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PubMed2005

Fibrillar assembly and stability of collagen coating on titanium for improved osteoblast responses.

Collagen, as a major constituent of human connective tissues, has been regarded as one of the most important biomaterials. As a coating moiety on Ti hard-tissue implants, the collagen has recently attracted a great deal of attention. This article reports the effects of fibrillar assembly and crosslinking of collagen on its chemical stability and the subsequent osteoblastic responses. The fibrillar self-assembly of collagen was carried out by incubating acid-dissolved collagen in an ionic-buffered medium at 37 degrees C. The degree of assembly was varied with the incubation time and monitored by the turbidity change. The differently assembled collagen was coated on the Ti and crosslinked with a carbodiimide derivative. The partially assembled collagen contained fibrils with varying diameters as well as nonfibrillar aggregates. On the other hand, the fully assembled collagen showed the complete formation of fibrils with uniform diameters of approximately 100-200 nm with periodic stain patterns within the fibrils, which are typical of native collagen fibers. Through this fibrillar assembly, the collagen coating had significantly improved chemical stability in both the saline and collagenase media. The subsequent crosslinking step also improved the stability of the collagen coating, particularly in the unassembled collagen. The fibrillar assembly and the crosslinking of collagen significantly influenced the osteoblastic cell responses. Without the assembly, the collagen layer on Ti adversely affected the cell attachment and proliferation. However, those cellular responses were improved significantly when the collagen was assembled to fibrils and the assembly degree was increased. After crosslinking the collagen coating, these cellular responses were significantly enhanced in the case of the unassembled collagen but were not altered much in the assembled collagen. Based on these observations, it is suggested that the fibrillar assembly and the crosslinking of collagen require careful considerations in the collagen administration as a coating moiety.

Cell Line↗
PubMed1993

Dynamics of in vitro assembly of 16 S rRNA into 30 S ribosomal subunits.

One of the important unsolved problems in the ribosome field is the molecular basis for the sequential and co-operative nature of ribosome assembly. As an approach to this problem, we have taken advantage of the temperature dependence of in vitro reconstitution and have used chemical probing methods to examine the conformation and reactivity of 16 S rRNA at successive stages during subunit assembly. One class of nucleotides displays reactivities similar to those observed in native 30 S particles when the RNA and protein are incubated in the absence of any heat step (0 degrees C effects). At 30 degrees C, where the assembly process takes 2 hours, other bases can be assigned to one of several additional kinetic classes, determined by the rate at which their chemical reactivities transit from levels observed in naked RNA to levels observed in fully assembled subunits: (1) fast (t1/2 = < 5 min at 30 degrees C); (2) slow (t1/2 = 15 to 30 min at 30 degrees C); (3) delayed slow (t1/2 = 30 to 60 min at 30 degrees C). Finally, several nucleotides display transient kinetics in their reactivities, showing increasing reactivity at early time points and becoming protected later in assembly; most of these effects correspond to residues that were previously shown to display reciprocal enhancement and protection patterns during step-wise in vitro assembly. These findings, together with our previous studies using purified individual proteins lead to the following conclusions: (1) there is a predominant 5' to 3' polarity to in vitro assembly, even though it is uncoupled from transcription; (2) portions of the central and 3' major domains fold into an active conformation only at a very late stage of assembly; (3) bases footprinted by late-assembling proteins, according to the 30 S subunit assembly map, show generally slower kinetics than residues footprinted by proteins that bind early in the assembly map, providing direct evidence for the sequential nature of the in vitro assembly process; (4) most proteins are associated with nucleotides that fall into more than one kinetic class, suggesting that assembly proceeds through multiple pathways, or that individual proteins interact sequentially with different regions of the RNA.

Cold Temperature↗
PubMed1986

Molecular assembly of tobacco mosaic virus in vitro.

TMV assembly starts with a specific interaction between the assembly origin on the RNA and a disk aggregate of coat protein. The assembly origin is located in the 30K protein cistron for common and tomato strains of TMV and in the coat protein cistron for cowpea strain of TMV and for CGMMV. All the assembly origins have three essential structures: a long base-paired hairpin loop structure; a target sequence, GAPuGUUG, at the top of the hairpin loop structure; and a tract where every third base is a purine. The protein aggregate responsible for the initiation of TMV assembly is a 20S disk, a two-layered aggregate of 34 protein subunits. The two layers of a disk open apart onto the central hole and this structure may be critical for the disk to interact with the assembly origin on the RNA. The target sequence may bind specifically to this structure. Although only a low concentration of 20S disks exists in the usual assembly condition, one disk is enough to initiate TMV assembly. TMV elongation proceeds in two directions. Elongation to the 5'-end proceeds rapidly by preferential incorporation of protein subunits (or A protein) and in 5-7 min gives rise to 260 nm intermediate particles whose 5'-end is coated. A model of elongation toward the 5'-end is shown in Fig. 15. Protected RNAs from nuclease digestion during the assembly reaction produce a banding pattern on gels by electrophoresis. The banding pattern reflects features of the RNA rather than protein that are used in the assembly reaction, since the pattern was the same for assembly between TMV-RNA and CGMMV protein subunits as for assembly between TMV-RNA and TMV protein containing 20S aggregates. The 20S aggregate in the assembly solution has a helical structure with 39 protein subunits rather than the disk structure. Rapid addition of 20S helical aggregates to the top of the growing rod seems to be impossible because of its topological complexity. Elongation toward the 3'-end does not start for at least the first 4 min after initiation. It probably cannot begin until the 5'-end RNA tail disappears into the intermediate rod. Elongation toward the 3'-end favors 20S aggregates as the protein source and gives rise to the full-length rods in about 30 min after the initiation. There are no topological difficulties in adding 20S helical aggregates to the protruding RNA tail.(ABSTRACT TRUNCATED AT 400 WORDS)

Genes, Viral↗
PubMed2001

Probabilistic approaches to the use of higher order clone relationships in physical map assembly.

UNLABELLED: Physical map assembly is the inference of genome structure from experimental data. Map assembly depends on the integration of diverse data including sequence tagged site (STS) marker content, clone sizing, and restriction digest fingerprints (RDF). As experimentally measured data, these are uncertain and error prone. Physical map assembly from error free data is straightforward and can be accomplished in linear time in the number of clones, but the assembly of an optimal map from error prone data is an NP-hard problem. We present an alternative approach to physical map assembly that is based on a probabilistic view of the data and seeks to identify those features of the map that can be reliably inferred from the available data. With this approach, we achieve a number of goals. These include the use of multiple data sources, appropriate representation of uncertainties in the underlying data, the use of clone length information in fingerprint map assembly, and the use of higher order information in map assembly. By higher order information, we mean relationships that are not expressible in terms of neighbouring clone relationships. These include triplet and multiple clone overlaps, the uniqueness of STS position, and fingerprint marker locations. In a probabilistic view of physical mapping, we assert that all of the many possible map assemblies are equally likely a priori. Given experimental data, we can only state which assemblies are more likely than others given the experimental observations. Parameters of interest are then derived as likelihood weighted averages over map assemblies. Ideally these averages should be sums or integrals over all possible map assemblies, but computationally this is not feasible for real-world map assembly problems. Instead, sampling is used to asymptotically approach the desired parameters. Software implementing our probabilistic approach to mapping has been written. Assembly of mixed RDF and STS maps containing up to 60 clones can be accomplished on a desktop PC with run times under an hour. AVAILABILITY: http://stl.wustl.edu/software/gibbsmap/.

Cloning, Molecular↗
PubMed1995

Xenopus lamin B3 has a direct role in the assembly of a replication competent nucleus: evidence from cell-free egg extracts.

Xenopus egg extracts which assemble replication competent nuclei in vitro were depleted of lamin B3 using monoclonal antibody L6 5D5 linked to paramagnetic beads. After depletion, the extracts were still capable of assembling nuclei around demembranated sperm heads. Using field emission in lens scanning electron microscopy (FEISEM) we show that most nuclei assembled in lamin B3-depleted extracts have continuous nuclear envelopes and well formed nuclear pores. However, several consistent differences were observed. Most nuclei were small and only attained diameters which were half the size of controls. In a small number of nuclei, nuclear pore baskets, normally present on the inner aspect of the nuclear envelope, appeared on its outer surface. Finally, the assembly of nuclear pores was slower in lamin B3-depleted extracts, indicating a slower overall rate of nuclear envelope assembly. The results of FEISEM were confirmed using conventional TEM thin sections, where again the majority of nuclei assembled in lamin B3-depleted extracts had well formed double unit membranes containing a high density of nuclear pores. Since nuclear envelope assembly was mostly normal but slow in these nuclei, the lamin content of 'depleted' extracts was investigated. While lamin B3 was recovered efficiently from cytosolic and membrane fractions by our procedure, a second minor lamin isoform, which has characteristics similar to those of the somatic lamin B2, remained in the extract. Thus it is likely that this lamin is necessary for nuclear envelope assembly. However, while lamin B2 did not co-precipitate with lamin B3 during immunodepletion experiments, several protein species did specifically associate with lamin B3 on paramagnetic immunobeads. The major protein species associated with lamin B3 migrated with molecular masses of 102 kDa and 57 kDa, respectively, on one-dimensional polyacrylamide gels. On two-dimensional O'Farrell gels the mobility of the 102 kDa protein was identical to the mobility of a major nuclear matrix protein, indicating a specific association between lamin B3 and other nuclear matrix proteins. Nuclei assembled in lamin B3-depleted extracts did not assemble a lamina, judged by indirect immunofluorescence, and failed to initiate semi-conservative DNA replication. However, by reinoculating depleted extracts with purified lamin B3, nuclear lamina assembly and DNA replication could both be rescued. Thus it seems likely that the inability of lamin-depleted extracts to assemble a replication competent nucleus is a direct consequence of a failure to assemble a lamina.

Animals↗
PubMed2006

Quantitative analysis of multi-component spherical virus assembly: scaffolding protein contributes to the global stability of phage P22 procapsids.

Assembly of the hundreds of subunits required to form an icosahedral virus must proceed with exquisite fidelity, and is a paradigm for the self-organization of complex macromolecular structures. However, the mechanism for capsid assembly is not completely understood for any virus. Here we have investigated the in vitro assembly of phage P22 procapsids using a quantitative model specifically developed to analyze assembly of spherical viruses. Phage P22 procapsids are the product of the co-assembly of 420 molecules of coat protein and approximately 100-300 molecules of scaffolding protein. Scaffolding protein serves as an assembly chaperone and is not part of the final mature capsid, but is essential for proper procapsid assembly. Here we show that scaffolding protein also affects the thermodynamics of assembly, and for the first time this quantitative analysis has been performed on a virus composed of more than one type of protein subunit. Purified coat and scaffolding proteins were mixed in varying ratios in vitro to form procapsids. The reactions were allowed to reach equilibrium and the proportion of the input protein assembled into procapsids or remaining as free subunits was determined by size exclusion chromatography and SDS-PAGE. The results were used to calculate the free energy contributions for individual coat and scaffolding proteins. Each coat protein subunit was found to contribute -7.2(+/-0.1)kcal/mol and each scaffolding protein -6.1(+/-0.2)kcal/mol to the stability of the procapsid. Because each protein interacts with two or more neighbors, the pair-wise energies are even less. The weak protein interactions observed in the assembly of procapsids are likely important in the control of nucleation, since an increase in affinity between coat and scaffolding proteins can lead to kinetic traps caused by the formation of too many nuclei. In addition, we find that adjusting the molar ratio of scaffolding to coat protein can alter the assembly product. When the scaffolding protein concentration is low relative to coat protein, there is a correspondingly low yield of proper procapsids. When the relative concentration is very high, too many nuclei form, leading to kinetically trapped assembly intermediates.

Bacteriophage P22↗
PubMed1999

Separate assembly and transport domains within the Gag precursor of Mason-Pfizer monkey virus.

Mason-Pfizer monkey virus (M-PMV), the prototypical type D retrovirus, assembles immature capsids within the cytoplasm of the cell prior to plasma membrane interaction. Several mutants of M-PMV Gag have been described which display altered transport, assembly, or both. In this report, we describe the use of an in vitro synthesis and assembly system to distinguish between defects in intracellular transport and the process of assembly itself for two previously described gag gene mutants. Matrix domain mutant R55W converts the type D morphogenesis of M-PMV particles into type C and has been hypothesized to alter the transport of Gag, redirecting it to the plasma membrane where assembly subsequently occurs. We show here that R55W can assemble in both the in vitro translation-assembly system and within inclusion bodies in bacteria and thus has retained the capacity to assemble in the cytoplasm. This supports the concept that R55 is located within a domain responsible for the transport of Gag to an intracellular site for assembly. In contrast, deletions within the p12 domain of M-PMV Gag had previously been shown to affect the efficiency of particle formation such that under low-level expression conditions, Gag would fail to assemble. We demonstrate here that the efficiency of assembly in the in vitro system mirrors that seen in cells under expression conditions similar to that of an infection. These results argue that the p12 domain of this D-type retrovirus plays a critical role in the membrane-independent assembly of immature capsids.

Amino Acid Sequence↗
PubMed1996

The concept of transcortical cell assemblies: a key to the understanding of cortical lateralization and interhemispheric interaction.

According to Hebb, elements of higher cognitive processes, such as concepts, words and mental images, are realized in the brain as cortical cell assemblies, i.e. large and strongly connected neuron populations that form functional units. Neurons belonging to such assemblies may be scattered over wide cortical areas, and some cell assemblies may even comprise neurons of both hemispheres (transcortical assemblies). If full activation (ignition) of an assembly leads to fast circulation of neuronal activity in the assembly, this process should be visible in high-frequency cortical responses. Some evidence will be reviewed that cell assembly ignition indeed leads to changes in high-frequency cortical responses which can be recorded in the EEG and MEG. Within the cell assembly-framework, the question of cortical laterality translates into the question of how neurons of transcortical assemblies are balanced between the hemispheres. This approach allows for different degrees of laterality. Recent evidence is summarized that the degree of laterality indeed differs between language units. For example, the cortical representation of certain words appears to be strongly lateralized to the left hemisphere while those of others are less lateralized. If neurons of both hemispheres are part of one assembly bihemispheric processing should lead to a processing advantage compared to processing in the dominant hemisphere alone. The latter appears to be the case for lexical processing, as revealed by recent behavioral studies. In conclusion, the cell assembly-framework suggests a more fine-grained description of the issue of cortical laterality; it is not appropriate to ask whether "modules" supporting higher cortical functions are located either in the left or right hemisphere. Rather, it appears fruitful to ask how the neurons of transcortical cell assemblies are balanced between the hemispheres.

Brain Mapping↗
PubMed2004

In vitro assembly properties of mutant and chimeric intermediate filament proteins: insight into the function of sequences in the rod and end domains of IF.

The factors and mechanisms regulating assembly of intermediate filament (IF) proteins to produce filaments with their characteristic 10 nm diameter are not fully understood. All IF proteins contain a central rod domain flanked by variable head and tail domains. To elucidate the role that different domains of IF proteins play in filament assembly, we used negative staining and electron microscopy (EM) to study the in vitro assembly properties of purified bacterially expressed IF proteins, in which specific domains of the proteins were either mutated or swapped between a cytoplasmic (mouse neurofilament-light (NF-L) subunit) and nuclear intermediate filament protein (human lamin A). Our results indicate that filament formation is profoundly influenced by the composition of the assembly buffer. Wild type (wt) mouse NF-L formed 10 nm filaments in assembly buffer containing 175 mM NaCl, whereas a mutant deleted of 18 NH2-terminal amino acids failed to assemble under similar conditions. Instead, the mutant assembled efficiently in buffers containing CaCl2 > or = 6 mM forming filaments that were 10 times longer than those formed by wt NF-L, although their diameter was significantly smaller (6-7 nm). These results suggest that the 18 NH2-terminal sequence of NF-L might serve two functions, to inhibit filament elongation and to promote lateral association of NF-L subunits. We also demonstrate that lengthening of the NF-L rod domain, by inserting a 42 aa sequence unique to nuclear IF proteins, does not compromise filament assembly in any noticeable way. Our results suggests that the known inability of nuclear lamin proteins to assemble into 10 nm filaments in vitro cannot derive solely from their longer rod domain. Finally, we demonstrate that the head domain of lamin A can substitute for that of NF-L in filament assembly, whereas substitution of both the head and tail domains of lamins for those of NF-L compromises assembly. Therefore, the effect of lamin A "tail" domain alone, or the synergistic effect of lamin "head" and the "tail" domains together, interferes with assembly into 10-nm filaments.

Amino Acid Sequence↗
PubMed1988

Actin assembly by lithium ions.

The ability of Li+ to promote the assembly of actin has been compared with the more common cations used in actin assembly assays, K+, Mg2+, and Ca2+. The principal assay of actin assembly utilized was fluorescence photobleaching recovery (FPR), from which it is possible to determine the fraction of actin protomers incorporated into filaments and the average diffusion coefficients of the filaments. In addition, critical concentrations of actin over a range of concentrations of all of these cations have been determined using an assay that involves sonication and dilution of assembled actin filaments containing trace amounts of pyrene-labeled actin. The results demonstrate that Li+ is a more potent promoter of actin assembly than is K+. The more rapid assembly of actin in the presence of Li+ is attributable to an increased rate of filament elongation. Filaments assembled in equivalent concentrations of Li+ or K+ have the same diffusion coefficients, and thus presumably the same average lengths. The critical concentration of actin is about three times less in the presence of Li+ than in the presence of an equal concentration of K+. Cytochalasin D accelerates the rate of Li+-promoted actin assembly and reduces slightly the total fraction of actin assembly. However, cytochalasin D causes less shortening of filaments in the presence of Li+ than in the presence of K+ or Mg2+. By the criteria of assembly kinetics and critical concentration, Li+ is much less potent as a promoter of actin assembly than either Mg2+ or Ca2+. These results are discussed in terms of the role of electrostatic forces in the actin assembly mechanism and in terms of possible relationships to therapeutic and toxicity mechanisms for Li+.

Actins↗
PubMed1997

Thermodynamic and structural analysis of microtubule assembly: the role of GTP hydrolysis.

Different models have been proposed that link the tubulin heterodimer nucleotide content and the role of GTP hydrolysis with microtubule assembly and dynamics. Here we compare the thermodynamics of microtubule assembly as a function of nucleotide content by van't Hoff analysis. The thermodynamic parameters of tubulin assembly in 30-100 mM piperazine-N,N'-bis(2-ethanesulfonic acid), 1 mM MgSO4, 2 mM EGTA, pH 6.9, in the presence of a weakly hydrolyzable analog, GMPCPP, the dinucleotide analog GMPCP plus 2 M glycerol, and GTP plus 2 M glycerol were obtained together with data for taxol-GTP/GDP tubulin assembly (GMPCPP and GMPCP are the GTP and GDP nucleotide analogs where the alpha beta oxygen has been replaced by a methylene, -CH2-). All of the processes studied are characterized by a positive enthalpy, a positive entropy, and a large, negative heat capacity change. GMPCP-induced assembly has the largest negative heat capacity change and GMPCPP has the second largest, whereas GTP/2 M glycerol- and taxol-induced assembly have more positive values, respectively. A large, negative heat capacity is most consistent with the burial of water-accessible hydrophobic surface area, which gives rise to the release of bound water. The heat capacity changes observed with GTP/2 M glycerol-induced and with taxol-induced assembly are very similar, -790 +/- 190 cal/mol/k, and correspond to the burial of 3330 +/- 820 A2 of nonpolar surface area. This value is shown to be very similar to an estimate of the buried nonpolar surface in a reconstructed microtubule lattice. Polymerization data from GMPCP- and GMPCPP-induced assembly are consistent with buried nonpolar surface areas that are 3 and 6 times larger. A linear enthalpy-entropy and enthalpy-free energy plot for tubulin polymerization reactions verifies that enthalpy-entropy compensation for this system is based upon true biochemical correlation, most likely corresponding to a dominant hydrophobic effect. Entropy analysis suggests that assembly with GTP/2 M glycerol and with taxol is consistent with conformational rearrangements in 3-6% of the total amino acids in the heterodimer. In addition, taxol binding contributes to the thermodynamics of the overall process by reducing the delta H degree and delta S degree for microtubule assembly. In the presence of GMPCPP or GMPCP, tubulin subunits associate with extensive conformational rearrangement, corresponding to 10% and 26% of the total amino acids in the heterodimer, respectively, which gives rise to a large loss of configurational entropy. An alternative, and probably preferable, interpretation of these data is that, especially with GMPCP-tubulin, additional isomerization or protonation events are induced by the presence of the methylene moiety and linked to microtubule assembly. Structural analysis shows that GTP hydrolysis is not required for sheet closure into a microtubule cylinder, but only increases the probability of this event occurring. Sheet extensions and sheet polymers appear to have a similar average length under various conditions, suggesting that the minimum cooperative unit for closure of sheets into a microtubule cylinder is approximately 400 nm long. Because of their low level of occurrence, sheets are not expected to significantly affect the thermodynamics of assembly.

Animals↗
PubMed1999

In vitro nuclear assembly with affinity-purified nuclear envelope precursor vesicle fractions, PV1 and PV2.

Nuclear envelope precursor vesicles were affinity purified from a Xenopus egg extract by a chromatin binding method. Vesicles bound to chromatin at 4 degrees C were dissociated with a high salt buffer and further fractionated into nuclear envelope precursor vesicle fractions 1 (PV1) and 2 (PV2) by differential centrifugation. PV1 contained larger vesicles. When chromatin was incubated in a Xenopus egg cytosol fraction supplemented with PV1, vesicles bound to chromatin, fused with each other, formed a bilayered nuclear envelope, and assembled into spherical small nuclei. However, the thus assembled nuclei did not grow to the normal size. Nuclear pore complexes were not found on the thus assembled nuclei. On the other hand, PV2 contained smaller vesicles. PV2 vesicles bound to chromatin, fused little with each other in the Xenopus egg cytosol fraction, and no nuclei were assembled. When PV1 supplemented with PV2 was used for the nuclear assembly reaction, the assembled nuclei grew to the normal size. Nuclear pore complexes existed in the thus assembled nuclear envelopes. These results suggested that 1) two vesicle populations, PV1 and PV2, are necessary for the assembly of normal sized nuclei, 2) PV1 contains a chromatin targeting molecule(s) and membrane fusion machinery, 3) PV2 contains a chromatin targeting molecule(s) and a molecule(s) necessary for nuclear pore complex assembly, and 4) PV1 has the ability to assemble a nuclear membrane, and PV2 is necessary for the assembly of nuclear pore complexes and for nuclei to grow to the normal size. An in vitro nuclear assembly system constituted with affinity-purified vesicle fractions, PV1 and PV2, was established.

Animals↗
PubMed1999

A theoretical model successfully identifies features of hepatitis B virus capsid assembly.

The capsids of most spherical viruses are icosahedral, an arrangement of multiples of 60 subunits. Though it is a salient point in the life cycle of any virus, the physical chemistry of virus capsid assembly is poorly understood. We have developed general models of capsid assembly that describe the process in terms of a cascade of low order association reactions. The models predict sigmoidal assembly kinetics, where intermediates approach a low steady state concentration for the greater part of the reaction. Features of the overall reaction can be identified on the basis of the concentration dependence of assembly. In simulations, and on the basis of our understanding of the models, we find that nucleus size and the order of subsequent "elongation" reactions are reflected in the concentration dependence of the extent of the reaction and the rate of the fast phase, respectively. The reaction kinetics deduced for our models of virus assembly can be related to the assembly of any "spherical" polymer. Using light scattering and size exclusion chromatography, we observed polymerization of assembly domain dimers of hepatitis B virus (HBV) capsid protein. Empty capsids assemble at a rate that is a function of protein concentration and ionic strength. The kinetics of capsid formation were sigmoidal, where the rate of the fast phase had second-power concentration dependence. The extent of assembly had third-power concentration dependence. Simulations based on the models recapitulated the concentration dependences observed for HBV capsid assembly. These results strongly suggest that in vitro HBV assembly is nucleated by a trimer of dimers and proceeds by the addition of individual dimeric subunits. On the basis of this mechanism, we suggest that HBV capsid assembly could be an important target for antiviral therapeutics.

Capsid↗
PubMed2003

Multiple assembly signals in gamma-aminobutyric acid (type A) receptor subunits combine to drive receptor construction and composition.

Mammalian gamma-aminobutyric acid type A (GABAA) receptors are constructed from a large repertoire of subunits (alpha1-alpha6, beta1-beta3, gamma1-gamma3, delta, epsilon, theta and pi) into a pentameric ion channel. GABA(A) receptor assembly occurs within the endoplasmic reticulum (ER) and involves interactions with chaperone molecules. Only specific subunit combinations can produce functional surface receptors (with a fixed stoichiometry); other subunit combinations are retained within the ER and degraded. Thus, receptor assembly occurs by defined pathways to limit the diversity of GABAA receptors. The key to understanding how receptor diversity is achieved and controlled is the identification of assembly signals capable of distinguishing between other subunit partners. Analysis of an assembly box in alpha1 (residues 57-68) has revealed an absolute requirement for this region in the assembly of alphabeta receptors. Furthermore, a selective requirement for a single amino acid (R66) is observed for the assembly of alpha1beta2, but not alpha1beta1 or alpha1beta3, receptors. In addition, we have characterized an assembly signal in the beta3 subunit that is capable of driving the assembly of beta3, gamma2beta3 and alpha1beta3 receptors. Interestingly, this signal does not appear to utilize the alpha1 assembly box, suggesting the presence of alternative assembly signals within the alpha1 subunit. Although this beta3 signal is sufficient to permit the formation of betagamma receptors it is not necessary, suggesting that alternative assembly signals also exist within the beta3 subunit. These findings support the belief that GABAA receptor assembly occurs via multiple defined pathways that may be determined by subunit availability.

Amino Acid Sequence↗
PubMed1991

Mutations in the three largest subunits of yeast RNA polymerase II that affect enzyme assembly.

Mutations in the three largest subunits of yeast RNA polymerase II (RPB1, RPB2, and RPB3) were investigated for their effects on RNA polymerase II structure and assembly. Among 23 temperature-sensitive mutations, 6 mutations affected enzyme assembly, as assayed by immunoprecipitation of epitope-tagged subunits. In all six assembly mutants, RNA polymerase II subunits synthesized at the permissive temperature were incorporated into stably assembled, immunoprecipitable enzyme and remained stably associated when cells were shifted to the nonpermissive temperature, whereas subunits synthesized at the nonpermissive temperature were not incorporated into a completely assembled enzyme. The observation that subunit subcomplexes accumulated in assembly-mutant cells at the nonpermissive temperature led us to investigate whether these subcomplexes were assembly intermediates or merely byproducts of mutant enzyme instability. The time course of assembly of RPB1, RPB2, and RPB3 was investigated in wild-type cells and subsequently in mutant cells. Glycerol gradient fractionation of extracts of cells pulse-labeled for various times revealed that a subcomplex of RPB2 and RPB3 appears soon after subunit synthesis and can be chased into fully assembled enzyme. The RPB2-plus-RPB3 subcomplexes accumulated in all RPB1 assembly mutants at the nonpermissive temperature but not in an RPB2 or RPB3 assembly mutant. These data indicate that RPB2 and RPB3 form a complex that subsequently interacts with RPB1 during the assembly of RNA polymerase II.

Amino Acid Sequence↗
PubMed1999

Effects of purinenucleotide analogues on microtubule assembly.

This minireview summarizes the syntheses of various purinenucleotide analogues and their effects on microtubule (Mt) assembly. 27 analogues were so far synthesized and, together with 3 analogues commercially available (ITP, XTP and dGTP), their effects on Microtubule assembly were investigated. The positions C2, C6, C8, and ribose moiety of purine nucleotides were modified or substituted. It was found that the microenvironments of the purine base and ribose moiety are important for the nucleotides to support Mt assembly. Introduction of amino group into position C2 of ATP, formation of 2-amino ATP, caused Mt assembly substantially. 2-Amino deoxy ATP and deoxy GTP are more potent than GTP in supporting assembly. The introduction of reactive thiol group into C6 (6-SH-GTP) largely reduces the activity of the analogue to support assembly. However, sequestering reactivity of the thiol group by association with methyl group largely recovers the ability of the analogue to promote assembly. Free rotation of the glycosidic linkage was found to be also innevitable in promoting assembly, as the introduction of sulfur atom between C8 of the purine base and C2' of the ribose moiety (formation of 8,2'-S-cyclo purine nucleotides) caused total inhibition. Purinenucleoside triphosphate supports assembly better than GTP but the deoxy-type analogues are totally inhibitory. 2-Amino-8-hydroxy ATP and other analogues support assembly much better than does GTP. However, their diphosphate analogues are totally incapable of supporting assembly. Introduction of a bulky fluorescent probes into C3' can be made to visualize the fluorescent signal in assembled Mts. Together with the suggestions proposed from electron chrystallography of zinc-induced tubulin sheets, interactions of the purine base and ribose moieties with surrounding amino acid residues are discussed.

Adenosine Diphosphate↗
PubMed2006

Interactions between progesterone and tumor necrosis factor-alpha in the regulation of primordial follicle assembly.

Follicle assembly is the process by which groups or "nests" of oocytes break down to form primordial follicles. The size of the primordial follicle pool is the major determinant of the reproductive lifespan of a female. Previously, progesterone (P(4)) has been shown to inhibit follicle assembly, while tumor necrosis factor-alpha (TNFalpha) has been shown to promote the apoptosis that is necessary for follicle assembly. The present study examines how TNFalpha and progesterone interact to regulate primordial follicle assembly. Ovaries were collected from newborn rats and placed in organ culture to examine the actions of P(4) and TNFalpha. P(4) was found to decrease primordial follicle assembly and increase the percentage of unassembled oocytes both in vitro and in vivo. TNFalpha treatment did not change the proportion of assembled follicles in cultured ovaries, but blocked the ability of P(4) to inhibit follicle assembly. Microarray analysis of the ovarian transcriptome revealed that progesterone treatment of the ovaries altered the expression of 513 genes with 132 only expressed after P(4) treatment and 16 only expressed in control ovaries. The majority of genes were up-regulated greater than twofold over control, with a small subset of 16 genes down-regulated. Categories of genes affected by P(4) are described including a group of extracellular signaling factors. The progesterone receptors expressed at the time of follicle assembly included the surface membrane progesterone receptors PGRMC1, PGRMC2, and RDA288. The nuclear genomic P(4) receptor was not expressed at appreciable levels. Progesterone increased the expression of several genes (TANK, NFkappaB, Bcl2l1, and Bcl2l2) involved in a signaling pathway that promotes cell survival and inhibits apoptosis. Observations indicate that P(4) acts through the surface membrane progesterone receptors to regulate primordial follicle assembly, and that TNFalpha can override the inhibitory actions of P(4) on follicle assembly. A major mechanism involved in the actions of P(4) is an increase in cell survival genes and inhibition of the apoptosis pathway. Observations provide insight into the hormonal regulation of primordial follicle assembly and lead to novel approaches to potentially manipulate follicle assembly and reproductive capacity.

Animals↗
PubMed2004

Physical mechanisms and biological significance of supramolecular protein self-assembly.

In living cells, chemical reactions of metabolism, information processing, growth and development are organized in a complex network of interactions. At least in part, the organization of this network is accomplished as a result of physical assembly by supramolecular scaffolds. Indeed, most proteins function in cells within the context of multimeric or supramolecular assemblies. With the increasing availability of atomic structures and molecular thermodynamics, it is possible to recast the problem of non-covalent molecular self-assembly from a unified perspective of structural thermodynamics and kinetics. Here, we present a generalized theory of self-assembly based on Wegner's kinetic model and use it to delineate three physical mechanisms of self-assembly: as limited by association of assembly units (nucleation), by association of monomers (isodesmic), and by conformational reorganization of monomers that is coupled to assembly (conformational). Thus, we discuss actin, tubulin, clathrin, and the capsid of icosahedral cowpea chlorotic mottle virus with respect to assembly of architectural scaffolds that perform largely mechanical functions, and pyruvate dehydrogenase, and RING domain proteins PML, arenaviral Z, and BRCA1:BARD1 with regard to assembly of supramolecular enzymes with metabolic and chemically directive functions. In addition to the biological functions made possible by supramolecular self-assembly, such as mesoscale mechanics of architectural scaffolds and metabolic coupling of supramolecular enzymes, we show that the physical mechanisms of self-assembly and their structural bases are biologically significant as well, having regulatory roles in both formation and function of the assembled structures in health and disease.

Actins↗