共查询到20条相似文献,搜索用时 0 毫秒
1.
Teschke CM 《Biochemistry》1999,38(10):2873-2881
Aggregation is a common side reaction in the folding of proteins which is likely due to inappropriate interactions of folding intermediates. In the in vivo folding of phage P22 coat protein, amino acid substitutions that cause a temperature-sensitive-folding (tsf) phenotype lead to the localization of the mutant coat proteins to inclusion bodies. Investigated here is the aggregation of wild-type (WT) coat protein and 3 tsf mutants of coat protein. The tsf coat proteins aggregated when refolded in vitro at high temperature. If the tsf coat proteins were refolded at 4 degrees C, they were able attain an assembly active state. WT coat protein, on the other hand, did not aggregate significantly even when folded at high temperature. The refolded tsf mutants exhibited altered secondary and tertiary structures and had an increased surface hydrophobicity, which may explain the increased propensity of their folding intermediates to aggregate. 相似文献
2.
Cysteine residues in the transmembrane regions of M13 procoat protein suggest that oligomeric coat proteins assemble onto phage progeny
下载免费PDF全文
![点击此处可从《Journal of bacteriology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The M13 phage assembles in the inner membrane of Escherichia coli. During maturation, about 2,700 copies of the major coat protein move from the membrane onto a single-stranded phage DNA molecule that extrudes out of the cell. The major coat protein is synthesized as a precursor, termed procoat protein, and inserts into the membrane via a Sec-independent pathway. It is processed by a leader peptidase from its leader (signal) peptide before it is assembled onto the phage DNA. The transmembrane regions of the procoat protein play an important role in all these processes. Using cysteine mutants with mutations in the transmembrane regions of the procoat and coat proteins, we investigated which of the residues are involved in multimer formation, interaction with the leader peptidase, and formation of M13 progeny particles. We found that most single cysteine residues do not interfere with the membrane insertion, processing, and assembly of the phage. Treatment of the cells with copper phenanthroline showed that the cysteine residues were readily engaged in dimer and multimer formation. This suggests that the coat proteins assemble into multimers before they proceed onto the nascent phage particles. In addition, we found that when a cysteine is located in the leader peptide at the -6 position, processing of the mutant procoat protein and of other exported proteins is affected. This inhibition of the leader peptidase results in death of the cell and shows that there are distinct amino acid residues in the M13 procoat protein involved at specific steps of the phage assembly process. 相似文献
3.
GroEL recognizes proteins that are folding improperly or that have aggregation-prone intermediates. Here we have used as substrates for GroEL, wildtype (WT) coat protein of phage P22 and 3 coat proteins that carry single amino acid substitutions leading to a temperature-sensitive folding (tsf) phenotype. In vivo, WT coat protein does not require GroEL for proper folding, whereas GroEL is necessary for the folding of the tsf coat proteins; thus, the single amino acid substitutions cause coat protein to become a substrate for GroEL. The conformation of WT and tsf coat proteins when in a binary complex with GroEL was investigated using tryptophan fluorescence, quenching of fluorescence, and accessibility of the coat proteins to proteolysis. WT coat protein and the tsf coat protein mutants were each found to be in a different conformation when bound to GroEL. As an additional measure of the changes in the bound conformation, the affinity of binding of WT and tsf coat proteins to GroEL was determined using a fluorescence binding assay. The tsf coat proteins were bound more tightly by GroEL than WT coat protein. Therefore, even though the proteins are identical except for a single amino acid substitution, GroEL did not bind these substrate polypeptides in the same conformation within its central cavity. Therefore, GroEL is likely to bind coat protein in a conformation consistent with a late folding intermediate, with substantial secondary and tertiary structure formed. 相似文献
4.
Construction of plasmids that produce phage P22 repressor 总被引:2,自引:0,他引:2
In a series of plasmid constructions, the c2 (repressor) gene of phage P22 was cloned in a multicopy plasmid and expressed at increasing level. The final result of these constructions is a plasmid that maintains a level of approx. 200 times as much repressor as is found in a lysogen. A series of increasingly virulent phage mutants was isolated by plating sequentially on host cells with increasing levels of repressor. The methods used in the constructions should be applicable to obtaining elevated expression of cloned genes in other systems. 相似文献
5.
Reconstitution of the thermostable trimeric phage P22 tailspike protein from denatured chains in vitro 总被引:6,自引:0,他引:6
Intermediates in the intracellular chain folding and association pathway of the P22 tailspike endorhamnosidase have been identified previously by physiological and genetic methods. Conditions have now been found for the in vitro refolding of this large (Mr = 215,000) oligomeric protein. Purified Salmonella phage P22 tailspikes, while very stable to urea in neutral solution, were dissociated by moderate concentrations of urea at acidic pH. The tailspike protein was denatured to unfolded polypeptide chains in 6 M urea, pH 3, as disclosed by analytical ultracentrifugation, fluorescence, and circular dichroism. Upon dilution into neutral buffer at 10 degrees C, the polypeptides fold spontaneously and associate to form trimeric tailspikes with high yield. Like native phage P22 tailspikes, the reconstitution product is resistant to denaturation by dodecyl sulfate in the cold and displays endorhamnosidase activity. Sedimentation coefficients, electrophoretic mobility, and fluorescence emission maxima of native and reconstituted tailspikes are identical within experimental error. By characterization of intermediates, localization of temperature-sensitive steps, and analysis of the effect of previously identified folding mutations, the reconstitution system described should allow comparison of in vivo and in vitro folding pathways of this large protein oligomer. 相似文献
6.
7.
Sequential interactions of structural proteins in phage phi 29 procapsid assembly. 总被引:7,自引:6,他引:1
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The mechanism of viral capsid assembly is an intriguing problem because of its fundamental importance to research on synthetic viral particle vaccines, gene delivery systems, antiviral drugs, chimeric viruses displaying antigens or ligands, and the study of macromolecular interactions. The genes coding for the scaffolding (gp7), capsid (gp8), and portal vertex (gp10) proteins of the procapsid of bacteriophage phi 29 of Bacillus subtilis were expressed in Escherichia coli individually or in combination to study the mechanism of phi 29 procapsid assembly. When expressed alone, gp7 existed as a soluble monomer, gp8 aggregated into inclusion bodies, and gp10 formed the portal vertex. Circular dichroisin spectrum analysis indicated that gp7 is mainly composed of alpha helices. When two of the proteins were coexpressed, gp7 and gp8 assembled into procapsid-like particles with variable sizes and shapes, gp7 and gp10 formed unstable complexes, and gp8 and gp10 did not interact. These results suggested that gp7 served as a bridge for gp8 and gp10. When gp7, gp8, and gp10 were coexpressed, active procapsids were produced. Complementation of extracts containing one or two structural components could not produce active procapsids, indicating that no stable intermediates were formed. A dimeric gp7 concatemer promoted the solubility of gp8 but was inactive in the assembly of procapsid or procapsid-like particles. Mutation at the C terminus of gp7 prevented it from interacting with gp8, indicating that this part of gp7 may be important for interaction with gp8. Coexpression of the portal protein (gp20) of phage T4 with phi 29 gp7 and gp8 revealed the lack of interaction between T4 gp20 and phi 29 gp7 and/or gp8. Perturbing the ratio of the three structural proteins by duplicating one or another gene did not reduce the yield of potentially infectious particles. Changing of the order of gene arrangement in plasmids did not affect the formation of active procapsids significantly. These results indicate that phi 29 procapsid assembly deviated from the single-assembly pathway and that coexistence of all three components with a threshold concentration was required for procapsid assembly. The trimolecular interaction was so rapid that no true intermediates could be isolated. This finding is in accord with the result of capsid assembly obtained by the equilibrium model proposed by A. Zlotnick (J. Mol. Biol. 241:59-67, 1994). 相似文献
8.
Though disulfide bonds are absent from P22 tailspike protein in its native state, a disulfide-bonded trimeric intermediate has been identified in the tailspike folding and assembly pathway in vitro. The formation of disulfide bonds is critical to efficient assembly of native trimers as mutations at C-terminal cysteines reduce or inhibit trimer formation. We investigated the effect of different redox folding environments on tailspike formation to discover if simple changes in reducing potential would facilitate trimer formation. Expression of tailspike in trxB cell lines with more oxidizing cytoplasms led to lower trimer yields; however, observed assembly rates were unchanged. In vitro, the presence of any redox buffer decreased the overall yield compared to non-redox buffered controls; however, the greatest yields of the native trimer were obtained in reducing rather than oxidizing environments at pH 7. Slightly faster trimer formation rates were observed in the redox samples at pH 7, perhaps by accelerating the reduction of the disulfide-bonded protrimer to the native trimer. These rates and the effects of the redox system were found to depend greatly on the pH of the refolding reaction. Oxidized glutathione (GSSG) trapped a tailspike intermediate, likely as a mixed disulfide. This trapped intermediate was able to form native trimer upon addition of dithiothreitol (DTT), indicating that the trapped intermediate is on the assembly pathway, rather than the aggregation pathway. Thus, the presence of redox agents interfered with the ability of the tailspike monomers to associate, demonstrating that disulfide associations play an important role during the assembly of this cytoplasmic protein. 相似文献
9.
Novel second-site suppression of a cold-sensitive defect in phage P22 procapsid assembly 总被引:2,自引:0,他引:2
The DNA packaging portal of the phage P22 procapsid is formed of 12 molecules of the 90,000 dalton gene 1 protein. The assembly of this dodecameric complex at a unique capsid vertex requires scaffolding subunits. The mechanism that ensures the location of the 12-fold symmetrical portal at only one of the 12 5-fold vertices of an icosahedral virus capsid presents a unique assembly problem, which, in some viruses, is solved by the portal also acting as initiator of procapsid assembly. Phage P22 procapsids, however, are formed in the absence of the portal protein. The 1-csH137 mutation prevents the incorporation of the portal protein into procapsids. In a mixed infection with cs+ phage, the mutant subunits are able to form functional portals, suggesting that the cold-sensitivity does not affect portal-portal interactions, but affects the interaction of portal subunits with some other molecular species involved in the initiation of portal assembly. Interestingly, the cs defect is suppressed by temperature-sensitive folding mutations at four sites in the P22 tailspike gene 9. The suppression is allele-specific; other tailspike tsf mutations fail to suppress the cs defect. Translation through a suppressor site is required for suppression. This observation is unexpected, since analysis of nonsense mutations in this gene indicates that it is not required for procapsid assembly. Examination of the nucleic acid sequences in the neighborhood of each of the suppressor sites shows significant sequence similarity with the scaffolding gene translational initiation region on the late message. This supports a previously proposed model, in which procapsid assembly is normally initiated in a region on the late messenger RNA that includes the gene 8 start site. By this model, the suppressor mutations may be acting through protein-RNA interactions, changing sequences that identify alternative or competing sites at which the mutant portal subunits may be organized for assembly into the differentiated vertex of the phage capsid. 相似文献
10.
P22 cro? mutants were isolated as one class of phage P22 mutants (cly mutants) that have a very high frequeney of lysogeny relative to wild-type P22. These mutants: (1) do not form plaques and over-lysogenize relative to wild-type P22 after infection of a wild-type Salmonella host; (2) are defective in anti-immunity; and (3) fail to turn off high-level synthesis of P22 c2-repressor after infection.P22 cro? mutations are recessive and map between the P22 c2 and c1 genes. P22 cro? mutations are suppressed by clear-plaque mutations in the c1 gene, one of which is simultaneously cy?. They are also suppressed, but incompletely, by mutations in the c2 (repressor) gene, especially those that do not completely abolish c2 gene function.Salmonella host mutants have been isolated that are permissive for the lytic growth of the P22 cro? mutants. 相似文献
11.
Olia AS Al-Bassam J Winn-Stapley DA Joss L Casjens SR Cingolani G 《Journal of molecular biology》2006,363(2):558-576
To infect and replicate, bacteriophage P22 injects its 43 kbp genome across the cell wall of Salmonella enterica serovar Typhimurium. The attachment of phage P22 to the host cell as well as the injection of the viral DNA into the host is mediated by the virion's tail complex. This 2.8 MDa molecular machine is formed by five proteins, which include the portal protein gp1, the adhesion tailspike protein gp9, and three tail accessory factors: gp4, gp10, gp26. We have isolated the tail accessory factor gp4 and characterized its structure and binding interactions with portal protein. Interestingly, gp4 exists in solution as a monomer, which displays an exceedingly low structural stability (Tm 34 degrees C). Unfolded gp4 is prone to aggregation within a narrow range of temperatures both in vitro and in Salmonella extracts. In the virion the thermal unfolding of gp4 is prevented by the interaction with the dodecameric portal protein, which stabilizes the structure of gp4 and suppresses unfolded gp4 from irreversibly aggregating in the Salmonella milieu. The structural stabilization of gp4 is accompanied by the concomitant oligomerization of the protein to form a ring of 12 subunits bound to the lower end of the portal ring. The interaction of gp4 with portal protein is complex and likely involves the distinct binding of two non-equivalent sets of six gp4 proteins. Binding of the first set of six gp4 equivalents to dodecameric portal protein yields a gp(1)12:gp(4)6 assembly intermediate, which is stably populated at 30 degrees C and can be resolved by native gel electrophoresis. The final product of the assembly reaction is a bi-dodecameric gp(1)12:gp(4)12 complex, which appears hollow by electron microscopy, suggesting that gp4 does not physically plug the DNA entry/exit channel, but acts as a structural adaptor for the other tail accessory factors: gp10 and gp26. 相似文献
12.
The interaction between the coat protein of the group I bacteriophage fr with its translational operator site is compared with the previously studied R17 interaction. The sequence of the two RNA binding sites differ by 2 of 20 nucleotides and two coat proteins by 17 of 129 amino acids. An analysis of the binding of fr coat protein to 24 operator variants revealed that the two proteins recognize operator sequences in virtually the same way. However, fr coat protein binds to nearly every RNA 6 to 14-fold tighter than R17 coat protein. Since the fr operator is a weaker binding variant and the fr coat protein shows a different temperature dependence of binding, it is unlikely that the two systems have different Kas in vivo. RNA fragments containing the operator sequences can initiate the capsid assembly with both fr and R17 coat protein. Surprisingly, the two coat proteins can form a mixed capsid in vitro. 相似文献
13.
Nonnative interactions between cysteines direct productive assembly of P22 tailspike protein
下载免费PDF全文
![点击此处可从《Biophysical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Nonnative disulfide bond formation can play a critical role in the assembly of disulfide bonded proteins. During the folding and assembly of the P22 tailspike protein, nonnative disulfide bonds form both in vivo and in vitro. However, the mechanism and identity of cysteine disulfide pairs remains elusive, particularly for P22 tailspike, which contains no disulfide bonds in its native, functional form. Understanding the interactions between cysteine residues is important for developing a mechanistic model for the role of nonnative cysteines in P22 tailspike assembly. Prior in vivo studies have suggested that cysteines 496, 613, and 635 are the most likely site for sulfhydryl reactivity. Here we demonstrate that these three cysteines are critical for efficient assembly of tailspike trimers, and that interactions between cysteine pairs lead to productive assembly of native tailspike. 相似文献
14.
The opdA gene of Salmonella typhimurium encodes an endoprotease, oligopeptidase A (OpdA). Strains carrying opdA mutations were deficient as hosts for phage P22. P22 and the closely related phages L and A3 formed tiny plaques on an opdA host. Salmonella phages 9NA, KB1, and ES18.h1 were not affected by opdA mutations. Although opdA strains displayed normal doubling times and were infected by P22 as efficiently as opdA+ strains, the burst size of infectious particles from an opdA host was less than 1/10 of that from an opdA+ host. This decrease resulted from a reduced efficiency of plating of particles from an opdA infection. In the absence of a functional opdA gene, most of the P22 particles are defective. To identify the target of OpdA action, P22 mutants which formed plaques larger than wild-type plaques on an opdA mutant lawn were isolated. Marker rescue experiments using cloned fragments of P22 DNA localized these mutations to a 1-kb fragment. The nucleotide sequence of this fragment and a contiguous region (including all of both P22 gene 7 and gene 14) was determined. The mutations leading to opdA independence affected the region of gene 7 coding for the amino terminus of gp7, a protein required for DNA injection by the phage. Comparison of the nucleotide sequence with the N-terminal amino acid sequence of gp7 suggested that a 20-amino-acid peptide is removed from gp7 during phage development. Further experiments showed that this processing was opdA dependent and rapid (half-life, less than 2 min) and occurred in the absence of other phage proteins. The opdA-independent mutations lead to mutant forms of gp7 which function without processing. 相似文献
15.
Eighteen single amino acid substitutions in phage P22 coat protein cause temperature-sensitive folding defects (tsf). Three intragenic global suppressor (su) substitutions (D163G, T166I and F170L), localized to a flexible loop, rescue the folding of several tsf coat proteins. Here we investigate the su substitutions in the absence of the original tsf substitutions. None of the su variant coat proteins displayed protein folding defects. Individual su substitutions had little effect on phage production in vivo; yet double and triple combinations resulted in a cold-sensitive (cs) phenotype, consistent with a defect in assembly. During virus assembly and maturation, conformational switching of capsid subunits is required when chemically identical capsid subunits form an icosahedron. Analysis of double- and triple-su phage-infected cell lysates by negative-stain electron microscopy reveals an increase in aberrant structures at the cs temperature. In vitro assembly of F170L coat protein causes production of polyheads, never seen before in phage P22. Purified procapsids composed of all of the su coat proteins showed defects in expansion, which mimics maturation in vitro. Our results suggest that a previously identified surface-exposed loop in coat protein is critical in conformational switching of subunits during both procapsid assembly and maturation. 相似文献
16.
Previous studies have shown that the assembly of the precursor shell (prohead) of bacteriophage P22 requires the copolymerization of the gene 5 coat protein with the gene 8 scaffolding protein. Removal of the scaffolding protein by mutation prevents efficient coat protein assembly, but some aberrant particles do form. We have now isolated these structures and characterized them with respect to morphology, protein composition, and small-angle X-ray scattering properties.The aberrant particles fall into three morphological classes, i.e. complex spirals and closed shells of two sizes. Small-angle X-ray scattering studies confirm that the larger particles are hollow shells with the radius of proheads (), and not of the mature virus (). These structures lack the inner shell of scaffolding protein found in proheads. The small particles have a radius of 195 Å, smaller than proheads, and appear to contain material, not scaffolding protein, within the outer shell.The aberrant particles contain two minor protein species, the gene 9 tail-spike protein, and an unidentified 67,000 molecular weight polypeptide, probably from the host. Neither is found in normal proheads. Removal of gene.9 product by mutation did not affect the formation of the aggregates. Fractionation of the morphological classes of particles revealed that the 67,000 molecular weight band was associated with the closed shells. It may be serving as a pseudo-initiator.Earlier studies had shown that treatment of proheads with sodium dodecyl sulfate in vitro resulted in loss of the scaffolding protein, and expansion of the shell to the mature radius of 285 Å. When the 8? prohead-sized shells were treated similarly, they also expanded to the mature-sized shell. These results support the idea that there are at least two stable states of the coat protein, one of which, the prohead form, is an obligatory precursor of the mature form. 相似文献
17.
The assembly of the precursor shells of bacteriophage P22 entails the co-polymerization of gene 5 coat protein with gene 8 scaffolding protein into double shell structures. During DNA encapsidation, the inner shell of scaffolding molecules dissociates and exits from the prohead. These molecules then recycle, catalyzing the assembly of newly synthesized coat protein to form new proheads (King and Casjens, 1974).Although gene 5 and gene 8 are adjacent on the phage chromosome, we find that the synthesis of the two proteins is differentially regulated. In productively infected cells, scaffolding protein is synthesized at a low rate relative to the coat protein. In contrast, cells that are infected with mutants blocked in DNA packaging and accumulate precursor shells synthesize scaffolding protein at a much higher rate. If a mutation is introduced into the coat protein gene, however, preventing shell assembly, the rate of scaffolding protein synthesis decreases to less than the wild-type rate.The experiments are consistent with models in which either continued synthesis of scaffolding protein depends upon co-polymerization with coat subunits, or soluble scaffolding subunits (but not assembled subunits) depress their own further synthesis. The finding that amber fragments of the scaffolding protein are synthesized at a very low rate is inconsistent with the second model. There is evidence, however, that fragments of the protein may have regulatory activity.The regulatory circuit couples scaffolding protein synthesis to morphogenesis. Gene dosage experiments show that regulation results in the maintenance of coat and scaffolding subunits in the proper ratio for shell assembly. 相似文献
18.
19.
Isolation of mutants in M13 coat protein that affect its synthesis, processing, and assembly into phage 总被引:9,自引:0,他引:9
The major coat protein (gene 8 protein) of bacteriophage M13 has been studied intensively as a model of membrane assembly, protein packing, and protein-DNA interactions. Because this protein is essential for assembly of the phage, very few mutants have been isolated. We have therefore cloned the gene 8 into a plasmid under control of the araB promoter. In the presence of arabinose, the cloned gene is expressed at a rate comparable to that in an M13-infected cell. Plasmid-derived procoat is inserted across the plasma membrane and processed to coat at a normal rate. The coat can support plaque formation by a defective M13 virus (M13am8) with an amber mutation in its procoat gene. This complementation assay was used to screen the mutagenized, cloned gene 8 for mutants which fail to make fully functional coat. Mutants were obtained which fail to synthesize procoat, which do not convert procoat to mature coat protein, or in which the coat protein is incapable of assembling into infectious virions. 相似文献
20.
Tokumoto U Nomura S Minami Y Mihara H Kato S Kurihara T Esaki N Kanazawa H Matsubara H Takahashi Y 《Journal of biochemistry》2002,131(5):713-719
The assembly of iron-sulfur (Fe-S) clusters is mediated by complex machinery which, in Escherichia coli, is encoded by the iscRSUA-hscBA-fdx-ORF3 gene cluster. Here, we demonstrate the network of protein-protein interactions among the components involved in the machinery. We have constructed (His)(6)-tagged versions of the components and identified their interacting partners that were co-purified from E. coli extracts with a Ni-affinity column. Direct associations of the defined pair of proteins were further examined in yeast cells using the two-hybrid system. In accord with the previous in vitro binding and kinetic experiments, interactions were observed for the combinations of IscS and IscU, IscU and HscB, IscU and HscA, and HscB and HscA. In addition, we have identified previously unreported interactions between IscS and Fdx, IscS and ORF3, IscA and HscA, and HscA and Fdx. We also found, by site-directed mutational analysis combined with the two-hybrid system, that two cysteine residues in IscU are essential for binding with HscB but not with IscS. Despite the complex network of interactions in various combinations of components, heteromultimeric complexes were not observed in our experiments except for the putative oligomeric form of IscU-IscS-ORF3. Thus, the sequential association and dissociation among the IscS, IscU, IscA, HscB, HscA, Fdx, and ORF3 proteins may be a critical process in the assembly of Fe-S clusters. 相似文献