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1.
Delayed Osmotic Effect on in Vitro Assembly of RuBisCO : Relationship to Large Subunit-Binding Protein Complex Dissociation 总被引:2,自引:1,他引:1
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Higher plant ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) cannot reassociate after dissociation, and its subunits do not assemble into active RuBisCO when synthesized in Escherichia coli. Newly synthesized subunits of RuBisCO are associated with a high molecular weight binding protein complex in pea chloroplasts. The immediate donor for large subunits which assemble into RuBisCO is a low molecular weight complex which may be derived from the high molecular weight binding protein complex. When the high molecular weight binding protein complex is diluted, it tends to dissociate, forming low molecular weight complexes. When the large subunit-binding protein complexes were examined after in organello protein synthesis, it was found that the low molecular weight complexes were more abundant when protein synthesis was carried out under hypotonic conditions. This increase in the assembly competent population of low molecular weight large subunit complexes can account for the increased amount of in vitro RuBisCO assembly which occurs under these conditions. The data indicate that the assembly of large subunits into RuBisCO is a function of the aggregation state of the large subunit binding protein complex during protein synthesis. This implies that the binding protein exerts its effects during or shortly after large subunit synthesis. 相似文献
2.
Panyukov YV Nemykh MA Rafikova ER Kurganov BI Yaguzhinsky LS Arutyunyan AM Drachev VA Dobrov EN 《The international journal of biochemistry & cell biology》2006,38(4):533-543
Ordered and amorphous protein aggregation causes numerous diseases. Tobacco mosaic virus coat protein for many decades serves as the classical model of ordered protein aggregation ("polymerization"). It was also found to be highly prone to heat-induced amorphous aggregation and the rate of this aggregation could be easily manipulated by changes in solution ionic strength and temperature. Here, we report that rapid amorphous aggregation of this protein can be induced at 25 degrees C in phosphate buffer by low micromolar (start at about 15 microM) concentrations of cationic surfactant cetyltrimethylammonium bromide. At equilibrium four surfactant molecules bound to the protein subunit. As judged by circular dichroism and fluorescence spectroscopy data, the coat protein molecules retained their native structure upon the cetyltrimethylammonium bromide induced aggregation. No aggregation was observed at the higher surfactant concentrations (above 300 microM). Micromolar concentrations of anionic surfactant sodium dodecylsulfate rapidly reversed the cetyltrimethylammonium bromide induced aggregation of the coat protein due to formation of mixed surfactant-surfactant micelles. Cetyltrimethylammonium bromide (100-300 microM) also induced the reversible intact tobacco mosaic virus virion aggregation. The possible liability to the cetyltrimethylammonium bromide induced amorphous aggregation of other ordered aggregate-producing proteins has been discussed. 相似文献
3.
Calpain is a heterodimeric Ca(2+)-dependent cysteine protease consisting of a large (80 kDa) catalytic subunit and a small (28 kDa) regulatory subunit. The effects of Ca(2+) on the enzyme include activation, aggregation, and autolysis. They may also include subunit dissociation, which has been the subject of some debate. Using the inactive C105S-80k/21k form of calpain to eliminate autolysis, we have studied its disassociation and aggregation in the presence of Ca(2+) and the inhibition of its aggregation by means of crystallization, light scattering, and sedimentation. Aggregation, as assessed by light scattering, depended on the ionic strength and pH of the buffer, on the Ca(2+) concentration, and on the presence or absence of calpastatin. At low ionic strength, calpain aggregated rapidly in the presence of Ca(2+), but this was fully reversible by EDTA. With Ca(2+) in 0.2 m NaCl, no aggregation was visible but ultracentrifugation showed that a mixture of soluble high molecular weight complexes was present. Calpastatin prevented aggregation, leading instead to the formation of a calpastatin-calpain complex. Crystallization in the presence of Ca(2+) gave rise to crystals mixed with an amorphous precipitate. The crystals contained only the small subunit, thereby demonstrating subunit dissociation, and the precipitate was highly enriched in the large subunit. Reversible dissociation in the presence of Ca(2+) was also unequivocally demonstrated by the exchange of slightly different small subunits between mu-calpain and m-calpain. We conclude that subunit dissociation is a dynamic process and is not complete in most buffer conditions unless driven by factors such as crystal formation or autolysis of active enzymes. Exposure of the hydrophobic dimerization surface following subunit dissociation may be the main factor responsible for Ca(2+)-induced aggregation of calpain. It is likely that dissociation serves as an early step in calpain activation by releasing the constraints upon protease domain I. 相似文献
4.
A single cyclic AMP-dependent protein kinase (EC 2.7.1.37) has been isolated from human platelets by using DEAE-cellulose ion-exchange chromatography and Sephadex G-150 gel filtration. The molecular weight of the protein kinase was estimated to be 86 490. In the presence of cyclic AMP, the protein kinase could be dissociated into a catalytic subunit of molecular weight 50 000, and either one regulatory subunit of molecular weight 110 000 or two regulatory subunits of molecular weights 110 000 and 38 100, depending on the pH used. Recombination of either of the regulatory subunits with the catalytic subunit restored cyclic AMP-dependency in the catalytic subunit. The apparent Km for ATP in the presence of 10 muM Mg2+ was 4 muM (plus cyclic AMP) and 4.3 muM (minus cyclic AMP). The concentration of cyclic AMP needed for half-maximal stimulation of the protein kinase was 0.172 muM and apparent dissociation constants of 3.7 nM (absence of MgATP) and 0.18 muM (presence of MgATP) were exhibited by the "protein kinase-cyclic AMP complex". The enzyme required Mg2+ for maximum activity and showed a pH optimum of 6.2 with histone as substrate. In addition to four major endogenous platelet protein acceptors of apparent molecular weights 45 000, 28000, 18 500, and 11 100, the platelet protein kinase also phosphorylated the exogenous acceptor proteins thrombin, collagen and histone, all capable of inducing platelet aggregation. Prothrombin, a nonaggregating agent, was not phosphorylated. 相似文献
5.
《Biochimica et Biophysica Acta (BBA) - Enzymology》1976,422(1):60-72
A single cyclic AMP-dependent protein kinase (EC 2.7.1.37) has been isolated from human platelets by using DEAE-cellulose ion-exchange chromatography and Sephadex G-150 gel filtration. The molecular weight of the protein kinase was estimated to be 86 490. In the presence of cyclic AMP, the protein kinase could be dissociated into a catalytic subunit of molecular weight 50 000, and either one regulatory subunit of molecular weight 110 000 or two regulatory subunits of molecular weights 110 000 and 38 100, depending on the pH used. Recombination of either of the regulatory subunits with the catalytic subunit restored cyclic AMP-dependency in the catalytic subunit.The apparent Km for ATP in the presence of 10 μM Mg2+ was 4 μM (plus cyclic AMP) and 4.3 μM (minus cyclic AMP). The concentration of cyclic AMP needed for half-maximal stimulation of the protein kinase was 0.172 μM and apparent dissociation constants of 3.7 nM (absence of MgATP) and 0.18 μM (presence of MgATP) were exhibited by the “protein kinase-cyclic AMP complex”. The enzyme required Mg2+ for maximum activity and showed a pH optimum of 6.2 with histone as substrate.In addition to four major endogenous platelet protein acceptors of apparent molecular weights 45 000, 28 000, 18 500, and 11 100, the platelet protein kinase also phosphorylated the exogenous acceptor proteins thrombin, collagen and histone, all capable of inducing platelet aggregation. Prothrombin, a nonaggregating agent, was not phosphorylated. 相似文献
6.
Qualitative and quantitative changes in the protein and glycoprotein components of the plasma membrane of the cellular slime mould Dictyostelium discoideum have been detected by analysis of sodium dodecyl sulphate-polyacrylamide gel electrophoretic patterns. The amounts of proteins of subunit molecular weight 220 000, 91 000, 63 000, 59 000, 56 000 increased during the acquisition of aggregation competence, while proteins of subunit molecular weight 82 000 and 22 000 decreased. The amounts of glycoproteins with apparent subunit molecular weights 285 000, 150 000, 137 000, 100 000, 53 000, 50 500 and 30 500 increased during differentiation while a 125 000 dalton component decreased dramatically in amount. The neutral and amino sugar composition of the plasma membrane was also analyzed and found to remain essentially unchanged during the first 12 h of differentiation. The major sugars were mannose, fucose, and glucosamine; galactose and galactosamine were also present, but in lower amounts. 相似文献
7.
Michael Taylor Helen Burress Tuhina Banerjee Supriyo Ray David Curtis Suren A. Tatulian Ken Teter 《PLoS pathogens》2014,10(2)
To generate a cytopathic effect, the catalytic A1 subunit of cholera toxin (CT) must be separated from the rest of the toxin. Protein disulfide isomerase (PDI) is thought to mediate CT disassembly by acting as a redox-driven chaperone that actively unfolds the CTA1 subunit. Here, we show that PDI itself unfolds upon contact with CTA1. The substrate-induced unfolding of PDI provides a novel molecular mechanism for holotoxin disassembly: we postulate the expanded hydrodynamic radius of unfolded PDI acts as a wedge to dislodge reduced CTA1 from its holotoxin. The oxidoreductase activity of PDI was not required for CT disassembly, but CTA1 displacement did not occur when PDI was locked in a folded conformation or when its substrate-induced unfolding was blocked due to the loss of chaperone function. Two other oxidoreductases (ERp57 and ERp72) did not unfold in the presence of CTA1 and did not displace reduced CTA1 from its holotoxin. Our data establish a new functional property of PDI that may be linked to its role as a chaperone that prevents protein aggregation. 相似文献
8.
N. E. Lamash 《Biology Bulletin》2001,28(1):107-109
A method for isolating a GTP-binding regulatory protein from starfish oocytes is described. The protein consists of three subunits with molecular weights of 40, 37, and about 8 kDa. It is shown that the 40-kDa subunit has a high GTPase activity and is susceptible to ADP-ribosylation by pertussis toxin. The latter property of this subunit proved to decrease upon its incubation with nonhydrolyzable GTP analogues. These data provide evidence that the plasma membrane of starfish oocytes contains a 40-kDa GTP-binding protein with properties characteristic of the alpha subunit of the inhibitory G
i
protein. The role of this protein in the transmembrane signal transmission from the 1-methyladenine receptor to intracellular effectors is discussed. 相似文献
9.
Eukaryotic ribosome assembly involves a plethora of factors, which ensure that a correctly folded ribosome contains all ribosomal protein components. Among these assembly factors, Yar1 has recently emerged as a molecular chaperone for ribosomal protein rpS3 of the small ribosomal subunit (40S) in yeast. In complex with its chaperone, rpS3 is imported into the nucleus and protected from aggregation. How rpS3 and other ribosomal proteins are initially sequestered and subsequently integrated into pre-ribosomal particles is currently poorly understood. Here, we present the crystal structure of yeast rpS3 in complex with its chaperone Yar1 at 2.8 Å resolution. The crystal structure rationalizes how Yar1 can protect rpS3 from aggregation while facilitating nuclear import and suggests a mechanism for a stepwise exchange of molecular partners that ribosomal proteins interact with during ribosome assembly. 相似文献
10.
Isolation of a Periplasmic Molecular Chaperone-Like Protein of Rhodobacter sphaeroides f. sp. denitrificans That Is Homologous to the Dipeptide Transport Protein DppA of Escherichia coli
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Masahiro Matsuzaki Yuso Kiso Isamu Yamamoto Toshio Satoh 《Journal of bacteriology》1998,180(10):2718-2722
A periplasmic protein has been found to prevent aggregation of the acid-unfolded dimethyl sulfoxide reductase (DMSOR), the periplasmic terminal reductase of dimethyl sulfoxide respiration in the phototroph Rhodobacter sphaeroides f. sp. denitrificans, in a manner similar to that of the Escherichia coli chaperonin GroEL (Matsuzaki et al., Plant Cell Physiol. 37:333–339, 1996). The protein was isolated from the periplasm of the phototroph. It had a molecular mass of 58 kDa and had no subunits. The sequence of 14 amino-terminal residues of the protein was completely identical to that of the periplasmic dipeptide transport protein (DppA) of E. coli. The 58-kDa protein prevented aggregation to a degree comparable to that of GroEL on the basis of monomer protein. The 58-kDa protein also decreased aggregation of guanidine hydrochloride-denatured rhodanese, a mitochondrial matrix protein, during its refolding upon dilution. The 58-kDa protein is a kind of molecular chaperone and could be involved in maintaining unfolded DMSOR, after secretion of the latter into the periplasm, in a competent form for its correct folding. 相似文献
11.
A lead-absorbing protein with superoxide dismutase activity from Streptomyces subrutilus 总被引:3,自引:0,他引:3
A lead binding protein was purified from the culture filtrate of Streptomyces subrutilus P5. The subunit and native molecular weights were estimated to be 28 and 55 kDa, respectively, indicating that the protein was composed of two identical subunits. The inhibition pattern, the metal content analysis and the EPR spectrum confirmed that the protein was a superoxide dismutase containing Fe and Zn (FeZnSOD). The protein precipitated immediately upon mixing with lead ions and the saturation number of lead ions was about 1100 lead atoms per subunit. Using this property, lead ions could be effectively removed from solutions. 相似文献
12.
When frozen plasma membranes isolated from maize seedling roots are thawed, a significant portion of GTP-binding activity goes into solution. The GTP-binding protein was purified by ion exchange chromatography on Mono-Q and gel filtration on Superose 6. Its molecular weight was estimated at 61 kDa by gel filtration. The same molecular weight was obtained upon solubilization of the GTP-binding protein with cholic acid followed by gel filtration in the presence of this detergent. SDS-PAGE demonstrated that the isolated GTP-binding protein consists of two types of subunit of molecular weights 27 kDa and 34 kDa. 相似文献
13.
1. Large quantities of human Factor XIII were prepared from ethanol precipitates of outdated human plasma. 2. Material homogeneous after chromatography on DEAE-cellulose was further resolved into two proteins, A and B, after filtration on Sepharose 6B. 3. Protein A has a molecular weight of 350000 and a subunit structure a(2)b(2) and is activated by thrombin and calcium. Protein B is inactive and probably has a subunit structure b(2). 4. Calcium causes protein A, after thrombin cleavage, to fragment to give protein B and a protein, containing only a' subunits, which is catalytically active. The latter protein slowly forms a misty precipitate which is still active and not cross-linked covalently. This confirms the suggestion of Schwartz et al. (1971) that catalytic activity is only associated with a' subunits. 5. Iodoacetate, which inhibits the enzyme, does not inhibit dissociation and aggregation of protein A. 6. The existence of two proteins and the fragmentation are possible explanations for the wide range of molecular weights given for Factor XIII in the literature. 相似文献
14.
Staurosporine inhibits agrin-induced acetylcholine receptor phosphorylation and aggregation 总被引:14,自引:9,他引:5
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《The Journal of cell biology》1994,125(3):661-668
Agrin, a protein that mediates nerve-induced acetylcholine receptor (AChR) aggregation at developing neuromuscular junctions, has been shown to cause an increase in phosphorylation of the beta, gamma, and delta subunits of AChRs in cultured myotubes. As a step toward understanding the mechanism of agrin-induced AChR aggregation, we examined the effects of inhibitors of protein kinases on AChR aggregation and phosphorylation in chick myotubes in culture. Staurosporine, an antagonist of both protein serine and tyrosine kinases, blocked agrin-induced AChR aggregation in a dose-dependent manner; 50% inhibition occurred at approximately 2 nM. The extent of inhibition was independent of agrin concentration, suggesting an effect downstream of the interaction of agrin with its receptor. Staurosporine blocked agrin-induced phosphorylation of the AChR beta subunit, which occurs at least in part on tyrosine residues, but did not reduce phosphorylation of the gamma and delta subunits, which occurs on serine/threonine residues. Staurosporine also prevented the agrin- induced decrease in the rate at which AChRs are extracted from intact myotubes by mild detergents. H-7, an antagonist of protein serine kinases, inhibited agrin-induced phosphorylation of the gamma and delta subunits but did not block agrin-induced phosphorylation of the AChR beta subunit, AChR aggregation, or the decrease in AChR extractability. The results provide support for the hypothesis that tyrosine phosphorylation of the beta subunit plays a role in agrin-induced AChR aggregation. 相似文献
15.
Konno T Morii T Shimizu H Oiki S Ikura K 《The Journal of biological chemistry》2005,280(17):17520-17525
Cross-linking of proteins catalyzed by tissue transglutaminase has been suggested to play key roles in a variety of cellular events, including cell apoptosis and human pathogenesis (e.g. polyglutamine and Alzheimer diseases). It has often been suggested that tissue transglutaminase enhances aggregation and precipitation of damaged or pathogenic proteins. To ascertain whether this is accurate, we investigated the effects of tissue transglutaminase-catalyzed modulation on the aggregation of structurally damaged and unfolded proteins. Our results indicated that the aggregation and precipitation of some unfolded proteins were inhibited by transglutaminasecatalyzed reaction, although the effect was strongly dependent upon the target protein species. To elucidate the molecular events underlying the inhibitory effect, extensive analysis was performed with regard to reduced beta-lactoglobulin using a number of techniques, including chromatography and spectroscopy. The results indicated that cross-linking yields high molecular weight soluble polymers but inhibits the growth of insoluble aggregates. The cross-linked beta-lactoglobulin retained stable secondary structures with a hydrophobic core. We concluded that the transglutaminase-catalyzed intermolecular cross-linking did not necessarily enhance protein aggregation but could sometimes have a suppressive effect. The results of the present study suggested that tissue transglutaminase modifies aggregation and deposition of damaged or pathogenic proteins in vivo in a wide variety of manners depending on the target protein species and solution conditions. 相似文献
16.
Alpha crystallin is an eye lens protein with a molecular weight of approximately 800 kDa. It belongs to the class of small heat shock proteins. Besides its structural role, it is known to prevent the aggregation of β- and γ-crystallins and several other proteins under denaturing conditions and is thus believed to play an important role in maintaining lens transparency. In this communication, we have investigated the effect of 2,2,2-trifluoroethanol (TFE) on the structural and functional features of the native α-crystallin and its two constituent subunits. A conformational change occurs from the characteristic β-sheet to the α-helix structure in both native α-crystallin and its subunits with the increase in TFE levels. Among the two subunits, αA-crystallin is relatively stable and upon preincubation prevents the characteristic aggregation of αB-crystallin at 20% and 30% (v/v) TFE. The hydrophobicity and chaperone-like activity of the crystallin subunits decrease on TFE treatment. The ability of αA-crystallin to bind and prevent the aggregation of αB-crystallin, despite a conformational change, could be important in protecting the lens from external stress. The loss in chaperone activity of αA-crystallin exposed to TFE and the inability of peptide chaperone—the functional site of αA-crystallin—to stabilize αB-crystallin at 20–30% TFE suggest that the site(s) involved in subunit interaction and chaperone-like function are quite distinct. 相似文献
17.
Remodeling of the conformational ensemble of the repeat domain of tau by an aggregation enhancer
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Elias Akoury Marco D. Mukrasch Jacek Biernat Katharina Tepper Valery Ozenne Eckhard Mandelkow Martin Blackledge Markus Zweckstetter 《Protein science : a publication of the Protein Society》2016,25(5):1010-1020
Misfolding of the microtubule‐associated protein Tau is a hallmark of Alzheimer disease and several other neurodegenerative disorders. Because of the dynamic nature of the Tau protein, little is known about the changes in Tau structure that occur during misfolding. Here we studied the structural consequences upon binding of the repeat domain of Tau, which plays a key role in pathogenic aggregation, to an aggregation enhancer. By combining NMR experiments with molecular simulations we show that binding of the aggregation enhancer polyglutamic acid remodels the conformational ensemble of Tau. Our study thus provides insight into an early event during misfolding of Tau. 相似文献
18.
The amount of a native protein reflects an equilibrium of protein synthesis, de novo folding and protein stability. Stress situations, like heat shock, or overproduction of a protein can cause an imbalance in this equilibrium, resulting in protein aggregation. Molecular chaperones control protein folding processes and protect misfolded proteins from aggregation in all cells. Since protein aggregation is frequently observed upon synthesis of heterologous proteins in E. coli, molecular chaperones have been applied in biotechnology by their co-overproduction with the desired protein. While increasing protein solubility in some cases, this approach has not been generally successful. Recent findings demonstrate, that protein aggregation, even in case of inclusion bodies, must not be a dead end in the life cycle of a protein. Such resolubilization of aggregated proteins is mediated by a bi-chaperone system consisting of ClpB and DnaK, the prokaryotic representatives of the Hsp100 and Hsp70 families. The disaggregation capacity of this bi-chaperone system has now been demonstrated in vitro and in vivo for a wide variety of aggregated proteins and offers a new perspective to increase the solubility of proteins of interest. 相似文献
19.
The heteroaggregate alpha-crystallin and homoaggregates of its subunits, alphaA- and alphaB-crystallins, function like molecular chaperones and prevent the aggregation of several proteins. Although modulation of the chaperone-like activity of alpha-crystallin by both temperature and chaotropic agents has been demonstrated in vitro, the mechanism(s) of its regulation in vivo have not been elucidated. The subunits of alpha-crystallin exchange freely, resulting in its dynamic and variable quaternary structure. Mixed aggregates of the alpha-crystallins and other mammalian small heat shock proteins (sHSPs) have also been observed in vivo. We have investigated the time-dependent structural and functional changes during the course of heteroaggregate formation by the exchange of subunits between homoaggregates of alphaA- and alphaB-crystallins. Native isoelectric focusing was used to follow the time course of subunit exchange. Circular dichroism revealed large tertiary structural alterations in the subunits upon subunit exchange and packing into heteroaggregates, indicating specific homologous and heterologous interactions between the subunits. Subunit exchange also resulted in quaternary structural changes as demonstrated by gel filtration chromatography. Interestingly, we found time-dependent changes in chaperone-like activity against the dithiothreitol-induced aggregation of insulin, which correlated with subunit exchange and the resulting tertiary and quaternary structural changes. Heteroaggregates of varying subunit composition, as observed during eye lens epithelial cell differentiation, generated by subunit exchange displayed differential chaperone-like activity. It was possible to alter chaperone-like activity of preexisting oligomeric sHSPs by alteration of subunit composition by subunit exchange. Our results demonstrate that subunit exchange and the resulting structural and functional changes observed could constitute a mechanism of regulation of chaperone-like activity of alpha-crystallin (and possibly other mammalian sHSPs) in vivo. 相似文献
20.
《Journal of molecular biology》2021,433(20):167182
The protein folding problem was apparently solved recently by the advent of a deep learning method for protein structure prediction called AlphaFold. However, this program is not able to make predictions about the protein folding pathways. Moreover, it only treats about half of the human proteome, as the remaining proteins are intrinsically disordered or contain disordered regions. By definition these proteins differ from natively folded proteins and do not adopt a properly folded structure in solution. However these intrinsically disordered proteins (IDPs) also systematically differ in amino acid composition and uniquely often become folded upon binding to an interaction partner. These factors preclude solving IDP structures by current machine-learning methods like AlphaFold, which also cannot solve the protein aggregation problem, since this meta-folding process can give rise to different aggregate sizes and structures. An alternative computational method is provided by molecular dynamics simulations that already successfully explored the energy landscapes of IDP conformational switching and protein aggregation in multiple cases. These energy landscapes are very different from those of ‘simple’ protein folding, where one energy funnel leads to a unique protein structure. Instead, the energy landscapes of IDP conformational switching and protein aggregation feature a number of minima for different competing low-energy structures. In this review, I discuss the characteristics of these multifunneled energy landscapes in detail, illustrated by molecular dynamics simulations that elucidated the underlying conformational transitions and aggregation processes. 相似文献