共查询到20条相似文献,搜索用时 15 毫秒
1.
E. A. Ermakova 《Biophysics》2006,51(2):202-208
A comparative study of the interaction of two RNases (binase and barnase) with the polypeptide inhibitor barstar was performed by Brownian dynamics simulation. It was demonstrated that this method adequately reproduced the dependence of the association rate on the pH of solution as well as the effect of mutations at individual amino acid residues on the inhibition of barnase by barstar. Two types of energy-favorable binase-barstar encounter complexes were found. In type I complex, the amino acid residues of the binase active center are involved in formation of the complex; in type II complex, the active center remains free. It is suggested that temporary binding of free barstar into type II complex competes with the inhibition reaction. Presumably, this explains the decrease in the rate of binase inhibition by barstar as compared with the analogous reaction of barnase. 相似文献
2.
Ermakova E 《Biophysical chemistry》2007,130(1-2):26-31
A comparative study of the competitive reactions-the association reaction of binase with polypeptide inhibitor barstar and the reaction of binase dimerization-has been performed by the Brownian dynamics simulation method. It was shown that three types of the binase dimers could be formed and the dimerization reaction could compete with the inhibition reaction. The first type of the dimers leaves the active centre of binase free. During the formation of the dimers of the second and the third types the active centre of one or both binase molecules is blocked and ribonuclease becomes partially or fully inactive. Brownian dynamics simulation shown, that the ratio of competitive reaction rates depends on pH and ionic strength of solution. 相似文献
3.
E B Chernokal'skaia E R Romakhina N P Balaban M R Sharipova V I Vershinina I B Leshchinskaia 《Nauchnye doklady vysshe? shkoly. Biologicheskie nauki》1992,(2):45-50
The structure gene of extracellular alkaline ribonuclease Bacillus intermedius (binase) has been cloned in E. coli cells in composition of pMT 316 plasmid carrying the inhibitor gene (barstar of barnase--binase structure homologue. The possibility to use such vector has been proved during the barstar action on binase catalytic activity. Using biochemical immunochemical analysis the expression of binase gene in E. coli cells has been confirmed. The recombinant clone E. coli which contains both plasmids simultaneously--carrying gene for barster and for benase has been produced. The given vector is suggested to be used for cloning of inhibitor gene to obtain a viable producer of alkaline intracellular ribonuclease. 相似文献
4.
Ermakova EA 《Biofizika》2006,51(2):242-249
A comparative study of the association of two ribonucleases, barnase and binase, with the polypeptide inhibitor barstar has been performed by the Brownian dynamics simulation method. It was shown that the method adequately reproduced the dependence of the association rate on pH and ionic strength of solution and the influence of mutations of some ribonuclease amino acids. Two types of energetically favorable complexes of binase-barstar encounter were determined. In the type I complex, the amino acids of binase active center take part in the complex formation. In the second complex, the active center is free. It was supposed that the temporary binding of barstar into complex of type II is competitive relative to the inhibition reaction. This can partially explain the decrease in the rate of binase inhibition as compared with the corresponding reaction of barnase. 相似文献
5.
Protasevich II Schulga AA Vasilieva LI Polyakov KM Lobachov VM Hartley RW Kirpichnikov MP Makarov AA 《FEBS letters》1999,445(2-3):384-388
The mechanism by which barnase and binase are stabilized in their complexes with barstar and the role of the Cys-40 residue of barstar in that stabilization have been investigated by scanning microcalorimetry. Melting of ribonuclease complexes with barstar and its Cys-82-Ala mutant is described by two 2-state transitions. The lower-temperature one corresponds to barstar denaturation and the higher-temperature transition to ribonuclease melting. The barstar mutation Cys-40-Ala, which is within the principal barnase-binding region of barstar, simplifies the melting to a single 2-state transition. The presence of residue Cys-40 in barstar results in additional stabilization of ribonuclease in the complex. 相似文献
6.
Znamenskaia LV Vershinina OA Vershinina VI Krasnov SI Kostrov SV Akimkina TV Leshchinskaia IB Hartley RW 《Molekuliarnaia genetika, mikrobiologiia i virusologiia》1999,(1):12-17
Plasmids with whole genes for ribonucleases from B. intermedius (binase) and B. pumilis (RNase Bp) assembled with the whole gene of barstar, a specific intracellular inhibitor, are constructed. The resultant plasmids pMZ55 and pMZ56 effectively express binase and RNase Bp genes in B. subtilis cells. A medium for maximum expression of RNase genes by recombinant strains is developed. The expression of binase and RNase Bp genes in B. subtilis cells is negatively regulated by exogenic inorganic phosphate. 相似文献
7.
Two types of dynamic solid-state NMR experiments-relaxation and 1D-MAS exchange-were combined for the investigation of the backbone dynamics of a 15% randomly 15N-enriched protein barstar in both free and binase-bound states. The main novelty of this work is a simultaneous quantitative processing of the results of these two types of experiments that we call Simultaneous Relaxation and Exchange Data Analysis (SREDA) approach. It extends the well-known model-free approach such that it permits to discriminate between various motional models (jumps between different sites, wobbling in a cone, etc.). This objective cannot be achieved by analyzing the relaxation or exchange data separately. The SREDA approach was applied to probe a modification of the average backbone dynamics of barstar upon forming a complex with another protein binase. T(1) and off-resonance T(1rho) relaxation times of 15N backbone nuclei were measured at three temperatures between 0 and 45 degrees C, 1D-MAS exchange (CODEX) data were obtained at room temperature within the mixing time range from 0.3 to 200 ms. It has been found that the barstar backbone participates in two molecular processes with correlation times in the 10(-9)-10(-7) and 10(-3)-10(-2) s ranges. Forming the complex with binase results in a significant decrease of the amplitudes of both motions, suggesting that the complex is a more rigid and stable structure than free barstar. 相似文献
8.
Hahnen E Znamenskaya L Koczan D Leshchinskaya I Hobom G 《Molecular & general genetics : MGG》2000,263(4):571-580
A second secreted ribonuclease, designated binase II, has been detected in Bacillus intermedius 7P, and its structural gene was cloned and sequenced. Unlike the well-known binase I, a 109-amino acid guanyl-specific enzyme,
the 292-residue binase II is closely related to the B. subtilis nuclease Bsn, in structure and in its enzymatic properties. Binase II is also insensitive to inactivation by barstar, an
inhibitor protein that is specific for guanyl-specific ribonucleases. While both B. intermedius enzymes are induced upon phosphate starvation, only the gene for binase I belongs to the pho regulon system and carries pho-box elements adjacent to its promoter sequence. The gene for binase II is similar to that for Bsn in lacking such elements.
The birB gene coding for binase II appears to be located next to the 3′-end of a ferric ion transport operon, with which it convergently
overlaps. This would allow attenuator control over binase II expression under conditions of starvation for ferric ions.
Received: 12 October 1999 / Accepted: 10 February 2000 相似文献
9.
Mitkevich VA Schulga AA Ermolyuk YS Lobachov VM Chekhov VO Yakovlev GI Hartley RW Nick Pace C Kirpichnikov MP Makarov AA 《Biophysical chemistry》2003,105(2-3):383-390
Differential scanning calorimetry was used to study the thermodynamics of denaturation of protein complexes for which the free energy stabilizing the complexes varied between -8 and -16 kcal/mol. The proteins studied were the ribonucleases barnase and binase, their inhibitor barstar and mutants thereof, and complexes between the two. The results are in good agreement with the model developed by Brandts and Lin for studying the thermodynamics of denaturation for tight complexes between two proteins which undergo two-state thermal unfolding transitions. 相似文献
10.
Aaron P. Yamniuk John A. Newitt Michael L. Doyle Fumio Arisaka Anthony M. Giannetti Preston Hensley David G. Myszka Fred P. Schwarz James A. Thomson Edward Eisenstein 《Journal of biomolecular techniques》2015,26(4):125-141
A significant challenge in the molecular interaction field is to accurately determine the stoichiometry and stepwise binding affinity constants for macromolecules having >1 binding site. The mission of the Molecular Interactions Research Group (MIRG) of the Association of Biomolecular Resource Facilities (ABRF) is to show how biophysical technologies are used to quantitatively characterize molecular interactions, and to educate the ABRF members and scientific community on the utility and limitations of core technologies [such as biosensor, microcalorimetry, or analytic ultracentrifugation (AUC)]. In the present work, the MIRG has developed a robust model protein interaction pair consisting of a bivalent variant of the Bacillus amyloliquefaciens extracellular RNase barnase and a variant of its natural monovalent intracellular inhibitor protein barstar. It is demonstrated that this system can serve as a benchmarking tool for the quantitative analysis of 2-site protein-protein interactions. The protein interaction pair enables determination of precise binding constants for the barstar protein binding to 2 distinct sites on the bivalent barnase binding partner (termed binase), where the 2 binding sites were engineered to possess affinities that differed by 2 orders of magnitude. Multiple MIRG laboratories characterized the interaction using isothermal titration calorimetry (ITC), AUC, and surface plasmon resonance (SPR) methods to evaluate the feasibility of the system as a benchmarking model. Although general agreement was seen for the binding constants measured using solution-based ITC and AUC approaches, weaker affinity was seen for surface-based method SPR, with protein immobilization likely affecting affinity. An analysis of the results from multiple MIRG laboratories suggests that the bivalent barnase-barstar system is a suitable model for benchmarking new approaches for the quantitative characterization of complex biomolecular interactions. 相似文献
11.
A. Yu. Rubina M. V. Tsybulskaya O. A. Zasedateleva S. B. Popletaeva M. A. Filippova V. I. Butvilovskaya S. A. Surzhikov A. A. Stomakhin A. S. Zasedatelev 《Molecular Biology》2012,46(4):596-604
The sequence specificity of protein—oligonucleotide interactions based on the example of binase interaction with oligodeoxyribonucleotides immobilized in biochip gel elements has been studied. Constants of the preferable binding of binase to the selected nucleotide sequences were measured. The GAGAGAG and GAGAGAGAG oligodeoxyribonucleotides, which specifically bind to binase, were used as molecular probes to develop affine sorptive media for binase isolation and concentration from diluted water solutions. The volume capacity of affinity sorbents with immobilized oligodeoxyribonucleotides was found to be 2.6 and 2.3 mg of binase per 1 mL of sorbent for GAGAGAG and GAGAGAGAG, respectively. It was shown that, after affinity chromatography and elution from sorptive media, the oligonucleotide specificity of binase corresponds to the specificity of the initial sample. 相似文献
12.
M. A. Efimova R. Shah Mahmud P. V. Zelenikhin M. I. Sabirova A. I. Kolpakov O. N. Ilinskaya 《Molecular Biology》2017,51(1):96-101
The experimental study identified the antiviral activity of Bacillus pumilus RNase (binase) against the reovirus of serotype 1/strain Lang. For the first time, it has been found that 50 μg/mL of binase effectively reduced the hemagglutinin and cytocidal activity of reovirus in Vero cell line. The preincubation of the enzyme with reovirus before infection of the cells inhibited the viral replication. To determine the stagedependent effect of reovirus reproduction upon binase inhibition, the infected cells were treated with binase or RNase A at different phases of the infectious cycle. The treatment of virus-infected cells has revealed that both enzymes have a maximal antiviral effect on the reovirus propagation during early phases of the reovirus reproduction cycle, with binase being more effective than RNase A. It has been hypothesized that the combined action of the oncolytic reovirus and binase is promising for the elimination of tumor cells carrying mutated RAS gene. 相似文献
13.
The crystal structure of the barstar mutants (Y29P) and (Y29D, Y30W) as well as that of the complexes of barstar(Y29P) with wild-type barnase and barnase(H102K) have been determined. These barstar mutants compensate for the dramatic loss of barnase-barstar interaction energy caused by a single mutation of the barnase active site His-102 to a lysine. The latter introduces an uncompensated charge in the pocket at the surface of barstar where Lys-102 is located. The analysis of the structures suggests a mechanism for this compensation based on the solvation of the charge of Lys-102. Additional compensation occurs through the formation of a hydrogen bond. 相似文献
14.
Nadezhda L. Mironova Irina Y. Petrushanko Olga A. Patutina Aexandra V. Sen’kova Olga V. Simonenko Vladimir A. Mitkevich Oleg V. Markov Marina A. Zenkova Alexander A. Makarov 《Cell cycle (Georgetown, Tex.)》2013,12(13):2120-2131
Exogenous ribonucleases are known to inhibit tumor growth via apoptosis induction in tumor cells, allowing to consider them as promising anticancer drugs for clinical application. In this work the antitumor potential of binase was evaluated in vivo and the mechanism of cytotoxic effect of binase on tumor cells was comprehensively studied in vitro. We investigated tumoricidal activity of binase using three murine tumor models of Lewis lung carcinoma (LLC), lymphosarcoma RLS40 and melanoma B-16. We show for the first time that intraperitoneal injection of binase at a dose range 0.1–5 mg/kg results in retardation of primary tumor growth up to 45% in LLC and RLS40 and inhibits metastasis up to 50% in LLC and RLS40 and up to 70% in B-16 melanoma. Binase does not exhibit overall toxic effect and displays a general systemic and immunomodulatory effects. Treatment of RLS40-bearing animals with binase together with polychemotherapy revealed that binase decreases the hepatotoxicity of polychemotherapy while maintaining its antitumor effect. It was demonstrated that the cytotoxic effect of binase is realized via the induction of the intrinsic and extrinsic apoptotic pathways. Activation of intrinsic apoptotic pathway is manifested by a drop of mitochondrial potential, increase in calcium concentration and inhibition of respiratory activity. Subsequent synthesis of TNF-α in the cells under the action of binase triggers extrinsic apoptotic pathway through the binding of TNF with cell-death receptors and activation of caspase 8. Thus binase is a potential anticancer therapeutics inducing apoptosis in cancer cells. 相似文献
15.
We used a novel charge optimization technique to study the small ribonuclease barnase and to analyze its interaction with a natural tight binding inhibitor, the protein barstar. The approach uses a continuum model to explicitly determine the charge distributions that lead to the most favorable electrostatic contribution to binding when competing desolvation and interaction effects are included. Given its backbone fold, barstar is electrostatically optimized for tight binding to barnase when compared with mutants where residues have been substituted with one of the 20 common amino acids. Natural proteins thus appear to use optimization of electrostatic interactions as one strategy for achieving tight binding. 相似文献
16.
Barnase and barstar. Expression of its cloned inhibitor permits expression of a cloned ribonuclease 总被引:26,自引:0,他引:26
R W Hartley 《Journal of molecular biology》1988,202(4):913-915
Barnase is the extracellular ribonuclease of Bacillus amyloliquefaciens and barstar its specific intracellular inhibitor. The gene for barstar has now been cloned and sequenced. When the wild-type gene for barnase is reconstructed from its previously cloned parts on the same plasmid as the barstar gene, the lethal effect of its expression is suppressed. A plasmid has been devised which directs the secretion of 100 mg per active barnase liter by Escherichia coli and another which provides large (500 to 1000 mg/l) yields of barstar. The structure of these plasmids and the derived 89 amino acid sequence of barstar are reported. 相似文献
17.
Sekatskii SK Favre M Dietler G Mikhailov AG Klinov DV Lukash SV Deyev SM 《Journal of molecular recognition : JMR》2010,23(6):583-588
Results of the single molecule force spectroscopy study of specific interactions between ribonuclease barnase and its inhibitor barstar are presented. Experimental data obtained for the force loading rate ranging 2-70 nN/s are well approximated by a single straight line, from which the dissociation barrier of the width of 0.12 nm and height of 0.75-0.85 × 10(-19)J can be inferred. The measured value of specific interaction does not depend on the NaCl concentration. This apparently contradicts the well-known dependence of the binding energy of this pair on the salt concentration, but such a "contradiction" is explained by the insensitivity of the force spectroscopy data to the relatively long-range electrostatic interaction. The latter essentially contributes to the value of barnase-barstar binding energy revealed by biochemical measurements, and it is exactly this electrostatic interaction which is influenced by the salt concentration. 相似文献
18.
Protein-protein interactions are very important in the function of a cell. Computational studies of these interactions have been of interest, but often they have utilized classical modelling techniques. In recent years, quantum mechanical (QM) treatment of entire proteins has emerged as a powerful approach to study biomolecular systems. Herein, we apply a semi-empirical divide and conquer (DC) methodology coupled with a dielectric continuum model for the solvent, to explore the contribution of electrostatics, polarization and charge transfer to the interaction energy between barnase and barstar in their complex form. Molecular dynamic (MD) simulation was performed to account for the dynamic behavior of the complex. The results show that electrostatics, charge transfer and polarization favor the formation of the complex. Our study shows that electrostatics dominates the interaction between barnase and barstar ( approximately 73%), while charge transfer and polarization are approximately 21% and approximately 6%, respectively. Close inspection of the polarization and charge-transfer effects on the charge distribution of the complex reveals the existence of two, well localized, regions in barstar. The first region includes the residues between P27 and Y47 and the second region is between N65 and D83. Since no such regions could be detected in barnase clearly suggests that barstar is well optimized for efficiently binding barnase. Furthermore, using our interaction energy decomposition scheme, we were able to identify all residues that have been experimentally determined to be important for the complex formation and to suggest other residues never have been investigated. This suggests that our approach will be useful as an aid in further understanding protein-protein contacts for the ultimate goal to produce successful inhibitors for protein complexes. 相似文献
19.
Petrushanko IIu Zelenikhin PV Mit'kevich VA Kleĭmenova AA Prasolov VS Makarov AA Il'inskaia ON 《Biofizika》2007,52(5):876-881
The effect of cationic microbial ribonuclease from Bacillus intermedius (binase) on normal precursors of myeloid cells of FDC-P1 mice and kit-transformed precursors expressing the receptor of the growth factor of stem cells has been studied by flow-through cytometry. Selective apoptogenic properties of binase toward kit-transformed cells were revealed. Viable kit-transformed cells responded to binase by an increase in the concentration of cytosolic calcium. The content of calcium in the cytosol of both cell types in which apoptosis was induced by binase decreased in a dose-dependent manner. The death of cells was not accompanied by a substantial decrease in the content of intracellular RNA. A possible mechanism of binase-induced effects, which involves changes in the expression of genes due to the interference of exogenous RNAse into the RNA interference, was considered. 相似文献
20.
pH dependence of the stability of barstar to chemical and thermal denaturation. 总被引:3,自引:2,他引:1
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R. Khurana A. T. Hate U. Nath J. B. Udgaonkar 《Protein science : a publication of the Protein Society》1995,4(6):1133-1144
Equilibrium unfolding of barstar with guanidine hydrochloride (GdnHCl) and urea as denaturants as well as thermal unfolding have been carried out as a function of pH using fluorescence, far-UV and near-UV CD, and absorbance as probes. Both GdnHCl-induced and urea-induced denaturation studies at pH 7 show that barstar unfolds through a two-state F<->U mechanism and yields identical values for delta GU, the free energy difference between the fully folded (F) and unfolded (U) forms, of 5.0 +/- 0.5 kcal.mol-1 at 25 degrees C. Thermal denaturation of barstar also follows a two-state F<->U unfolding transition at pH 7, and the value of delta GU at 25 degrees C is similar to that obtained from chemical denaturation. The pH dependence of denaturation by GdnHCl is complex. The Cm value (midpoint of the unfolding transition) has been used as an index for stability in the pH range 2-10, because barstar does not unfold through a two-state transition on denaturation by GdnHCl at all pH values studied. Stability is maximum at pH 2-3, where barstar exists in a molten globule-like form that forms a large soluble oligomer. The stability decreases with an increase in pH to 5, the isoelectric pH of the protein. Above pH 5, the stability increases as the pH is raised to 7. Above pH 8, it again decreases as the pH is raised to 10. The decrease in stability from pH 7 to 5 in wild-type (wt) barstar, which is shown to be characterized by an apparent pKa of 6.2 +/- 0.2, is not observed in H17Q, a His 17-->Gln 17 mutant form of barstar. This decrease in stability has therefore been correlated with the protonation of His 17 in barstar. The decrease in stability beyond pH 8 in wt barstar, which is characterized by an apparent pKa of 9.2 +/- 0.2, is not detected in BSCCAA, the Cys 40 Cys 82-->Ala 40 Ala 82 double mutant form of barstar. Thus, this decrease in stability has been correlated with the deprotonation of at least one of the two cysteines present in wt barstar. The increase in stability from pH 5 to 3 is characterized by an apparent pKa of 4.6 +/- 0.2 for wt barstar and BSCCAA, which is similar to the apparent pKa that characterizes the structural transition leading to the formation of the A form. The use of Cm as an index of stability has been supported by thermal denaturation studies.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献