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PubMed1999

Characterization of a protein complex containing spliceosomal proteins SAPs 49, 130, 145, and 155.

SF3b is a U2 snRNP-associated protein complex essential for spliceosome assembly. Although evidence that SF3b contains the spliceosomal proteins SAPs 49, 130, 145, and 155 has accumulated, a protein-mediated association between all of these proteins has yet to be directly demonstrated. Here we report the isolation of a cDNA encoding SAP 130, which completes the cloning of the putative SF3b complex proteins. Using antibodies to SAP 130 and other putative SF3b components, we showed that SAPs 130, 145, and 155 are present in a protein complex in nuclear extracts and that these proteins associate with one another in purified U2 snRNP. Moreover, SAPs 155 and 130 interact with each other (directly or indirectly) within this complex, and SAPs 49 and 145 are known to interact directly with each other. Thus, together with prior work, our studies indicate that SAPs 49, 130, 145, and 155 are indeed components of SF3b. The Saccharomyces cerevisiae homologs of SAPs 49 and 145 are encoded by essential genes. We show here that the S. cerevisiae homologs of SAPs 130 and 155 (scSAP 130/RSE1 and scSAP 155, respectively) are also essential. Recently, the SF3b proteins were found in purified U12 snRNP, which functionally substitutes for U2 snRNP in the minor spliceosome. This high level of conservation, together with the prior observation that the SF3b proteins interact with pre-mRNA very close to the branch site, suggest that the SF3b complex plays a critical role near or at the spliceosome catalytic core.

Adaptor Proteins, Signal Transducing↗
PubMed1978

Filter-binding assay for covalent DNA-protein complexes: adenovirus DNA-terminal protein complex.

A rapid, simple, and quantitative filter-binding assay using glass fiber filters has been developed to detect the convalent adenovirus DNA-terminal protein complex. The assay is unusually sensitive because binding of protein-free DNA generally is less than 0.1%. Binding of the adenovirus complex to filters is mediated by terminal protein. We have found that: (i) the adenovirus complex binds maximally to filters in NaCl at concentrations higher than 0.2 M; (ii) noncovalent complexes between protein-free DNA and adenovirus proteins bind to filters in salt at concentrations lower than 0.4 M but not in concentrations higher than 0.7 M; and (iii) protein-free DNA alone binds to filters in guanidine.hydrochloride at concentrations higher than 0.8 M. By varying the ionic conditions, "all or none" modulation of these interactions can be achieved.

Adenoviridae↗
PubMed2000

Involvement of iron (ferric) reduction in the iron absorption mechanism of a trivalent iron-protein complex (iron protein succinylate).

Iron protein succinylate is a non-toxic therapeutic iron compound. We set out to characterise the structure of this compound and investigate the importance of digestion and intestinal reduction in determining absorption of the compound. The structure of the compound was investigated by variable temperature Mössbauer spectroscopy, molecular size determinations and kinetics of iron release by chelators. Intestinal uptake was determined with radioactive compound force fed to mice. Reduction of the compound was determined by in vitro incubation with intestinal fragments. The compound was found to contain only ferric iron, present as small particles including sizes below 10 nm. The iron was released rapidly to chelators. Digestion with trypsin reduced the molecular size of the compound. Intestinal absorption of the compound was inhibited by a ferrous chelator (ferrozine), indicating that reduction to ferrous iron may be important for absorption. The native compound was a poor substrate for duodenal reduction activity, but digestion with pepsin, followed by pancreatin, released soluble iron complexes with an increased reduction rate. We conclude that iron protein succinylate is absorbed by a mechanism involving digestion to release soluble, available ferric species which may be reduced at the mucosal surface to provide ferrous iron for membrane transport into enterocytes.

Animals↗
PubMed1998

High performance density gradient electrophoresis of subcellular organelles, protein complexes and proteins.

A density gradient electrophoresis (DGE) apparatus (2.2 x, 14 cm) was constructed for the rapid separation of milligram quantities of proteins. By using binary buffers according to Bier (Electrophoresis 1993, 14, 1011-1018) proteins were rate-zonally separated in less than 60 min. Acidic proteins were separated in a pH 8.6, 56 microS/cm buffer, and basic proteins in a pH 5.4, 76 microS/cm buffer. Thus the A (pI 5.15) and B (pI 5.30) forms of beta-lactoglobulin as well as the sialylated glycoforms of apotransferrin were well separated at pH 8.6. The isoforms of myoglobin (pI 6.9 and 7.35, respectively), RNAse A (pI 9.45) and cytochrome c (pI 10.0) and lysozyme (pI 11) were separated at pH 5.4 within 80 min. On a 7 cm DGE column, subcellular organelles derived from HeLa cells were separated in standard electrophoresis buffer (655 microS/cm) for 90 min at 10 mA. Using a new low conductivity buffer (193 microS/cm) 20 min was sufficient to separate late endosomes, lysosomes, endoplasmic reticulum, early endosomes, plasma membrane, clathrin-coated pits, proteasomes, and clathrin-coated vesicles within a single run directly from a postnuclear supernatant.

Cell Fractionation↗
PubMed2001

Spin-column isolation of DNA-protein interactions from complex protein mixtures for AFM imaging.

Applications of atomic force microscopy (AFM) to investigate structural-functional interactions between DNA and proteins, at the molecular level, should prove valuable for gaining a better understanding of gene expression. Specific genomic DNA-protein interactions occur within a sea of intracellular proteins. Successful AFM imaging requires isolating the specific DNA-protein complex free of background protein contamination. Using spin-column chromatography, we report the successful isolation and AFM imaging of transcription factor DNA complexes from DNA molecules incubated with crude cell lysates. This method should be applicable for the isolation and imaging of other specific DNA-protein complexes pertinent to functional genomic research.

Chromatography, Gel↗
PubMed1980

Fractionation of Thylakoid Membranes with the Nonionic Detergent Octyl-beta-d-glucopyranoside: RESOLUTION OF CHLOROPHYLL-PROTEIN COMPLEX II INTO TWO CHLOROPHYLL-PROTEIN COMPLEXES.

The detergent octyl-beta-d-glucopyranoside (30 millimolar in 2 millimolar Tris-maleate, pH 7.0) preferentially extracts complexes containing protein and chlorophylls a plus b (CP) from spinach, leaving a residue highly enriched in CP I (P700-chlorophyll a protein). Use of the detergent results in a relatively gentle extraction since little free chlorophyll is formed and since sodium dodecyl sulfate-gel electrophoresis (on 10% acrylamide) of the extract also reveals the presence of two minor chlorophyll a complexes (apparent molecular weight, 47,000 and 43,000) instead of the usual single complex. The major complex preserved is CP 64, a chlorophyll a/b complex (apparent molecular weight, 64,000) which is an oligomer of another chlorophyll a/b complex, CP 27, the light-harvesting complex (apparent molecular weight, 27,000). Dissociation of each complex reveals two polypeptides (molecular weight, 32,000 and 28,000) and limited proteolysis confirms that those of CP 64 have the same structure as those of CP 27. An additional chlorophyll a/b complex (apparent molecular weight, 29,000) is clearly separable from CP 27, and differs from it and CP 64 in having a higher chlorophyll a/b ratio and a single polypeptide (molecular weight, 29,000) which differs structurally from those of the other complexes.

Journal Article↗
PubMed2005

High-resolution diffraction from crystals of a membrane-protein complex: bacterial outer membrane protein OmpC complexed with the antibacterial eukaryotic protein lactoferrin.

Crystals of the complex formed between the outer membrane protein OmpC from Escherichia coli and the eukaryotic antibacterial protein lactoferrin from Camelus dromedarius (camel) have been obtained using a detergent environment. Initial data processing suggests that the crystals belong to the hexagonal space group P6, with unit-cell parameters a = b = 116.3, c = 152.4 A, alpha = beta = 90, gamma = 120 degrees. This indicated a Matthews coefficient (VM) of 3.3 A3 Da(-1), corresponding to a possible molecular complex involving four molecules of lactoferrin and two porin trimers in the unit cell (4832 amino acids; 533.8 kDa) with 63% solvent content. A complete set of diffraction data was collected to 3 A resolution at 100 K. Structure determination by molecular replacement is in progress. Structural study of this first surface-exposed membrane-protein complex with an antibacterial protein will provide insights into the mechanism of action of OmpC as well as lactoferrin.

Animals↗
PubMed1990

Cap binding protein complex that restores protein synthesis in heat-shocked Ehrlich cell lysates contains highly phosphorylated eIF-4E.

Cell-free protein-synthesizing systems derived from Ehrlich ascites tumor cells that have been exposed to elevated temperatures retain the inhibition of translation that is seen at the cellular level. A multisubunit cap binding protein complex able to restore protein synthesis in these cell free systems was purified from Ehrlich ascites tumor cells via affinity chromatography using m7GTP-Sepharose and fast protein liquid chromatography on Mono Q. The purified complex contains an Mr 220,000 polypeptide (p220) and an Mr 28,000 polypeptide (p28), both of which are components of eukaryotic initiation factor 4F (eIF-4F). p28 is identical to eIF-4E. Restoring activity was relatively free of the Mr 46,000 polypeptide (p46) that is the third component of eIF-4F and does not appear to be dependent on its presence. p28 associated in a complex with p220 is 85% phosphorylated; however, the majority of p28 is not associated with p220, and this free form is only about 50% phosphorylated. The correlation between association of p28 with p220 and high levels of p28 phosphorylation suggests a possible role for phosphorylation in association of p220 with p28.

Animals↗
PubMed1980

[Structural organization of membranes reconstituted from phospholipids and subchromatophore pigment-protein complexes].

Pigment--protein complexes of the P870 reaction centers and complexes of the bacteriochlorophyll light-harvesting antenna were isolated from the chromatophores of the non-sulfur purple bacterium Rhodospirillum rubrum by solubilization with detergents. The proteoliposomes containing the reaction centers or reaction centers and the light-harvesting antenna as well as liposomes formed from phospholipids were obtained by a self-assembly procedure using seya bean phospholipids. The freeze-fracture study showed that the proteoliposomes contain a large amount of globular particles. The particles incorporated into the two types of the proteoliposomes were distinguished in size. The globules of the reaction center and antenna complexes were bigger in size than the reaction center globules. The globular structures were not found in the liposomal membranes. The liposomes formed in the absence of the pigment--protein complexes were predominantly the multilamellar vesicles. The proteoliposomes were mostly represented as monolamellar membrane vesicles. The spatial arrangement of the reaction center complexes in the membranes is discussed.

Bacterial Chromatophores↗
PubMed1999

Nondenaturing electrophoresis. Fractionating of photosynthetic pigment--protein complexes and blood plasma proteins.

Efficient polyacrylamide gel electrophoresis of labile proteins and protein complexes is reviewed. If only 0.001-0.01% SDS is dissolved in the electrode buffers, the detergent does not exhibit denaturing activity and guarantees high quality of electrophoresis. Even the structure and oxygen-producing activity of the labile photosystem PS2 are preserved after electrophoretic separation of photosynthetic pigment--protein complexes from Anacystis nidulans R2 or other cyanobacteria. The overall spectra of absorption or fluorescence of isolated pigment--protein complexes are equal to the corresponding spectra of the photosynthetic membrane. The distribution of chlorophyll molecules between the components of the photosynthetic apparatus coincides in spectral analysis data and gel fraction densitometry. More than 15 electrophoretic fractions of pigment--protein complexes of chloroplasts from green algae and higher plants were observed including some fractions of PS1, some spectrally different forms of light harvesting pigment--protein complexes, and their oligomers. High resolving capacity of electrophoresis was demonstrated by separation of plasma proteins. Low denaturing activity and low thermal dissipation of the electrode buffer solution allow the use of large diameter tubes (3.5 and 8 cm) in polyacrylamide gel electrophoresis. The cell destruction time and the membrane dissolving time are minimized. The method of electrophoretic staining of the gels was tested.

Blood Proteins↗
PubMed1985

Mutants of Sweetclover (Melilotus alba) Lacking Chlorophyll b: Studies on Pigment-Protein Complexes and Thylakoid Protein Phosphorylation.

Mutants of sweetclover (Melilotus alba) with defects in the nuclear ch5 locus were examined. Using thin-layer chromatography and absorption spectroscopy, three of these mutants were found to lack chlorophyll (Chl) b. One of these three mutants, U374, possessed thylakoid membranes lacking the three Chl b-containing pigment-protein complexes (AB-1, AB-2, and AB-3) while still containing A-1 and A-2, Chl a complexes derived from photosystems I and II, respectively. Complete solubilization and denaturation of the thylakoid proteins from this mutant revealed very little apoprotein from the Chl b-containing light-harvesting complexes, the major thylakoid proteins in normal plants. The normal and mutant sweetclover plants had active thylakoid protein kinase activities and numerous polypeptides were labeled following incubation with [gamma-(32)P]ATP. With the U374 mutant, however, there was very little detectable label co-migrating with the light-harvesting complex apoproteins on polyacrylamide gels. The Chl b-deficient chlorina-f2 mutant of barley (Hordeum vulgare) also had an active protein kinase activity capable of phosphorylating numerous polypeptides, including ones migrating with the same mobility as the light-harvesting complex apoproteins. These results indicate that the sweetclover mutants may be useful systems for studies on the function and organization of Chl b in thylakoid membranes of higher plants.

Journal Article↗
PubMed2003

Interaction and assembly of murine pre-replicative complex proteins in yeast and mouse cells.

Eukaryotic cells coordinate chromosome duplication by the assembly of protein complexes at origins of DNA replication by sequential binding of member proteins of the origin recognition complex (ORC), CDC6, and minichromosome maintenance (MCM) proteins. These pre-replicative complexes (pre-RCs) are activated by cyclin-dependent kinases and DBF4/CDC7 kinase. Here, we carried out a comprehensive yeast two-hybrid screen to establish sequential interactions between two individual proteins of the mouse pre-RC that are probably required for the initiation of DNA replication. The studies revealed multiple interactions among ORC subunits and MCM proteins as well as interactions between individual ORC and MCM proteins. In particular CDC6 was found to bind strongly to ORC1 and ORC2, and to MCM7 proteins. DBF4 interacts with the subunits of ORC as well as with MCM proteins. It was also demonstrated that CDC7 binds to different ORC and MCM proteins. CDC45 interacts with ORC1 and ORC6, and weakly with MCM3, -6, and -7. The three subunits of the single-stranded DNA binding protein RPA show interactions with various ORC subunits as well as with several MCM proteins. The data obtained by yeast two-hybrid analysis were paradigmatically confirmed in synchronized murine FM3A cells by immunoprecipitation of the interacting partners. Some of the interactions were found to be cell-cycle-dependent; however, most of them were cell-cycle-independent. Altogether, 90 protein-protein interactions were detected in this study, 52 of them were found for the first time in any eukaryotic pre-RC. These data may help to understand the complex interplay of the components of the mouse pre-RC and should allow us to refine its structural architecture as well as its assembly in real time.

Animals↗
PubMed1987

Protein precipitation assay for quantitation of tannins: determination of protein in tannin-protein complex.

A protein precipitation method for the determination of tannins has been developed. The protein in the tannin-protein complexes was measured using the ninhydrin assay of amino acids released by alkaline hydrolysis of the complex. Standard protein and the complex were hydrolyzed with 13.5 N NaOH at 120 degrees C for 20 min and the amino acids released were measured with ninhydrin. Tannins did not interfere in the determination of protein by ninhydrin assay. The bovine serum albumin (BSA) precipitated (y; mg) increased linearly with increase in tannic acid (x) from 0.2 to 0.9 mg (y = 2.598x - 0.258). The protein precipitation capacities (mg BSA precipitated/g dry wt) measured by the method for young and mature leaves of oaks were Quercus incana (young, 42.21; mature, 79.51), Q. ilex (young, 1.86; mature, 1.86), and Q. semecarpifolia (young, 733.54; mature, 304.32). The method can provide valuable information on the mechanisms of protein-tannin interactions and nutritional and physiological significances of tannins.

Animals↗
PubMed2002

A role for the Saccharomyces cerevisiae RENT complex protein Net1 in HMR silencing.

Silencing in the yeast Saccharomyces cerevisiae is known in three classes of loci: in the silent mating-type loci HML and HMR, in subtelomeric regions, and in the highly repetitive rDNA locus, which resides in the nucleolus. rDNA silencing differs markedly from the other two classes of silencing in that it requires a DNA-associated protein complex termed RENT. The Net1 protein, a central component of RENT, is required for nucleolar integrity and the control of exit from mitosis. Another RENT component is the NAD(+)-dependent histone deacetylase Sir2, which is the only silencing factor known to be shared among the three classes of silencing. Here, we investigated the role of Net1 in HMR silencing. The mutation net1-1, as well as NET1 expression from a 2micro-plasmid, restored repression at silencing-defective HMR loci. Both effects were strictly dependent on the Sir proteins. We found overexpressed Net1 protein to be directly associated with the HMR-E silencer, suggesting that Net1 could interact with silencer binding proteins and recruit other silencing factors to the silencer. In agreement with this, Net1 provided ORC-dependent, Sir1-independent silencing when artificially tethered to the silencer. In contrast, our data suggested that net1-1 acted indirectly in HMR silencing by releasing Sir2 from the nucleolus, thus shifting the internal competition for Sir2 from the silenced loci toward HMR.

Cell Cycle Proteins↗
PubMed2004

Predicting co-complexed protein pairs using genomic and proteomic data integration.

BACKGROUND: Identifying all protein-protein interactions in an organism is a major objective of proteomics. A related goal is to know which protein pairs are present in the same protein complex. High-throughput methods such as yeast two-hybrid (Y2H) and affinity purification coupled with mass spectrometry (APMS) have been used to detect interacting proteins on a genomic scale. However, both Y2H and APMS methods have substantial false-positive rates. Aside from high-throughput interaction screens, other gene- or protein-pair characteristics may also be informative of physical interaction. Therefore it is desirable to integrate multiple datasets and utilize their different predictive value for more accurate prediction of co-complexed relationship. RESULTS: Using a supervised machine learning approach--probabilistic decision tree, we integrated high-throughput protein interaction datasets and other gene- and protein-pair characteristics to predict co-complexed pairs (CCP) of proteins. Our predictions proved more sensitive and specific than predictions based on Y2H or APMS methods alone or in combination. Among the top predictions not annotated as CCPs in our reference set (obtained from the MIPS complex catalogue), a significant fraction was found to physically interact according to a separate database (YPD, Yeast Proteome Database), and the remaining predictions may potentially represent unknown CCPs. CONCLUSIONS: We demonstrated that the probabilistic decision tree approach can be successfully used to predict co-complexed protein (CCP) pairs from other characteristics. Our top-scoring CCP predictions provide testable hypotheses for experimental validation.

Computational Biology↗
PubMed1997

Binding of mammalian ribosomal protein complex P0.P1.P2 and protein L12 to the GTPase-associated domain of 28 S ribosomal RNA and effect on the accessibility to anti-28 S RNA autoantibody.

We have investigated binding of rat ribosomal proteins to the "GTPase domain" of 28 S rRNA and its effect on accessibility to the anti-28 S autoantibody, which recognizes a unique tertiary structure of this RNA domain. Ribosomal protein L12 and P protein complex (P complex) consisting of P0, P1, and P2 both bound to the GTPase domain of rat 28 S rRNA in a buffer containing Mg2. Chemical footprinting analysis of their binding sites revealed that the P complex mainly protected a conserved internal loop region comprising residues 1855-1861 and 1920-1922, whereas L12 protected an adjacent helix region encompassing residues 1867-1878 and 1887-1899. These sites are close to but distinct from the binding site for anti-28 S antibody determined previously. The bindings of P complex and L12 increased the anti-28 S accessibility, as revealed by gel retardation and quantitative immunoprecipitation analyses. In a Mg2+-eliminated condition, the RNA failed to bind to either anti-28 S or L12 but assembled into a complex under their coexistence. However, the RNA retained a property of binding to the P complex even in the absence of Mg2+, and this binding conferred high anti-28 S accessibility. These results indicated that the bindings of the P complex and L12 to their respective sites influenced the GTPase domain to increase the accessibility to anti-28 S. A possible RNA conformation adjusted by the protein bindings is discussed.

Animals↗
PubMed2000

Rational design of faster associating and tighter binding protein complexes.

A protein design strategy was developed to specifically enhance the rate of association (k(on)) between a pair of proteins without affecting the rate of dissociation (k(off)). The method is based on increasing the electrostatic attraction between the proteins by incorporating charged residues in the vicinity of the binding interface. The contribution of mutations towards the rate of association was calculated using a newly developed computer algorithm, which predicted accurately the rate of association of mutant protein complexes relative to the wild type. Using this design strategy, the rate of association and the affinity between TEM1 beta-lactamase and its protein inhibitor BLIP was enhanced 250-fold, while the dissociation rate constant was unchanged. The results emphasize that long range electrostatic forces specifically alter k(on), but do not effect k(off). The design strategy presented here is applicable for increasing rates of association and affinities of protein complexes in general.

Algorithms↗