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PubMed1996

Identification of the subunit loci in the extracellular multisubunit hemoglobin from annelid Perinereis aibuhitensis.

Giant hemoglobin (Hb) from Perinereis aibuhitensis is made of several types of protein components including single-chain globin (a), disulfide-bridged globin trimer (A-b-B), disulfide-bridged dimers of nonglobin chain (or linkers; L1-L1, L2-L2, and L1-L2), and oligomers of L1-L2 [(L1-L2)n]. The intact form of this giant Hb is a two-tiered hexagonal structure composed of 12 identical units, or so-called submultiples (six submultiples to a tier). To obtain a view of the three-dimensional architectural arrangement of these components in the intact form, we identified the subunit loci by using two mutually complementary chemical modifications and a colloidal gold labeling technique. Using the chemical modifications, we discovered that (i) linkers L1-L2 and L2-L2 were located at the exterior of the Hb, (ii) linker L1-L1 and globin a were buried in the interior, and (iii) linker (L1-L2)n and globin trimer A-b-B were located at both exterior and interior loci. The labeling with an L2-specific colloidal gold revealed the predominant loci of L2 at the outer and inner boundaries between neighboring submultiples in a hexagonal form. By combining these results with those from our previous reports [S. Ebina, K. Matsubara, K. Nagayama, M. Yamaki, and T. Gotoh (1995) Proc. Natl. Acad. Sci. USA 92, 7367-7371; K. Matsubara, M. Yamaki, Nagayama, H. Ishii, K. Imai, T. Gotoh, and S. Ebina (1996), in press], we deduced the following conclusions concerning the Hb architecture. The L1-L1 chains perhaps together with (L1-L2)n chains form a scaffold on which submultiples assemble into a two-tiered hexagonal arrangement, probably by connecting the carbohydrates in globin a. The L1-L2 and L2-L2 chains reinforce the connections of the submultiples by binding carbohydrates, perhaps those carbohydrates in globin A. We proposed to call this type of non-protein-dependent structural level as seen in such a carbohydrate-glued protein aggregate "protein-plus structure."

Animals↗
PubMed2003

Expression and biological role of laminin-1.

Of the approximately 15 laminin trimers described in mammals, laminin-1 expression seems to be largely limited to epithelial basement membranes. It appears early during epithelial morphogenesis in most tissues of the embryo, and remains present as a major epithelial laminin in some adult tissues. Previous organ culture studies with embryonic tissues have suggested that laminin-1 is important for epithelial development. Recent data using genetically manipulated embryonic stem (ES) cells grown as embryoid bodies provide strong support for the view of a specific role of laminin-1 in epithelial morphogenesis. One common consequence of genetic ablation of FGF signaling, beta1-integrin or laminin gamma1 chain expression in ES cells is the absence of laminin-1, which correlates with failure of BM assembly and epiblast differentiation. Partial but distinct rescue of epiblast differentiation has been achieved in all three mutants by exogenously added laminin-1. Laminin-1 contains several biologically active modules, but several are found in beta1 or gamma1 chains shared by at least 11 laminins. However, the carboxytermini of the alpha chains contain five laminin globular (LG) modules, distinct for each alpha chain. There is increasing evidence for a particular role of alpha1LG4 binding to its receptors for epithelial tubulogenesis. The biological roles of this and other domains of laminin-1 are currently being explored by genetic means. The pathways controlling laminin-1 synthesis have remained largely unknown, but recent advances raise the possibility that laminin-1 and collagen IV synthesis can be regulated by pro-survival kinases of the protein kinase B/Akt family.

Animals↗
PubMed2001

Cavity-directed synthesis of labile silanol oligomers within self-assembled coordination cages.

This paper introduces a concept that is referred to as cavity-directed synthesis by showing the selective oligomerization of trialkoxysilanes, RSi(OMe)3 (7), in self-assembled hollow compounds. Pd(II)-linked coordination hosts (cage, bowl, or tube) are found to strictly control the oligomerization of 7 (R = 2-naphthyl) in such a way that their optimal guests are produced in their cavities. Thus, within coordination tube 1, one molecule of 7 is accommodated and subsequently hydrolyzed to give silanetriol RSi(OH)3 (4). Under ordinary aqueous conditions, this reactive compound undergoes rapid polycondensation (so-called sol-gel condensation) leading to Si-O networks. Within the cavity of 1, however, 4 remains very stable and the polycondensation is completely suppressed. On the other hand, coordination bowl 2 and cage 3 give its dimers RSi(OH)2OSi(OH)2R (5) and cyclic trimers [RSi(OH)O]3 (6), respectively. X-ray crystallographic studies clearly show that the cavity size and the shape of 1, 2, and 3 nicely fit with those of 4, 5, and 6, respectively, demonstrating that the cavities strictly direct the oligomerizaion reaction of 7.

Journal Article↗
PubMed2005

Biochemical analysis of the EJC reveals two new factors and a stable tetrameric protein core.

The multiprotein exon junction complex (EJC) is deposited on mRNAs upstream of exon-exon junctions as a consequence of pre-mRNA splicing. In mammalian cells, this complex serves as a key modulator of spliced mRNA metabolism. To date, neither the complete composition nor the exact assembly pathway of the EJC has been entirely elucidated. Using in vitro splicing and a two-step chromatography procedure, we have purified the EJC and analyzed its components by mass spectrometry. In addition to finding most of the known EJC factors, we identified two novel EJC components, Acinus and SAP18. Heterokaryon analysis revealed that SAP18 is a shuttling protein whereas Acinus is restricted to the nucleus. In MS2 tethering assays Acinus stimulated gene expression at the RNA level, while MLN51, another EJC factor, stimulated mRNA translational efficiency. Using tandem affinity purification (TAP) of proteins overexpressed in HeLa cells, we demonstrated that Acinus binds directly to another EJC component, RNPS1, while stable association of SAP18 to form the trimeric apoptosis and splicing associated protein (ASAP) complex requires both Acinus and RNPS1. Using the same methodology, we further identified what appears to be the minimal stable EJC core, a heterotetrameric complex consisting of eIF4AIII, Magoh, Y14, and MLN51.

Carrier Proteins↗
PubMed1989

DNA primase-DNA polymerase alpha assembly from mouse FM3A cells. Purification of constituting enzymes, reconstitution, and analysis of RNA priming as coupled to DNA synthesis.

The mouse DNA primase-DNA polymerase alpha complex can be resolved with buffer containing 50% ethylene glycol (Suzuki, M., Enomoto, T., Hanaoka, F., and Yamada, M. (1985) J. Biochem. (Tokyo) 98, 581-584). The dissociated primase and DNA polymerase alpha have been purified sufficiently that there was no cross-contamination with each other. By the use of thus isolated DNA primase and DNA polymerase alpha in addition to DNA primase-DNA polymerase alpha complex, we have studied primer RNA synthesis and DNA elongation separately as well as the coupled reaction of the initiation and elongation of DNA chains. In the absence of deoxyribonucleoside triphosphates, the isolated primase synthesized oligoribonucleotides of an apparent length of 7-11 nucleotides (monomeric oligomer) and multiples of a modal length of 9-10 nucleotides (multimeric oligomer) and fd phage single-stranded circular DNA. Monomeric and dimeric oligomers were synthesized processively, and trimeric and larger oligomers were produced by repeated cycles of processive synthesis. The primase complexed with DNA polymerase alpha mainly synthesized monomeric and a small amount of dimeric oligomers. In the presence of deoxyribonucleoside triphosphates at concentrations above 10 microM, the DNA primase-DNA polymerase alpha complex exclusively synthesized monomeric oligomers only, which were utilized as primers for DNA synthesis. On the other hand, the products synthesized by the isolated primase were qualitatively unchanged as compared with those synthesized in the absence of DNA precursors. When the synthesis of oligomers by the isolated primase was coupled with DNA elongation by the addition of the primase-free DNA polymerase alpha, the synthesis of dimeric oligomers was inhibited as a result of efficient DNA elongation from monomeric oligomers.

Animals↗
PubMed2006

Correction of pulmonary abnormalities in Sftpd-/- mice requires the collagenous domain of surfactant protein D.

Surfactant protein D (SP-D) is a member of the collectin family of innate defense proteins. Members of this family share four distinct structural domains: an N-terminal cross-linking domain, a collagenous domain, a neck region, and a carbohydrate recognition domain. In this study, the function of the collagenous domain was evaluated by expressing a SP-D collagen deletion mutant protein (rSftpdCDM) in wild type and SP-D null mice (Sftpd(-/-)). rSftpdCDM formed disulfide-linked trimers that further oligomerized into higher order structures. The mutant protein effectively bound carbohydrate and aggregated bacteria in vitro. Whereas rSftpdCDM did not disrupt pulmonary morphology or surfactant phospholipid levels in wild type mice, the mutant protein failed to rescue the emphysema or enlarged foamy macrophages that are characteristic of Sftpd(-/-) mice. Moreover, rSftpdCDM partitioned with small aggregate surfactant in a manner similar to SP-D, but rSftpdCDM did not correct the abnormal surfactant ultrastructure or phospholipid levels observed in Sftpd(-/-) mice. In contrast, rSftpdCDM completely corrected viral clearance and the abnormal inflammatory response that occurs following pulmonary influenza A challenge in Sftpd(-/-) mice. Our findings indicate that the collagen domain of SP-D is not required for assembly of disulfide-stabilized oligomers or the innate immune response to viral pathogens. The collagen domain of SP-D is required for the regulation of pulmonary macrophage activation, airspace remodeling, and surfactant lipid homeostasis.

Animals↗
PubMed2001

Drebrin particles: components in the ensemble of proteins regulating actin dynamics of lamellipodia and filopodia.

Drebrin, an actin-binding 70-kDa protein with an unusually slow SDS-PAGE mobility corresponding to approximately 120 kDa, containing a proline-rich, profilin-binding motif, had originally been reported from neuronal cells, but recently has also been found in diverse other kinds of tissues and cell lines. In biochemical analyses of various cells and tissues, employing gel filtration, sucrose gradient centrifugation, immunoprecipitation and -blotting, we have identified distinct states of soluble drebrin: a approximately 4S monomer, an 8S, ca. 217-kDa putative trimer, a 13S and a > 20S oligomer. In the 8S particles only [35S]methionine-labelled drebrin but no other actin-binding protein has been detected in stoichiometric amounts. By immunofluorescence and immunoelectron microscopy, drebrin-positive material often appeared as "granules" up to 400 nm in diameter, in some cell types clustered near the Golgi apparatus or in lamellipodia, particularly at leading edges, or in dense-packed submembranous masses at tips (acropodia) or ruffles of leading edges, in filopodia and at plaques of adhering junctions. We conclude that these drebrin complexes and drebrin-rich structures allow the build-up and maintenance of high local drebrin concentrations in strategic positions for the regulation of actin filament assembly, thereby contributing to cell motility and morphology, in particular local changes of plasticity and the formation of protrusions.

Actins↗
PubMed2001

Self-assembled helices from 2,2'-biimidazoles.

2,2'-Biimidazoles were synthesized by palladium(0)-catalyzed coupling of 2-iodoimidazoles bearing an alkyl and an ester group at the 4- and 5-positions, respectively. The products were found to be fluorescent and moderately soluble in organic solvents. Three biimidazoles were subjected to single crystal X-ray diffraction analysis. In all three instances, adjacent molecules were found to be bound together in the solid state by pairs of N-H...N hydrogen bonds, forming twisted ribbon-like columns which resemble double helices. The amount of helical twist observed between neighboring biimidazole subunits in these helices varies with the identity of the alkyl and ester groups; in two cases it is approximately 60 degrees, whereas in the third it is about 90 degrees. Mass spectra of six different biimidazoles display ions with masses corresponding to dimers; this indicates that these compounds retain some affinity for each other in the gas phase. The three most soluble biimidazoles also show mass spectrometric peaks ascribable to trimers and tetramers. The solution-phase aggregation tendencies of these latter three compounds were studied by vapor pressure osmometry. In each case, the apparent molecular weight in 1,2-dichloroethane solution is higher than would be expected for free monomers.

Computer Graphics↗
PubMed1997

Structural analyses of gp45 sliding clamp interactions during assembly of the bacteriophage T4 DNA polymerase holoenzyme. II. The Gp44/62 clamp loader interacts with a single defined face of the sliding clamp ring.

The phage T4 gp45 sliding clamp is a ring-shaped replication accessory protein that is mounted onto DNA in an ATP-dependent manner by the gp44/62 clamp loader. In the preceding paper (Pietroni, P., Young, M. C., Latham, G. J., and von Hippel, P. H. (1997) J. Biol. Chem. 272, 31666-31676), two gp45 mutants were exploited to probe interactions of the sliding clamp ring with the gp44/62 loading machinery at various steps during the clamp loading process. In this report, these studies are extended to examine the polarity of the binding interaction between gp45 and gp44/62. Three different gp45 mutants containing a single cysteine in three topographically distinct positions were used. Several different reporter groups, including extrinsic fluorophores, a photo-cross-linker, and a biotin linker for use in a novel "streptavidin interference assay," were covalently attached to these cysteine residues. Since gp45 is a trimeric protein, these three different mutations allowed us to probe up to nine distinct local environments along the surface of the sliding clamp in the presence and absence of other replication proteins. The results show that the gp44/62-ATP clamp loader complex binds exclusively to the C-terminal (S19C) face of the gp45 ring.

Adenosine Triphosphate↗
PubMed2003

Polymer complexes. XXXX. Supramolecular assembly on coordination models of mixed-valence-ligand poly[1-acrylamido-2-(2-pyridyl)ethane] complexes.

The build-up of polymer metallic supramolecules based on homopolymer (1-acrylamido-2-(2-pyridyl)ethane (AEPH)) and ruthenium, rhodium, palladium as well as platinum complexes has been pursued with great interest. The homopolymer shows three types of coordination behaviour. In the mixed valence paramagnetic trinuclear polymer complexes [(11)+(12)] in the paper and in mononuclear polymer complexes (1)-(5) it acts as a neutral bidentate ligand coordinating through the N-pyridine and NH-imino atoms, while in the mixed ligand diamagnetic poly-chelates, which are obtained from the reaction of AEPH with PdX2 and KPtCl4 in the presence of N-heterocyclic base consisting of polymer complexes (9)+(10), and in monouclear compounds (6)-(8), it behaves as a monobasic bidentate ligand coordinating through the same donor atoms. In mononuclear compounds (13)+(14) it acts as a monobasic and neutral bidentate ligand coordinating only through the same donor atoms. Monomeric distorted octahedral or trimeric chlorine-bridged, approximately octahedral structures are proposed for these polymer complexes. The poly-chelates are of 1:1, 1:2 and 3:2 (metal-homopolymer) stoichiometry and exhibit six coordination. The values of ligand field parameters were calculated. The homopolymer and their polymer complexes have been characterized physicochemically.

Chelating Agents↗
PubMed1996

Peptide interaction with a class I major histocompatibility complex-encoded molecule: allosteric control of the ternary complex stability.

Thermodynamics and kinetics of interaction between a soluble class I MHC heterodimer composed of the H-2Kd heavy chain (H) and human beta 2microglobulin (beta 2m) with a dansylated peptide series based on residues 147-155 of influenza virus nucleoprotein sequence were studied by means of real-time fluorescence measurements. Peptide-heterodimer binding is a second-order process with specific rates practically independent of peptide structure (3-5 x 10(6) M-1 s-1). The ternary complex assembly involves a rate-limiting step of beta 2m association with H to yield an unstable heterodimer (tau < or = 5 s, 37 degrees C). Peptide binding provides a positive feedback enhancing H's affinity for beta 2m, thus stabilizing the ternary complex. The latter decays by either peptide or beta 2m dissociation. The first-order rate constants of peptide dissociation (0.5 x 10(-2))-(0.4 x 10(-3)) s-1, 37 degrees C) depend on their structures and are faster than that of beta 2m dissociation. The former process decreases the H affinity for beta 2m and induces their dissociation. This dissociation, in turn, drastically lowers H affinity for peptide. Thus, these three components produce a system which is stable as a trimer. This behavior is rationalized by the functional requirements of class I molecules: Peptide structure determines the ternary complex's lifetime, and peptide rebinding on the cell surface is rendered unlikely by the limited stability of the empty heterodimers and the very low peptide affinity of the heavy chains.

Allosteric Regulation↗
PubMed2006

TRP1 interacting PDZ-domain protein GIPC forms oligomers and is localized to intracellular vesicles in human melanocytes.

PDZ proteins coordinate assembly of protein complexes that participate in diverse biological processes. GIPC is a multifunctional PDZ protein that interacts with several soluble and membrane proteins. Unlike most PDZ proteins, GIPC contains single PDZ domain and the mechanisms by which GIPC mediates its actions remain unclear. We investigated the possibility that in lieu of multiple PDZ domains, GIPC forms multimers. Here, we demonstrate that GIPC can bind to itself and that the PDZ domain is involved in GIPC-GIPC interaction. Gel filtration, sucrose gradient centrifugation and chemical cross-linking showed that whereas bulk of cytosolic GIPC was present as monomer, oligomers with an estimated molecular mass corresponding to GIPC homotrimer were readily detectable in the membrane fraction. Modeling of GIPC PDZ domain showed feasibility of trimerization. Immunogold electron microscopy showed that GIPC is present in clusters near vesicles. Our data suggest that oligomers of GIPC mediate its functions in melanocytes.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Requirement for GP64 to drive efficient budding of Autographa californica multicapsid nucleopolyhedrovirus.

Budded virions (BV) of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) contain a major envelope glycoprotein (GP64) that is present on the plasma membrane of infected cells. GP64 is acquired by virions during budding through the plasma membrane, the final step in assembly of the budded virion at the cell surface. Previous studies (S. A. Monsma, A. G. P. Oomens, and G. W. Blissard (1996). J. Virol. 70, 4607-4616) showed that insertional inactivation of the AcMNPV gp64 gene resulted in a virus unable to move from cell to cell and nonlethal to orally infected Trichoplusia ni larvae. To determine whether GP64 is involved in virion budding, we measured BV production from Sf9 cells infected with a gp64null virus. Sf9 cells infected with gp64null virus vAc64- were pulse labeled, and progeny BV were isolated on equilibrium sucrose gradients and quantified. BV production from vAc64- was reduced to approximately 2% of that from wild-type AcMNPV. Thus the GP64 protein is important for efficient virion budding. To determine whether the highly charged 7-amino acid cytoplasmic tail domain (CTD) of GP64 was required for virion production, we generated a series of GP64 constructs containing C-terminal truncations or substitutions. Modified forms of GP64 were analyzed in transfected cells and in recombinant viruses in which the wild-type gp64 gene was replaced with a modified gp64. Deletion of 1-7 amino acids from the CTD did not affect GP64 trimerization, protein transport to the cell surface, or membrane fusion activity. However, deletions of 11 or 14 amino acids, which removed the CTD and portions of the predicted transmembrane (TM) domain, were trimerized but were present at lower levels on the cell surface due to shedding of these truncated proteins. Comparisons of growth curves and quantitative measurements of labeled progeny BV production from recombinant viruses expressing either wild-type or mutant GP64 proteins showed that deletion of the 7-residue CTD only moderately reduced the production of infectious virions ( approximately 50%). However, deletions of the C terminal 11 or 14 amino acids had more substantial effects. Removal of the C terminal 11 amino acids reduced titers of infectious virus by 78-96% and labeled progeny virions were reduced by 91-92%. Removal of 14 amino acids from the C terminus resulted in an approximately 98% reduction in progeny BV and a virus that was apparently incapable of efficient propagation in cell culture. Thus the GP64 CTD is not essential for production of infectious BV, but removal of the CTD results in a measurable reduction in budding efficiency. Deletion of the CTD plus small portions of the transmembrane domain resulted in shedding of GP64, reduced surface levels, and a dramatic reduction in the production of BV. Together, these data indicate that GP64 is an important and limiting factor in BV production.

Amino Acid Sequence↗
PubMed2005

Noncovalent Shiga-like toxin assemblies: characterization by means of mass spectrometry and tandem mass spectrometry.

Shiga-like toxin 1 (SLTx), produced by enterohemorrhagic strains of Escherichia coli (EHEC), belongs to a family of structurally and functionally related AB(5) protein toxins that are associated with human disease. EHEC infection often gives rise to hemolytic colitis, while toxin-induced kidney damage is one of the major causes of hemolytic uremic syndrome (HUS) and acute renal failure in children. As such, an understanding and analysis of the noncovalent interactions that maintain the quaternary structure of this toxin are fundamentally important since such interactions have significant biochemical and medical implications. This paper reports on the analysis of the noncovalent homopentameric complex of Shiga-like toxin B chain (SLTx-B(5)) using electrospray ionization (ESI) triple-quadrupole (QqQ) mass spectrometry (MS) and tandem mass spectrometry (MS/MS) and the analysis of the noncovalent hexameric holotoxin (SLTx-AB(5)) using ESI time-of-flight (TOF) MS. The triple-quadrupole analysis revealed highly charged monomer ions dissociate from the multiprotein complex to form dimer, trimer, and tetramer product ions, which were also seen to further dissociate. The ESI-TOFMS analysis of SLTx-AB(5) revealed the complex remained intact and was observed in the gas phase over a range of pHs. Theses findings demonstrate that the gas-phase structure observed for both the holotoxin and the isoloated B chains correlates well with the structures reported to exist in the solution phase for these proteins. Such analysis provides a rapid screening technique for assessing the noncovalent structure of this family of proteins and other structurally related toxins.

Hydrogen-Ion Concentration↗
PubMed2000

Integrins as receptors for laminins.

Laminins are a family of trimeric glycoproteins present in the extracellular matrix and the major constituents of basement membranes. Integrins are alpha beta transmembrane receptors that play critical roles in both cell-matrix and cell-cell adhesion. Several members of the integrin family, including alpha 1 beta 1, alpha 2 beta 1, alpha 3 beta 1, alpha 6 beta 1, alpha 7 beta 1 and alpha 6 beta 4 heterodimers serve as laminin receptors on a variety of cell types. This review summarizes recent advances in understanding the involvement of individual integrins in cell interactions with laminins and the roles of laminin-binding integrins in adhesion-mediated events in vertebrates, including embryonic development, cell migration and tumor cell invasiveness, cell proliferation and differentiation, as well as basement membrane assembly. We discuss the regulation of integrin function via alternative splicing of cytoplasmic domains of alpha and beta subunits of the integrin receptors for laminins and present examples of functional collaboration between laminin-binding integrins and non-integrin laminin receptors. Advances in our understanding of the laminin-binding integrins continue to demonstrate the essential roles these receptors play in maintaining cell polarity and tissue architecture.

Animals↗
PubMed1982

Hemocyanins in spiders, XV. The role of the individual subunits in the assembly of Eurypelma hemocyanin.

The role of the seven different subunits in the quaternary structure of the 24-meric (37 S) hemocyanin of the tarantula, Eurypelma californicum, was studied by reassembly experiments. Individual subunits and combinations of 2, 3, 4, etc. different subunits were incubated in a total concentration of 1-2 mg/ml overnight in Tris buffer, pH 7.5, omitting divalent cations. The reassembly mixtures were then analyzed by thinlayer gel filtration, electron microscopy, analytical ultracentrifugation, and polyacrylamide gel electrophoresis. At least at the low protein concentration employed, none of the isolated monomeric subunits (a, d, e, f, g) is capable of hexamer are likewise ineffective. The minimal number of monomers required for hexamer formation is three, but only about half of the 10 possible combinations was effective. The best results were obtained with a + f + g. Four and five monomers always yielded hexamers, although the composition of the latter is not known. The heterodimer bc is a prerequisite to go beyond the hexameric state. bc alone forms tetramers and large, strand-like aggregates. a shows a high affinity towards bc, trimers and tetramers being formed. Out of all combinations of bc and two monomers, only one (bc + a + g) produced hexamers or heptamers. With three monomers plus bc, dodecamers and larger structures (but no 24-mers) were obtained, but only if also a was present (bc + a + f + g being the best combination). If all subunits minus one monomer were combined, the hexamer/heptamer level was not exceeded if a was omitted. 24-mers were formed in appreciable yield only in one case, namely if e was absent. However, this reassembly product turned out to be unstable. To obtain stable 24-meric hemocyanin, the complete set of subunits had to be present. The roles of the different subunits are defined as follows: bc is a "core" subunit which forms the inter-hexamer link within each dodecameric half. It serves also in inter-dodecamer bonding. The artificial homodimers bb and cc are ineffective in this respect. bc must be supplemented by a which is needed for dodecamer stabilization. f is also required to link two dodecamers, while g serves to stabilize this bridge. d and e are required to finish off and to stabilize the 24-mer. A model is proposed showing some neighbourhood relationships of the subunits within the hemocyanin 24-mer, and a pathway of reassembly is discussed.

Animals↗
PubMed2005

Protein oligomerization through domain swapping: role of inter-molecular interactions and protein concentration.

Domain swapping has been shown to be an important mechanism controlling multiprotein assembly and has been suggested recently as a possible mechanism underlying protein aggregation. Understanding oligomerization via domain swapping is therefore of theoretical and practical importance. By using a symmetrized structure-based (Gō) model, we demonstrate that in the free-energy landscape of domain swapping, a large free-energy barrier separates monomeric and domain-swapped dimeric configurations. We investigate the effect of finite monomer concentration, by implementing a new semi-analytical method, which involves computing the second virial coefficient, a thermodynamic indicator of inter-molecular interactions. This method, together with the symmetrized structure-based (Gō) model, minimizes the need for expensive many-protein simulations, providing a convenient framework to investigate concentration effect. Finally, we perform direct simulations of domain-swapped trimer formation, showing that this modeling approach can be used for higher-order oligomers.

Dimerization↗
PubMed2006

Spatial assembly and RNA binding stoichiometry of a LINE-1 protein essential for retrotransposition.

LINE-1, or L1, is a highly successful retrotransposon in mammals, comprising 17% and 19% of the human and mouse genomes, respectively. L1 retrotransposition and hence amplification requires the protein products of its two open reading frames, ORF1 and ORF2. The sequence of the ORF1 protein (ORF1p) is not related to any protein with known function. ORF1p has RNA binding and nucleic acid chaperone activities that are both required for retrotransposition. Earlier studies have shown that ORF1p forms a homotrimer with an asymmetric dumbbell shape, in which a rod separates a large end from a small end. Here, we determine the topological arrangement of monomers within the homotrimer by comparing atomic force microscopy (AFM) images of the full ORF1p with those of truncations containing just the N or C-terminal regions. In addition, AFM images of ORF1p bound to RNA at high protein/RNA molar ratios show that ORF1p can form tightly packed clusters on RNA, with binding occurring at the C-terminal domain. The number of bound ORF1p trimers increases with increasing length of the RNA, revealing that the binding site size is about 50 nt, a value confirmed by nitrocellulose filter binding under stoichiometric conditions. These results are consistent with a role for ORF1p during L1 retrotransposition that includes both coating the RNA and acting as a nucleic acid chaperone. Furthermore, these in vitro L1 ribonucleoprotein particles provide insight into the structure of the L1 retrotransposition intermediate.

Animals↗