrate, 3-phosphoglycerate (3-PGri), to the isolated N-terminal fragment, could be detected but the presence of the complementary C-terminal fragment led to restoration of the substrate binding ability of the N domain. Thus, refolding of the isolated N-terminal fragment yields a highly flexible, globular, potentially productive intermediate with non-native secondary structure and highly exposed hydrophobic clusters, which favour dimerization.

[Effect of ADP and GroES on interaction of molecular chaperonin GroEL with non-native lysozyme]. / Vliianie ADP i GroES na vzaimodeistvie molekuliarnogo shaperonina GroEL s nenativnym lizotsimom.

The interaction of the molecular chaperonin GroEL with fluorescein-labeled lysozyme in the presence of high concentrations of thiol reagent--dithiothreitol (DTT) has been studied. In case of high concentrations of DTT lysozyme loses the native conformation due to the disruption of the intramolecular disulfide bonds stabilizing its structure and effectively aggregates. It has been shown that in the presence of high concentrations of DTT and two-fold molar excess of GroEL the lysozyme tightly interacts with GroEL that essentially decreases the efficiency of its aggregation. The addition of ADP to the complex of GroEL with nonnative lysozyme noticeably decreases the interaction of the chaperonin with nonnative protein target resulting in some increase of the efficiency of its aggregation. However, the addition of the co-chaperonin GroES together with ADP (i.e. the formation of the complex of GroEL with GroES) leads to drastic weakness of the interaction of GroEL with nonnative lysozyme and the efficiency of its aggregation becomes comparable with that in the absence of GroEL.

Refolding kinetics of pig muscle and yeast 3-phosphoglycerate kinases and of their proteolytic fragments.

The time course of refolding of both pig muscle and yeast 3-phosphoglycerate kinase (molecular masses about 47 kDa), as well as their proteolytic C-terminal fragments (30 and 33 kDa, respectively) has been investigated. Very similar refolding kinetics (with half-time between 80-120 s, at 20 degrees C) were observed by fluorescence and ultraviolet absorbance spectroscopy, as well as by activity measurements, for the intact enzyme from both sources. This time course appears not to depend on the time the protein spends in the unfolded state, i.e. it is certainly not controlled by proline isomerization. Furthermore, after removal of a large N-terminal part (molecular mass of about 18 kDa for pig muscle enzyme or 13 kDa for yeast enzyme) of the molecule by proteolysis, refolding of the remaining C-terminal fragment of both proteins follows kinetics virtually indistinguishable from those of the intact protein molecule.