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Results for “Protein Multimerization”

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At least 19 recordsLinked to original sources

PubMed1990

Adenovirus DNA-binding protein forms a multimeric protein complex with double-stranded DNA and enhances binding of nuclear factor I.

The 72-kilodalton adenovirus DNA-binding protein (DBP) binds to single-stranded DNA as well as to RNA and double-stranded DNA and is essential for the replication of viral DNA. We investigated the binding of DBP to double-stranded DNA by gel retardation analysis. By using a 114-base-pair DNA fragment, five or six different complexes were observed by gel retardation. The mobility of these complexes is dependent on the DBP concentration, suggesting that the complexes arise by sequential binding of DBP molecules to the DNA. In contrast to binding to single-stranded DNA, the binding of DBP to double-stranded DNA appears to be noncooperative. DBP binds to linear DNA as well as to circular DNA, while linear DNA containing the adenovirus terminal protein was also recognized. No specificity for adenovirus origin sequences was observed. To study whether the binding of DBP could influence initiation of DNA replication, we analyzed the effect of DBP on the binding of nuclear factor I (NFI) and NFIII, two sequence-specific origin-recognizing proteins that enhance initiation. At subsaturating levels of NFI, DBP increases the rate of binding of NFI considerably, while no effect was seen on NFIII. This stimulation of NFI binding is specific for DBP and was not observed with another protein (NFIV), which forms a similar DNA-multimeric protein complex. In agreement with enhanced NFI binding, DBP stimulates initiation of adenovirus DNA replication in vitro especially strongly at subsaturating NFI concentrations. We explain our results by assuming that DBP forms a complex with origin DNA that promotes formation of an alternative DNA structure, thereby facilitating the binding of NFI as well as the initiation of DNA replication via NFI.

Adenoviruses, Human↗

PubMed1997

Effect of mutations in vesicle-associated membrane protein (VAMP) on the assembly of multimeric protein complexes.

The assembly of multimeric protein complexes that include vesicle-associated membrane protein 2 (VAMP-2) and the plasma membrane proteins syntaxin 1A and synaptosome-associated protein of 25 kDa (SNAP-25) are thought to reflect the biochemical correlates of synaptic vesicle targeting, priming, or fusion. Using a variety of protein-protein interaction assays and a series of deletion and point mutations, we have investigated the domains of VAMP-2 required for the formation of binary complexes with either syntaxin 1A or SNAP-25 and ternary complexes with both syntaxin 1A and SNAP-25. Deletions within the central conserved domain of VAMP-2 eliminated binding to either syntaxin 1A or both syntaxin 1A and SNAP-25. Although all of the deletion mutants were able to form ternary complexes, only some of these complexes were resistant to denaturation in sodium dodecyl sulfate. These results demonstrate that cooperative interactions result in the formation of at least two biochemically distinct classes of ternary complex. Two point mutations previously shown to have effects on the intracellular trafficking of VAMP-2 (M46A, reduced endocytosis and sorting to synaptic vesicles; N49A, enhanced sorting to synaptic vesicles) lie within a domain required for both syntaxin 1A and SNAP-25 binding. Syntaxin 1A and SNAP-25 binding was reduced by the M46A mutation and enhanced by the N49A mutation, suggesting that a correlation exists between the membrane-trafficking phenotype of the two VAMP-2 point mutants and their competence to form complexes with either syntaxin 1A or SNAP-25.

Animals↗

PubMed2004

Analysis of the stability of multimeric proteins by effective DeltaG and effective m-values.

Analyzing the stability of a multimeric protein is challenging because of the intrinsic difficulty in handling the mathematical model for the folded multimer-unfolded monomer equilibrium. To circumvent this problem, we introduce the concept of effective stability, DeltaGeff (= -RTlnKeff), where Keff is the equilibrium constant expressed in monomer units. Analysis of the denaturant effect on DeltaGeff gives new insight into the stability of multimeric proteins. When a multimeric protein is mostly folded, the dependence of effective stability on denaturant concentration (effective m-value) is simply the m-value of its monomeric unit. However, when the protein is mostly unfolded, its stability depends on denaturant concentration with the m-value of its multimeric form. We also find that the effective m-value at the Cm is a good approximation of the apparent m-value determined by fitting the equilibrium unfolding data from multimeric proteins with a two-state monomer model. Moreover, when the m-value of a monomeric unit is estimated from its size, the effective stability of a multimeric protein can be determined simply from Cm and this estimated m-value. These simple and intuitive approaches will allow a facile analysis of the stability of multimeric proteins. These analyses are also applicable for high-throughput analysis of protein stability on a proteomic scale.

Models, Chemical↗

PubMed1987

Properties of a multimeric protein complex from chloroplasts possessing potential activities of NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase.

A homogeneous multimeric protein isolated from the green alga, Scenedesmus obliquus, has both latent phosphoribulokinase activity and glyceraldehyde-3-phosphate dehydrogenase activity. The glyceraldehyde-3-phosphate dehydrogenase was active with both NADPH and NADH, but predominantly with NADH. Incubation with 20 mM dithiothreitol and 1 mM NADPH promoted the coactivation of phosphoribulokinase and NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase, accompanied by a decrease in the glyceraldehyde-3-phosphate dehydrogenase activity linked to NADH. The multimeric enzyme had a Mr of 560,000 and was of apparent subunit composition 8G6R. R represents a subunit of Mr 42,000 conferring phosphoribulokinase activity and G a subunit of 39,000 responsible for the glyceraldehyde-3-phosphate dehydrogenase activity. On SDS-PAGE the Mr-42,000 subunit comigrates with the subunit of the active form of phosphoribulokinase whereas that of Mr-39,000 corresponds to that of NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase. The multimeric enzyme had a S20,W of 14.2 S. Following activation with dithiothreitol and NADPH, sedimenting boundaries of 7.4 S and 4.4 S were formed due to the depolymerization of the multimeric protein to NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase (4G) and active phosphoribulokinase (2R). It has been possible to isolate these two enzymes from the activated preparation by DEAE-cellulose chromatography. Prolonged activation of the multimeric protein by dithiothreitol in the absence of nucleotide produced a single sedimenting boundary of 4.6 S, representing a mixture of the active form of phosphoribulokinase and an inactive dimeric form of glyceraldehyde-3-phosphate dehydrogenase. Algal thioredoxin, in the presence of 1 mM dithiothreitol and 1 mM NADPH, stimulated the depolymerization of the multimeric protein with resulting coactivation of phosphoribulokinase and NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase. Light-induced depolymerization of the multimeric protein, mediated by reduced thioredoxin, is postulated as the mechanism of light activation in vivo. Consistent with such a postulate is the presence of high concentrations of the active forms of phosphoribulokinase and NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase in extracts from photoheterotrophically grown algae. By contrast, in extracts from the dark-grown algae the multimeric enzyme predominates.

Chlorophyta↗

PubMed2003

Functional genomic dissection of multimeric protein families in zebrafish.

The study of multimeric protein function in the postgenomicera has become complicated by the discovery of multiple isoforms for each subunit of those proteins. A correspondingly large number of potential isoform combinations offer the multicellular organism a constellation of protein assemblies from which to generate a variety of functions across different cells, tissues, and organs. At the same time, the multiplicity of potential subunit isoform combinations presents a significant challenge when attempting to dissect the functions of particular isoform combinations. Biochemical and cell culture methods have brought us to a significant state of understanding of multimeric proteins but are unable to answer questions of function within the context of the many tissues and developmental stages of the multicellular organism. Answering those questions can be greatly facilitated in model systems in which expression can be determined over time, in the context of the whole organism, and in which hypomorphic function of each subunit can be studied individually and in combination. Fortunately, the potential for high-throughput in situ hybridization studies and antisense-based reverse genetic knockdowns in zebrafish offers exciting opportunities to meet this challenge. Some of these opportunities, along with cautions of interpretation and gaps in the existing technologies, are discussed in the context of ongoing investigations of the dimeric Na,K-ATPases and heterotrimeric G proteins.

Animals↗

PubMed2002

Thermodynamic stability measurements on multimeric proteins using a new H/D exchange- and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry-based method.

We recently reported on a new H/D exchange- and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry-based technique, termed SUPREX, that removes several important limitations associated with measuring the thermodynamic stability of proteins. In contrast to conventional spectroscopy-based techniques for characterizing the equilibrium unfolding behavior of proteins, SUPREX is amenable to the thermodynamic analysis of both purified and unpurified proteins using mg to ng quantities of material. Here we report on the application of SUPREX to the analysis of multimeric protein systems. Included in this work are the SUPREX results we obtained in studies on six model multimeric proteins including the GCN4p1 dimer, the coil-V(a)L(d) trimer, the 4-oxalocrotonate tautomerase (4-OT) hexamer, the Trp repressor (TrpR) dimer, the Arc repressor (ArcR) dimer, and an ArcR mutant (the (DOA20)ArcR) dimer which contained two destabilizing mutations including an Asp to Ala mutation at position 20 and an amide to ester bond mutation between amino acid (aa) residues 19 and 20. As part of the work described here, we present a new method for the analysis of SUPREX data that is generally applicable to both monomeric and multimeric protein systems. Our results on the model proteins in this study indicate that this new method can be used to determine folding free energies for proteins with the accuracy and precision of conventional spectroscopy-based methods.

Bacterial Proteins↗

PubMed2001

Tail-vein injection of mannan-binding lectin DNA leads to high expression levels of multimeric protein in liver.

The human plasma protein mannan-binding lectin (MBL) is an essential part of the innate immune defense system. Low levels of MBL are associated with recurrent infections and other clinically significant signs of a compromised immune defense. Previous studies have addressed the possibility of reconstitution therapy by the use of recombinant or plasma-derived protein. Natural MBL is a multimeric protein, which consists of up to 18 identical polypeptide chains. Synthesis by in vitro methods of MBL with the proper multimeric structure is difficult. We here report that mice obtain MBL levels comparable to those found in normal human plasma when injected with an MBL expression construct as naked plasmid DNA contained in a large volume of physiologic salt solution. The expression was confined to the liver and high MBL expression levels were obtained with less than 5% of the liver cells transfected. The multimeric structure of the MBL found in plasma of injected mice was similar to that of natural MBL. Thus, liver expression following injection of naked DNA is an alternative to reconstitution therapy with a protein having a complex quaternary structure.

Animals↗

PubMed2005

SR-rich motif plays a pivotal role in recombinant SARS coronavirus nucleocapsid protein multimerization.

The nucleocapsid (N) protein of SARS coronavirus (SARS-CoV) is reported to function in encapsidating the viral genomic RNA into helical nucleocapsid, and its self-association is believed to be vital in coating the viral genomic RNA. Characterization of SARS-CoV N multimerization may thereby help us better understand the coronavirus assembly. In the current work, using the yeast two-hybrid technique, an unexpected interaction between residues 1-210 and 211-290 (central region) of the SARS-CoV N protein was detected, and SPR results further revealed that the SR-rich motif (amino acids 183-197) of SARS-CoV N protein is responsible for such an interaction. Chemical cross-linking and gel-filtration analyses indicated that the residues 283-422 of the SARS-CoV N protein have multimeric ability, although the full-length N protein is prone to exist predominantly as dimers. In addition, the multimeric ability of the C-terminal domain of SARS-CoV N protein could be weakened by the SR-rich motif interaction with the central region (amino acids 211-290). All of these data suggested that the SR-rich motif of the SARS-CoV N protein might play an import role in the transformation of the SARS-CoV N protein between the dimer and multimer during its binding to its central region for self-association or dissociation. This current paper will hopefully provide some new ideas in studying SARS-CoV N multimerization.

Amino Acid Motifs↗

PubMed1989

HeLa nuclear protein recognizing DNA termini and translocating on DNA forming a regular DNA-multimeric protein complex.

Employing an exonuclease III protection assay we detected a protein in crude HeLa nuclear extracts binding, with apparent sequence specificity, to molecular ends of adenovirus type 2 (Ad2) DNA. This protein, designated nuclear factor IV (NFIV), was purified to homogeneity and was shown to be a hetero-dimer of 72,000 and 84,000 Mr. Binding to terminal Ad2 sequences was strongly enhanced by the presence of either of the sequence-specific DNA-binding proteins nuclear factor I and nuclear factor III. These proteins appeared to function as blockades for translocation of NFIV on DNA, thus producing apparent sequence specificity. In the absence of such a blockade, NFIV moved freely, without energy input, on any double-stranded DNA forming a regular DNA-multimeric protein complex as shown by methidiumpropyl EDTA footprinting and electron microscopy. Binding is completely dependent upon the presence of molecular ends. Evidence was obtained for a two-step mechanism in which termini are recognized by NFIV and used as a starting point for subsequent translocation. The possible functions of the protein in adenovirus DNA replication and in cellular processes requiring DNA termini are discussed.

Base Sequence↗

PubMed2000

Chain-selective isotopic labeling for NMR studies of large multimeric proteins: application to hemoglobin.

Multidimensional, multinuclear NMR has the potential to elucidate the mechanisms of allostery and cooperativity in multimeric proteins under near-physiological conditions. However, NMR studies of proteins made up of non-equivalent subunits face the problem of severe resonance overlap, which can prevent the unambiguous assignment of resonances, a necessary step in interpreting the spectra. We report the application of a chain-selective labeling technique, in which one type of subunit is labeled at a time, to carbonmonoxy-hemoglobin A (HbCO A). This labeling method can be used to extend previous resonance assignments of key amino acid residues, which are important to the physiological function of hemoglobin. Among these amino acid residues are the surface histidyls, which account for the majority of the Bohr effect. In the present work, we report the results of two-dimensional heteronuclear multiple quantum coherence (HMQC) experiments performed on recombinant (15)N-labeled HbCO A. In addition to the C2-proton (H epsilon(1)) chemical shifts, these spectra also reveal the corresponding C4-proton (H delta(2)) resonances, correlated with the N epsilon(2) and N delta(1) chemical shifts of all 13 surface histidines per alpha beta dimer. The HMQC spectrum also allows the assignment of the H delta(1), H epsilon(1), and N epsilon(1) resonances of all three tryptophan residues per alpha beta dimer in HbCO A. These results indicate that heteronuclear NMR, used with chain-selective isotopic labeling, can provide resonance assignments of key regions in large, multimeric proteins, suggesting an approach to elucidating the solution structure of hemoglobin, a protein with molecular weight 64.5 kDa.

Binding Sites↗

PubMed2002

Lumenal protein multimerization in the distal secretory pathway/secretory granules.

Differences in protein solubility appear to play an important role in lumenal protein trafficking through Golgi/post-Golgi compartments. Recent advances indicate that multimeric protein assembly is one of the factors regulating the efficiency of protein storage within secretory granules, by mechanisms that, with slight modification, might be considered to represent the culmination of a process of Golgi cisternal maturation.

Animals↗

PubMed2002

Versatile cloning system for construction of multimeric proteins for use in atomic force microscopy.

This manuscript introduces a versatile system for construction of multimeric proteins to be used as substrates for atomic force microscopy. The construction makes use of a cassette system that allows modules to be cut and ligated in any combination in eight different positions. The modules can be sequenced in situ after construction. A three-module fragment can be produced that is of a size amenable to structural and biophysical analysis to check the effect of placing a protein into a multimeric construct. We show that if the parent titin modules are retained in a construct, they can act both as linkers and as an internal standard for the force measurements. Proteins that cannot be expressed solubly in an eight-module homopolymer have been expressed and subject to force measurements using this system.

Cloning, Molecular↗

PubMed2000

Unfolding of multimeric proteins in presence of denaturants. A case study of helianthinin from Helianthus annuus L.

Helianthinin (11S), a multisubunit protein from Sunflower Seeds (Helianthus annuus L.) dissociates to its monomer (2S) through a trimeric (7S) intermediate as a function of guanidine hydrochloride (GuHCl) and guanidine thiocyanate (GuHSCN) concentration. Measurements of viscosity, velocity sedimentation patterns and spectroscopic parameters of the protein in presence of these denaturants both at equilibrium and as a function of time clearly suggest that the dissociation, unfolding and aggregation of this multimeric protein occur sequentially. The unfolding of the protein in guanidinium salts has two transitions with the first transition occurring between native to intermediate and the second transition occurring between intermediate state and unfolded state. The midpoint concentration for the major transition is 1.75 M for GuHCl and 0.8 M for GuHSCN. Evaluation of this data suggests that during the process of denaturation the simultaneous unfolding of acidic and basic subunits of the protein takes place. At intermediate concentrations of denaturant namely 1.6 M of GuHCl or 1.1 M of GuHSCN the aggregation of the protein was found to be maximum. The results suggests the possibility of a mechanism for the dissociation, denaturation and unfolding of multimeric proteins in presence of the chemical denaturants.

2S Albumins, Plant↗

PubMed1996

Theoretical basis for differential scanning calorimetric analysis of multimeric proteins.

A new general equation simulating irreversible DSC transitions of multimeric proteins was developed. The equation put forward here is the result of an improved mathematical re-elaboration of the classical Lumry-Eyring models, where no restrictive a priori assumptions are made on the kinetic constraints of the denaturation process, or on the enthalpy of the final denatured state. In order to test the wide applicability of this new effective theoretical tool, a series of DSC transitions were simulated with the aim of determining the effects of all relevant thermodynamic, kinetic or experimental parameters on the shape of DSC profiles. Moreover, the classical equations used widely in DSC investigations for the calculus in both kinetic parameters and changes of molecularity, were studied in the light of the model developed here, highlighting, in each case, their rather limited applicability. The new approach proposed in this article was applied to study the thermal denaturation of an hexameric protein (Glucosamine-6-phosphate deaminase), putting in evidence the practical applicability of the theoretical equations developed.

Journal Article↗

PubMed2005

Two-dimensional Blue Native/sodium dodecyl sulfate gel electrophoresis for analysis of multimeric proteins in platelets.

Two-dimensional (2-D) Blue Native/SDS gel electrophoresis combines a first-dimensional separation of monomeric and multimeric proteins in their native state with a second denaturing dimension. These high-resolution 2-D gels aim at identifying multiprotein complexes with respect to their subunit composition. We applied this method for the first time to analyze two human platelet subproteomes: the cytosolic and the microsomal membrane protein fraction. Solubilization of platelet membrane proteins was achieved with the nondenaturing detergent n-dodecyl-beta-D-maltoside. To validate native solubilization conditions, we demonstrated the correct assembly of the Na,K-ATPase, a functional multimeric transmembrane protein, when expressed in Xenopus oocytes. We identified 63 platelet proteins after in-gel tryptic digestion of 58 selected protein spots and liquid chromatography-coupled tandem mass spectrometry. Nine proteins were detected for the first time in platelets by a proteomic approach. We also show that this technology efficiently resolves several known membrane and cytosolic multiprotein complexes. Blue Native/SDS gel electrophoresis is thus a valuable procedure to analyze specific platelet subproteomes, like the membrane(-bound) protein fraction, by mass spectrometry and immunoblotting and could be relevant for the study of protein-protein interactions generated following platelet activation.

Animals↗

PubMed2006

The cysteine string protein multimeric complex.

Cysteine string protein (CSPalpha) is a member of the cellular folding machinery that is located on regulated secretory vesicles. We have previously shown that CSPalpha in association with Hsc70 (70kDa heat shock cognate protein) and SGT (small glutamine-rich tetratricopeptide repeat domain protein) is a guanine nucleotide exchange factor (GEF) for G(alphas). Association of this CSPalpha complex with N-type calcium channels, a channel key in coupling calcium influx with synaptic vesicle exocytosis, triggers tonic G protein inhibition of the channels. Syntaxin 1A, a plasma membrane SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) critical for neurotransmission, coimmunoprecipitates with the CSPalpha/G protein/N-type calcium channel complex, however the significance of syntaxin 1A as a component of this complex remains unknown. In this report, we establish that syntaxin 1A interacts with CSPalpha, Hsc70 as well as the synaptic protein interaction (synprint) region of N-type channels. We demonstrate that huntingtin(exon1), a putative biologically active fragment of huntingtin, displaces both syntaxin 1A and CSPalpha from N-type channels. Identification of the protein components of the CSPalpha/GEF system is essential in establishing its precise role in synaptic transmission.

Animals↗

PubMed1992

Polycomb and polyhomeotic are constituents of a multimeric protein complex in chromatin of Drosophila melanogaster.

The polycomb group (Pc-G) genes are responsible for maintaining the repressed state of homeotic genes during development. It has been suggested that the Pc-G exerts its transcriptional control by regulating higher order chromatin structure. In particular, the finding of genetic and molecular similarities to components involved in heterochromatin formation, led to the proposal that homeotic genes are permanently repressed by mechanisms similar to those responsible for heterochromatin compaction. Because of synergistic effects, Pc-G gene products are thought to act in a multimeric complex. Using immunoprecipitation we show that two members of the Pc-G, Polycomb and polyhomeotic, are constituents of a soluble multimeric protein complex. Size fractionation indicates that a large portion of the two proteins are found in a distinct complex of molecular weight 2-5 x 10(6) Da. During embryogenesis the two proteins show the same spatial distribution. In addition, by double-immunofluorescence labelling we can demonstrate that Polycomb and polyhomeotic have exactly the same binding patterns on polytene chromosomes of larval salivary glands. We propose that some Pc-G proteins act in multimeric complexes to compact the chromatin of stably repressed genes like the homeotic regulators.

Animals↗

PubMed2003

Adenovirus dodecahedron allows large multimeric protein transduction in human cells.

Adenovirus dodecahedron is a virus-like particle composed of only two viral proteins of human adenovirus serotype 3 that are responsible for virus attachment and internalization. We show here that this dodecameric particle, devoid of genetic information, efficiently penetrates human cells and can deliver large multimeric proteins such as immunoglobulins.

Adenoviruses, Human↗