共查询到20条相似文献,搜索用时 15 毫秒
1.
Dithiobis (succinimidyl propionate) has been used to cross-link sarcoplasmic reticulum microsome proteins. Although the 100,000 dalton calcium stimulated ATPase and the 60,000 dalton calcium-binding protein calsequestrin were readily cross-linked to form homopolymers, no heteropolymer formation between these two proteins were detected. The 90,000 dalton protein A1 which is always observed in our preparations appeared to preferrentially form dimers on cross-linking. When calsequestrin was solubilized using 0.1 mg deoxycholate/mg protein, this protein was not cross-linked even at dithiobis(succinimidyl propionate) concentrations ten times those used to cross-link this protein in the intact membrane. In a similar manner the deoxycholate-solubilized ATPase (0.5 mg deoxycholate/mg protein) was not cross-linked by dithiobis (succinimidyl propionate). These results suggest that the state of aggregation of the sarcoplasmic reticulum proteins may be modified when solubilized in detergents such as deoxycholate. When the 100,000 dalton ATPase polypeptide was cleaved with trypsin to two fragments with molecular weights of approximately 55,000, these could be readily cross-linked. The fragments were capable of forming polymers with either other 55,000 dalton fragments or with the 100,000 dalton ATPase. The 29,000 and 22,000 dalton fragments, produced by further tryptic cleavage of the 55,000 dalton fragments, were not cross-linked at dithiobis (succinimidyl propionate) concentrations which readily cross-linked the 55,000 dalton fragments. Thus tryptic cleavage of the ATPase to fragments smaller than 55,000 dalton altered associations made by the ATPase in the membrane. 相似文献
2.
The microscopic structures of natural rubber (NR) and deproteinized NR (DPNR) were investigated by means of small-angle neutron scattering (SANS), small-angle X-ray scattering (SAXS), and atomic force microscopy (AFM). They were compared to those of isoprene rubber (IR), which is a synthetic analogue of NR in terms of chemical structure without any non-rubber components like proteins. Comparisons of the structure and mechanical properties of NR, DPNR, and IR lead to the following conclusions. (i) The well-known facts, for example, the outstanding green strength of NR and strain-induced crystallization, are due not much to the presence of proteins but to other components such as the presence of phospholipids and/or the higher stereoregularity of NR. It also became clear the naturally residing proteins accelerate the upturn of stress at low strain. The protein phases work as cross-linking sites and reinforcing fillers in the rubbery matrix. (ii) The microscopic structures of NR were successfully reproduced by SANS intensity functions consisting of squared-Lorentz and Lorentz functions, indicating the presence of inhomogeneities in bulk and thermal concentration fluctuations in swollen state, respectively. On the other hand, IR rubbers were homogeneous in bulk. (iii) The inhomogeneities in NR are assigned to protein aggregates of the order of 200 A or larger. Although these aggregates are larger in size as well as in volume fraction than those of cross-link inhomogeneities introduced by cross-linking, they are removed by deproteinization. (iv) Swelling of both NR and IR networks introduces gel-like concentration fluctuations whose mesh size is of the order of 20 A. 相似文献
3.
When Escherichia coli 30 S ribosomal subunits are reacted with protein-protein bifunctional reagents, a number of protein pairs as well as aggregates containing three or more ribosomal proteins are formed. In the present study we have purified one of the protein pairs obtained by reaction of 30 S ribosomal subunits with either radioactive or nonradioactive dimethylsuberimidate. Following molecular weight determination and ammonolysis, the pair was shown to consist of ribosomal proteins S5 and S8. The "native" structure of the complex was surmised from its capacity to be reconstituted into a biologically active 30 S ribosomal subunit. From peptide maps and primary structure determination of various peptides it was demonstrated that the cross-linking bond between ribosomal proteins S5 and S8 involves primarily the residues Lys-93 of protein S8 and the COOH-terminal lysine (Lys-166) of ribosomal protein S5. This result is substantiated by the finding that a mutant carrying an altered S5 lacking the COOH-terminal lysine yields a greatly reduced amount of S5-S8 cross-link. In addition to the points of cross-linking it was found that Lys-30, Lys-68, and Lys-86 of S8 and Lys-5 of S5 react with dimethylsuberimidate, indicating that these residues are available for reaction and suggesting their topographical localization on the ribosomal surface. 相似文献
4.
The dityrosine bond (DT) is an oxidative covalent cross-link between two tyrosines. DT cross-linking is increasingly identified as a marker of oxidative stress, aging and disease, and has been detected in diverse pathologies. While DT cross- linked proteins have been documented, the consequences of the DT link on the structure and function of the so modified proteins are yet to be understood. With this in view, we have studied the properties of intermolecular DT-dimers of four proteins of diverse functions, namely the enzyme ribonuclease A, the signal protein calmodulin, and the eye lens proteins alpha- and gamma B-crystallins. We find that DT is formed through radical reactions and type I photosensitization (including .OH, O2- and OONO-), but not by 1O2 and NO, (which modify his, trp and met more readily). Tyr residues on the surface of the protein make DT bonds (intra- and intermolecular) most readily and preferentially. The conformation of each of these DT-dimers, monitored by spectroscopy, is seen not to be significantly altered in comparison to that of the parent monomer, but the structural stability of the DT cross-linked molecule is lower than that of the parent native monomer. The DT-dimer is denatured at a lower temperature, and at lower concentrations of urea or guanidinium chloride. The effect of DT-cross-linking on the biological activities of these proteins was next studied. The enzymatic activity of the DT-dimer of ribonuclease A is not lost but lowered. DT-dimerization of lens alpha-crystallin did not significantly affect the chaperone-like ability; it inhibits the self-aggregation and precipitation of target proteins just as well as the parent, unmodified alpha-crystallin does. DT-dimerization of gamma B-crystallin is however seen to lead to more ready aggregation and precipitation, a point of interest in cataract. In the case of calmodulin, we could generate both intermolecular and intramolecular DT cross-linking, and study both the DT-dimer and DT-monomer. The DT-dimer binds smooth muscle light chain kinase and also Ca2+, but less efficiently and over a broad concentration range than the native monomer. The intramolecular DT-monomer is weaker in all these respects, presumably since it is structurally more constrained. These results suggest that DT cross-linking of globular proteins weakens their structural stability and compromises (though does not abolish) their biological activity, both of which are pathologically relevant. The intramolecular DT cross-link would appear to lead to more severe structural and functional consequences. 相似文献
5.
Clustering of integral membrane proteins of the human erythrocyte membrane stimulates autologous IgG binding, complement deposition, and phagocytosis. 总被引:10,自引:0,他引:10
Damaged or old erythrocytes are cleared rapidly from circulation. Because several common biochemical lesions can induce the clustering of integral membrane proteins, we have proposed that formation of microscopic protein aggregates in the membrane might constitute a cell surface marker that promotes removal of the defective/senescent cells. We demonstrate here that treatments that cluster integral membrane proteins in erythrocytes (1 mM ZnCl2, 1 mM acridine orange, and 0.35 microM melittin) induce autologous IgG binding, complement fixation, and phagocytosis by human monocytes in vitro. Removal of the clustering agents prior to incubation in autologous serum or cross-linking of cell surface proteins before addition of clustering agents prohibited the above response, while cross-linking after treatment with the clustering agents preserved the response even if the clustering agents were later removed. Furthermore, subsequent reversal of the chemical cross-link maintaining the clustered distribution also reversed the induction of IgG binding, complement deposition, and phagocytosis. Finally, by deleting or inactivating different steps in the phagocytosis pathway, the chronology of steps was shown to be: (i) integral protein clustering, (ii) IgG binding, (iii) complement deposition, and (iv) phagocytosis. 相似文献
6.
Stabilization of vasopressin-induced membrane events by bifunctional imidoesters 总被引:1,自引:1,他引:0
下载免费PDF全文
![点击此处可从《The Journal of cell biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
《The Journal of cell biology》1981,89(2):261-266
Vasopressin increases the water permeability of the luminal membrane of the toad bladder epithelial cell. This change in permeability correlates with the occurrence in luminal membranes of intramembrane particle aggregates, which may be the sites for transmembrane water flow. Withdrawal of vasopressin is ordinarily associated with a rapid reduction of water flow to baseline values and a simultaneous disappearance of the particle aggregates. The bifunctional imidoesters dithiobispropionimidate (DTBP) and dimethylsuberimidate (DMS), which cross-link amino groups in membrane proteins and lipids, slow the return of water flow to baseline after vasopressin withdrawal. Cross- linking is maximal at pH 10, and is reduced as pH is lowered. Freeze- fracture studies show persistence of luminal membrane particle aggregates in cross-linked bladders and a reduction in their frequency as water flow diminishes. Fusion of aggregate-containing cytoplasmic tubular membrane structures with the luminal membrane is also maintained by the imidoesters. Reductive cleavage of the central S-S bond of DTBP by beta-mercaptoethanol reverses cross-linking, permitting resumption of the rapid disappearance of the vasopressin effect. Bladders that have undergone DTBP cross-linking and beta- mercaptoethanol reduction respond to a second stimulation by vasopressin. Thus, the imidoesters provide a physiologic and reversible means of stabilizing normally rapid membrane events. 相似文献
7.
The recombinant two-on-two hemoglobin from the cyanobacterium Synechoccocus sp. PCC 7002 (S7002 rHb) is a bishistidine hexacoordinate globin capable of forming a covalent cross-link between a heme vinyl and a histidine in the C-terminal helix (H helix). Of the two heme axial histidines, His46 (in the E helix, distal side) and His70 (in the F helix, proximal histidine), His46 is displaced by exogenous ligands. S7002 rHb can be readily prepared as an apoglobin (apo-rHb), a non-cross-linked hemichrome (ferric iron and histidine axial ligands, rHb-R), and a cross-linked hemichrome (rHb-A). To determine the effects of heme binding and subsequent cross-linking, apo-rHb, rHb-R, and rHb-A were subjected to thermal denaturation and 1H/2H exchange. Interpretation of the latter data was based on nuclear magnetic resonance assignments obtained with uniformly 15N- and 13C,15N-labeled proteins. Apo-rHb was found to contain a cooperative structural core, which was extended and stabilized by heme binding. Cross-linking resulted in further stabilization attributed mainly to an unfolded-state effect. Protection factors were higher at the cross-link site and near His70 in rHb-A than in rHb-R. In contrast, other regions became less resistant to exchange in rHb-A. These included portions of the B and E helices, which undergo large conformational changes upon exogenous ligand binding. Thus, the cross-link readjusted the dynamic properties of the heme pocket. 1H/2H exchange data also revealed that the B, G, and H helices formed a robust core regardless of the presence of the heme or cross-link. This motif likely encompasses the early folding nucleus of two-on-two globins. 相似文献
8.
《Micron and Microscopica Acta》1988,19(4):213-226
Exposure to formaldehyde brings about small but readily detectable changes in the staining behaviour of collagen fibrils. These changes can be interpreted in chemical terms by comparing fibril staining patterns with artificial patterns computer-generated from sequence data. Positive staining with phosphotung-state (where heavy metal is confined to anions), shows that most of the lysyl and hydroxylysyl side-chains lose their charge character as a result of formaldehyde treatment and cease to take up staining ions. The charge character of arginyl (and probably histidyl) residues is unaltered and these residues continue to react with stain. Acidic residues are also unaffected. These results accord with biochemical evidence that the initial reaction between proteins and formaldehyde leading to subsequent cross-linking involves modification of ε-amino (and α-amino) groups. They show too that the secondary condensation producing the actual cross-link does not alter the charge character of the second group, at least when it is on an arginyl (or histidyl) side-chain.Formaldehyde-induced changes in stain deposition can also be detected after negative staining, although they are slight compared with those brought about by glutaraldehyde. Unlike glutaraldehyde, formaldehyde introduces no bulky polymeric adducts into the fibril structure, and the conspicuous stain-excluding bands seen in negative staining patterns following glutaraldehyde fixation are absent after exposure to formaldehyde. For this reason, where chemical fixation is used to stabilize macromolecules and supramolecular aggregates prior to negative staining and high resolution electron optical imaging, formaldehyde would seem to be preferable to glutaraldehyde. Data from fibril staining patterns and from thermal stability measurements (made on collagen gels) show that formaldehyde fixation does not preclude a subsequent reaction with glutaraldehyde.As with other fixatives, there is reduced accessibility to stain after formaldehyde treatment. Accessibility is least in the overlap zone where the denser packing of collagen molecules provides greater opportunities for intermolecular cross-linking. Gel electrophoresis confirms that formaldehyde-induced cross-links in fibrils are predominantly intermolecular. 相似文献
9.
10.
Ritchie H Lawrie LC Crombie PW Mosesson MW Booth NA 《The Journal of biological chemistry》2000,275(32):24915-24920
In this study, we identified lysine residues in the fibrinogen Aalpha chain that serve as substrates during transglutaminase (TG)-mediated cross-linking of plasminogen activator inhibitor 2 (PAI-2). Comparisons were made with alpha(2)-antiplasmin (alpha(2)-AP), which is known to cross-link to lysine 303 of the Aalpha chain. A 30-residue peptide containing Lys-303 specifically competed with fibrinogen for cross-linking to alpha(2)-AP but not for cross-linking to PAI-2. Further evidence that PAI-2 did not cross-link via Lys-303 was the cross-linking of PAI-2 to I-9 and des-alphaC fibrinogens, which lack 100 and 390 amino acids from the C terminus of the Aalpha chain, respectively. PAI-2 or alpha(2)-AP was cross-linked to fibrinogen and digested with trypsin or endopeptidase Glu-C, and the resulting peptides analyzed by mass spectrometry. Peptides detected were consistent with tissue TG (tTG)-mediated cross-linking of PAI-2 to lysines 148, 176, 183, 457 and factor XIIIa-mediated cross-linking of PAI-2 to lysines 148, 230, and 413 in the Aalpha chain. alpha(2)-AP was cross-linked only to lysine 303. Cross-linking of PAI-2 to fibrinogen did not compete with alpha(2)-AP, and the two proteins utilized different lysines in the Aalpha chain. Therefore, PAI-2 and alpha(2)-AP can cross-link simultaneously to the alpha polymers of a fibrin clot and promote resistance to lysis. 相似文献
11.
Self-assembly of complex structures is commonplace in biology but often poorly understood. In the case of the actin cytoskeleton, a great deal is known about the components that include higher order structures, such as lamellar meshes, filopodial bundles, and stress fibers. Each of these cytoskeletal structures contains actin filaments and cross-linking proteins, but the role of cross-linking proteins in the initial steps of structure formation has not been clearly elucidated. We employ an optical trapping assay to investigate the behaviors of two actin cross-linking proteins, fascin and α-actinin, during the first steps of structure assembly. Here, we show that these proteins have distinct binding characteristics that cause them to recognize and cross-link filaments that are arranged with specific geometries. α-Actinin is a promiscuous cross-linker, linking filaments over all angles. It retains this flexibility after cross-links are formed, maintaining a connection even when the link is rotated. Conversely, fascin is extremely selective, only cross-linking filaments in a parallel orientation. Surprisingly, bundles formed by either protein are extremely stable, persisting for over 0.5 h in a continuous wash. However, using fluorescence recovery after photobleaching and fluorescence decay experiments, we find that the stable fascin population can be rapidly competed away by free fascin. We present a simple avidity model for this cross-link dissociation behavior. Together, these results place constraints on how cytoskeletal structures assemble, organize, and disassemble in vivo. 相似文献
12.
The covalent cross-linking of cell wall proteins into the cell wall glucan/chitin matrix is an important step in the biogenesis of the fungal cell wall. We demonstrate that the Neurospora crassa DFG5 (NCU03770) and DCW1 (NCU08127) enzymes function in vivo to cross-link glycoproteins into the cell wall. Mutants lacking DFG5 or DCW1 release slightly elevated levels of cell wall proteins into their growth medium. Mutants lacking both DFG5 and DCW1 have substantially reduced levels of cell wall proteins in their cell walls and release large amounts of known cell wall proteins into the medium. DFG5 and DCW1 are members of the GH76 family of glycosyl hydrolases, which have specificity to recognize and cleave α-1,6-mannans. A model for incorporation of glycoproteins into the cell wall through the α-1,6-mannan core of the N-linked galactomannan is presented. In this model, DFG5 and DCW1 recognize the N-linked galactomannan present on glycoproteins and cross-link it into the cell wall glucan/chitin matrix. 相似文献
13.
The increased interest in chemical cross-linking for probing protein structure and interaction has led to a large increase in literature describing new cross-linkers and search programs. However, this has not led to a corresponding increase in the analysis of large and complex proteins. A major obstacle is that the new cross-linkers are either not readily available and/or have a low reactivity. In combination with aging search programs that are slow and have low sensitivity, or new search programs that are described but not released, these efforts do little to advance the field of cross-linking. Here we present a method pipeline for chemical cross-linking, using two standard cross-linkers, BS3 and BS2G, combined with our freely available CrossWork search program. By this approach we generate cross-link data sufficient to derive structural information for large and complex proteins. CrossWork searches batches of tandem mass-spectrometric data, and identifies cross-linked and non-cross-linked peptides using a standard PC. We tested CrossWork by searching mass-spectrometric datasets of cross-linked complement factor C3 against small (1 protein) and large (1000 proteins) search spaces, and show that the resulting distance constraints agree with the established structures. We further investigated the structure of the multi-domain ERp72, and combined the individual domains of ERp72 into a single structure. 相似文献
14.
The dityrosine bond (DT) is an oxidative covalent cross-link between two tyrosines. DT cross-linking is increasingly identified as a marker of oxidative stress, aging and disease, and has been detected in diverse pathologies. While DT cross- linked proteins have been documented, the consequences of the DT link on the structure and function of the so modified proteins are yet to be understood. With this in view, we have studied the properties of intermolecular DT-dimers of four proteins of diverse functions, namely the enzyme ribonuclease A, the signal protein calmodulin, and the eye lens proteins alpha- and gamma B-crystallins. We find that DT is formed through radical reactions and type I photosensitization (including OH, O2
– and OONO–), but not by 1O2 and NO2
– (which modify his, trp and met more readily). Tyr residues on the surface of the protein make DT bonds (intra- and intermolecular) most readily and preferentially. The conformation of each of these DT-dimers, monitored by spectroscopy, is seen not to be significantly altered in comparison to that of the parent monomer, but the structural stability of the DT cross-linked molecule is lower than that of the parent native monomer. The DT-dimer is denatured at a lower temperature, and at lower concentrations of urea or guanidinium chloride. The effect of DT-cross-linking on the biological activities of these proteins was next studied. The enzymatic activity of the DT-dimer of ribonuclease A is not lost but lowered. DT-dimerization of lens alpha-crystallin did not significantly affect the chaperone-like ability; it inhibits the self-aggregation and precipitation of target proteins just as well as the parent, unmodified alpha-crystallin does. DT-dimerization of gamma B-crystallin is however seen to lead to more ready aggregation and precipitation, a point of interest in cataract. In the case of calmodulin, we could generate both intermolecular and intramolecular DT cross-linking, and study both the DT-dimer and DT-monomer. The DT-dimer binds smooth muscle light chain kinase and also Ca2+, but less efficiently and over a broad concentration range than the native monomer. The intramolecular DT-monomer is weaker in all these respects, presumably since it is structurally more constrained. These results suggest that DT cross-linking of globular proteins weakens their structural stability and compromises (though does not abolish) their biological activity, both of which are pathologically relevant. The intramolecular DT cross-link would appear to lead to more severe structural and functional consequences. 相似文献
15.
Binding of proteins to DNA is usually considered 1D with one protein bound to one DNA molecule. In principle, proteins with multiple DNA binding domains could also bind to and thereby cross-link different DNA molecules. We have investigated this possibility using high-mobility group A1 (HMGA1) proteins, which are architectural elements of chromatin and are involved in the regulation of multiple DNA-dependent processes. Using direct stochastic optical reconstruction microscopy (dSTORM), we could show that overexpression of HMGA1a-eGFP in Cos-7 cells leads to chromatin aggregation. To investigate if HMGA1a is directly responsible for this chromatin compaction we developed a DNA cross-linking assay. We were able to show for the first time that HMGA1a can cross-link DNA directly. Detailed analysis using point mutated proteins revealed a novel DNA cross-linking domain. Electron microscopy indicates that HMGA1 proteins are able to create DNA loops and supercoils in linearized DNA confirming the cross-linking ability of HMGA1a. This capacity has profound implications for the spatial organization of DNA in the cell nucleus and suggests cross-linking activities for additional nuclear proteins. 相似文献
16.
Analysis of multiple forms of nuclear factor I in human and murine cell lines. 总被引:10,自引:4,他引:10
下载免费PDF全文
![点击此处可从《Molecular and cellular biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Nuclear factor I (NFI) is a group of related site-specific DNA-binding proteins that function in adenovirus DNA replication and cellular RNA metabolism. We have measured both the levels and forms of NFI that interact with a well-characterized 26-base-pair NFI-binding site. Five different NFI-DNA complexes were seen in HeLa nuclear extracts by using a gel mobility shift (GMS) assay. In addition, at least six forms of NFI were shown to cross-link directly to DNA by using a UV cross-linking assay. The distinct GMS complexes detected were composed of different subspecies of NFI polypeptides as assayed by UV cross-linking. Different murine cell lines possessed varying levels and forms of NFI binding activity, as judged by nitrocellulose filter binding and GMS assays. The growth state of NIH 3T3 cells affected both the types of NFI-DNA complexes seen in a GMS assay and the forms of the protein detected by UV cross-linking. 相似文献
17.
The structural consequences of MgADP binding at the vicinity of the ATPase-related thiol SH1 (Cys-707) have been examined by subjecting myosin subfragment 1, premodified at SH2 (Cys-697) with N-ethylmaleimide (NEM), to reaction with the bifunctional reagent p-phenylenedimaleimide (pPDM) in the presence and absence of MgADP. By monitoring the changes in the Ca2(+)-ATPase activity as a function of reaction time, it appears that the reagent rapidly modifies SH1 irrespective of whether MgADP is present or not. In the absence of nucleotide, only extremely low levels of cross-linking to the 50-kDa middle segment of S1 can be detected, while in the presence of MgADP substantial cross-linking to this segment is observed. A similar cross-link is also formed if MgADP is added subsequent to the reaction of the SH2-NEM-pre-modified S1 with pPDM in the absence of nucleotide. Isolation of the labeled tryptic peptide from the cross-linked adduct formed with [14C]pPDM, and subsequent partial sequence analyses, indicates that the cross-link is made from SH1 to Cys-522. Moreover, it appears that this cross-link results in the trapping of MgADP in this S1 species. These data suggest that the binding of MgADP results in a change in the structure of S1 in the vicinity of the SH1 thiol relative to the 50-kDa "domain" which enables Cys-522 to adopt the appropriate configuration to enable it to be cross-linked to SH1 by pPDM. 相似文献
18.
Arthur J L Cooper Thomas M Jeitner Vittorio Gentile John P Blass 《Neurochemistry international》2002,40(1):53-67
Protein aggregates are a hallmark of Huntington's disease (HD) and other inherited neurodegenerative diseases caused by an elongated (CAG)(n) repeat in the genome and to a corresponding increase in the size of the Q(n) domain in the expressed protein. When the protein associated with HD (huntingtin) contains <35 Q repeats disease does not occur. However, an n>/=40 leads to disease. Some investigators have proposed that aggregates in the nuclei of affected cells are toxic, but other workers have suggested that the aggregates may be neutral or even protective. Whether or not they are toxic, an understanding of the processes whereby the aggregates develop may shed light on the neuropathological processes involved in the (CAG)(n)/Q(n)-expansion disorders. Q(n) domains have a tendency to non-covalently self align as 'polar zippers' rendering them less soluble, but evidence that such polar zippers occur in the aggregates in intact HD brain has so far been limited. The human brain contains at least three Ca(2+)-dependent enzymes (transglutaminases, TGases) that catalyze protein cross-linking reactions, namely TGase 1, TGase 2 (tissue transglutaminase, tTGase) and TGase 3. Q(n) aggregates have been found by several groups to be excellent substrates of tTGase. Moreover, the activity toward the Q(n) domains increases greatly as n is increased to 40 or beyond. tTGase mRNA and total TGase activity are elevated in HD brain. Moreover, some evidence suggests that Ca(2+) homeostasis is disrupted in HD brain. We propose that the combination of increased huntingtin (or huntingtin fragment containing the Q(n) domain) in the nucleus, increased the ability of the Q(n) domains to act as substrate, increased Ca(2+) levels and increased inherent TGase activity all contribute to increased cross-linking of proteins in HD brain. At first the proteasome machinery can recognize and degrade the cross-linked proteins, but over time the proteasome machinery may be overwhelmed and protein aggregates will accumulate. 相似文献
19.
Nuclear mRNA precursors are spliced by a large macromolecular complex called the spliceosome which contains, in most eucaryotes, five small nuclear RNAs (snRNAs) each in the form of a small ribonucleoprotein particle (the U1, U2, U5, and U4/U6 snRNPs). Although secondary structures have been derived for all five spliceosomal snRNAs based on phylogenetic, biochemical, and genetic data, little tertiary structure information is available. Here we use the general cross-linking reagent nitrogen mustard [bis-(2-chloroethyl)methylamine] to detect tertiary interactions within U2 snRNA. After the cross-linking of deproteinized HeLa nuclear extract, two intramolecularly cross-linked U2 species with anomalous electrophoretic mobility can be detected (X-U2#1 and X-U2#2). The 3' and 5' boundaries of each cross-link were determined by rapid enzymatic RNA sequencing of end-labeled RNA. X-U2#1 is cross-linked between the region U41-U55 and G105 or G106, X-U2#2 between U53 and G97 or G98. We then tested the ability of the two cross-linked species to bind snRNP proteins in vitro (in nuclear extract or S100) and in vivo (in Xenopus oocytes). X-U2#2 reconstituted efficiently both in vitro and in vivo but X-U2#1 did not, as judged by immunoprecipitation with antibodies specific for Sm- and U2-specific proteins. Since the cross-link in X-U2#2 involves the Sm binding site but does not block snRNP assembly, our data strongly suggest that the Sm binding site lies on the surface of the native snRNP. 相似文献
20.
Interactions between VirB9 and VirB10 membrane proteins involved in movement of DNA from Agrobacterium tumefaciens into plant cells. 总被引:3,自引:1,他引:2
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 11 VirB proteins from Agrobacterium tumefaciens are predicted to form a membrane-bound complex that mediates the movement of DNA from the bacterium into plant cells. The studies reported here on the possible VirB protein interactions in such a complex demonstrate that VirB9 and VirB10 can each form high-molecular-weight complexes after treatment with a chemical cross-linker. Analysis of nonpolar virB mutants showed that the formation of the VirB10 complexes does not occur in a virB9 mutant and that VirB9 and VirB10 are not components of the same cross-linked complex. VirB9, when stabilized by the concurrent expression of VirB7, was shown to be sufficient to permit VirB10 to cross-link into its usual high-molecular-weight forms in the absence of other Vir proteins. Randomly introduced single point mutations in virB9 resulted in Agrobacterium strains with severely attenuated virulence. Although some of the mutants contained wild-type levels of VirB9 and displayed an unaltered VirB9 cross-linking pattern, VirB10 cross-linking was drastically reduced. We conclude that specific amino acid residues in VirB9 are necessary for interaction with VirB10 resulting in the capacity of VirB10 to participate in high-molecular-weight complexes that can be visualized by chemical cross-linking. 相似文献