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At least 667 records · Page 37Linked to original sources
PubMed2005

An assembly landscape for the 30S ribosomal subunit.

Self-assembling macromolecular machines drive fundamental cellular processes, including transcription, messenger RNA processing, translation, DNA replication and cellular transport. The ribosome, which carries out protein synthesis, is one such machine, and the 30S subunit of the bacterial ribosome is the preeminent model system for biophysical analysis of large RNA-protein complexes. Our understanding of 30S assembly is incomplete, owing to the challenges of monitoring the association of many components simultaneously. Here we have developed a method involving pulse-chase monitored by quantitative mass spectrometry (PC/QMS) to follow the assembly of the 20 ribosomal proteins with 16S ribosomal RNA during formation of the functional particle. These data represent a detailed and quantitative kinetic characterization of the assembly of a large multicomponent macromolecular complex. By measuring the protein binding rates at a range of temperatures, we find that local transformations throughout the assembling subunit have similar but distinct activation energies. Thus, the prevailing view of 30S assembly as a pathway proceeding through a global rate-limiting conformational change must give way to one in which the assembly of the complex traverses a landscape dotted with various local conformational transitions.

Kinetics↗
PubMed2006

Centriole assembly in Caenorhabditis elegans.

Centrioles are necessary for flagella and cilia formation, cytokinesis, cell-cycle control and centrosome organization/spindle assembly. They duplicate once per cell cycle, but the mechanisms underlying their duplication remain unclear. Here we show using electron tomography of staged C. elegans one-cell embryos that daughter centriole assembly begins with the formation and elongation of a central tube followed by the peripheral assembly of nine singlet microtubules. Tube formation and elongation is dependent on the SAS-5 and SAS-6 proteins, whereas the assembly of singlet microtubules onto the central tube depends on SAS-4. We further show that centriole assembly is triggered by an upstream signal mediated by SPD-2 and ZYG-1. These results define a structural pathway for the assembly of a daughter centriole and should have general relevance for future studies on centriole assembly in other organisms.

Animals↗
PubMed2004

Stimuli-responsive polypeptide vesicles by conformation-specific assembly.

In biology, lipids are well known for their ability to assemble into spherical vesicles. Proteins, in particular virus capsids, can also form regular vesicle-like structures, where the precise folding and stable conformations of many identical subunits directs their self-assembly. Functionality present on these subunits also controls their disassembly within the cellular environment, for example, in response to a pH change. Here, we report the preparation of diblock copolypeptides that self-assemble into spherical vesicular assemblies whose size and structure are dictated primarily by the ordered conformations of the polymer segments, in a manner similar to viral capsid assembly. Furthermore, functionality was incorporated into these molecules to render them susceptible to environmental stimuli, which is desirable for drug-delivery applications. The control of assembly and function exhibited in these systems is a significant advance towards the synthesis of materials that can mimic the precise three-dimensional assembly found in proteins.

Hydrogen-Ion Concentration↗
PubMed2025

A chromosome-level genome assembly of Guimi No. 2 (Actinidia chinensis).

In this study, we report a high-quality chromosome-level genome assembly of Actinidia chinensis var. chinensis 'Guimi No. 2'. This cultivar, discovered in Guizhou karst ecosystems, exhibits resistance to Pseudomonas syringae pv. actinidiae (Psa). Using a combination of MGI short-read sequencing, PacBio HiFi long-read sequencing, and Hi-C technology, we generated a genome assembly of 608.43 Mb with a contig N50 of 20.70 Mb, and 99.70% of the assembly was successfully anchored onto 29 pseudochromosomes. The quality value (QV) and the LTR Assembly Index (LAI) of the assembled genome were 72.23 and 10.10. The BUSCO analysis indicated that the genome assembly and gene model prediction were 98.40% and 96.56% complete, respectively. A total of 251.15 Mb of repetitive sequences and 45,986 protein-coding genes were annotated. This genome assembly provides critical insights into A. chinensis's genomic architecture and serves as a foundational resource for elucidating disease resistance mechanisms against Psa, while enabling comparative phylogenomic studies across the Actinidia genus.

Actinidia↗
PubMed2006

Chitosan-mediated in situ biomolecule assembly in completely packaged microfluidic devices.

We report facile in situ biomolecule assembly at readily addressable sites in microfluidic channels after complete fabrication and packaging of the microfluidic device. Aminopolysaccharide chitosan's pH responsive and chemically reactive properties allow electric signal-guided biomolecule assembly onto conductive inorganic surfaces from the aqueous environment, preserving the activity of the biomolecules. A transparent and nonpermanently packaged device allows consistently leak-free sealing, simple in situ and ex situ examination of the assembly procedures, fluidic input/outputs for transport of aqueous solutions, and electrical ports to guide the assembly onto the patterned gold electrode sites within the channel. Both in situ fluorescence and ex situ profilometer results confirm chitosan-mediated in situ biomolecule assembly, demonstrating a simple approach to direct the assembly of biological components into a completely fabricated device. We believe that this strategy holds significant potential as a simple and generic biomolecule assembly approach for future applications in complex biomolecular or biosensing analyses as well as in sophisticated microfluidic networks as anticipated for future lab-on-a-chip devices.

Biosensing Techniques↗
PubMed2005

Mobilization of the iron centre in IscA for the iron-sulphur cluster assembly in IscU.

The biogenesis of iron-sulphur clusters requires the co-ordinated delivery of both iron and sulphur. It is now clear that sulphur in iron-sulphur clusters is derived from L-cysteine by cysteine desulphurases. However, the iron donor for the iron-sulphur cluster assembly still remains elusive. Our previous studies indicated that Escherichia coli IscA, a member of the iron-sulphur cluster assembly machinery, is an iron-binding protein that can provide iron for the iron-sulphur cluster assembly in a proposed scaffold IscU. To determine how the iron centre in IscA is transferred for the iron-sulphur cluster assembly in IscU, we explore the mobility of the iron centre in IscA. The UV-visible and EPR measurements show that L-cysteine, but not IscU, is able to mobilize the iron centre in IscA and make the iron available for the iron-sulphur cluster assembly in IscU. Other related biological thiols such as N-acetyl-L-cysteine or reduced glutathione have no effect on the iron centre of IscA, suggesting that L-cysteine is unique in mobilizing the iron centre of IscA. Nevertheless, L-cysteine alone is not sufficient to transfer the iron from IscA to IscU. Both L-cysteine and cysteine desulphurase (IscS) are required for the IscA-mediated assembly of iron-sulphur clusters in IscU. The results suggest that L-cysteine may have two distinct functions in the biogenesis of iron-sulphur clusters: to mobilize the iron centre in IscA and to provide sulphur via cysteine desulphurase (IscS) for the iron-sulphur cluster assembly in IscU.

Carrier Proteins↗
PubMed1983

Evidence for the sequential assembly of cytochrome oxidase subunits in rat liver mitochondria.

The assembly of cytochrome oxidase was studied in isolated rat liver mitochondria and isolated rat hepatocytes labelled in vitro with L-[35S]methionine. This was achieved by studying the temporal association of radioactive subunits which are immunoabsorbed with antibodies against subunits I, II and the holoenzyme. Antibodies against the holoenzyme were shown to be highly specific for subunit V. The results show that subunit I appears in the holoenzyme late in the assembly process. No radioactive subunit I is absorbed with antiserum against subunit II or the holoenzyme (subunit V) after a 30 min pulse in either isolated mitochondria or hepatocytes. However, both antisera absorb radioactive subunits I after a 150 min chase in isolated hepatocytes. This was confirmed using antibodies against subunit I, which absorbed only radioactive subunit I after a 30 min pulse but absorbed radioactive subunits I-III and VI after a 150 min chase. Thus, the late assembly of radioactive subunit I is explained by a temporal sequence in the assembly process and not by the presence of a large, non-radioactive pool of subunit I. Using the above approach and the three specific antisera, the following temporal sequence in the assembly of cytochrome oxidase was established. Subunits II and III assemble rapidly with each other or with cytoplasmically translated subunit VI. This complex of three peptides in turn assembles slowly with subunit I or with the other cytoplasmically translated subunits. The early association of subunit VI with the mitochondrially translated subunits II and III suggests a possible role of the former in integration of the holoenzyme.

Animals↗
PubMed1985

Microtubules and nucleoside diphosphate kinase. Comparison of kinetics of GTP- and CTP-induced assembly.

The kinetics of assembly of MAP2-tubulin microtubule protein were examined as a function of the GTP concentration in order to test the hypothesis that CTP-induced assembly results from the generation of GTP by nucleoside diphosphate kinase. These studies show that (a) there is no assembly below a minimum GTP concentration and that this represents a nucleation requirement, (b) the rate of elongation is inconsistent with a single assembly-species, and (c) the elongation rate increases markedly as the GTP concentration is raised, although GTP is not absolutely required for elongation. These assembly kinetics have been compared with those with increasing CTP concentrations, by using microtubule protein containing a very low nucleoside diphosphate kinase activity of known substrate specificity. Neither nucleation nor the observed rates of elongation can be attributed to the formation of GTP, either (a) in terms of the generation of free GTP and subsequent binding to tubulin or (b) by the direct charging of GDP bound to the tubulin exchangeable site. The results show that nucleoside diphosphate kinase is not required for CTP-induced microtubule assembly, and suggest that CTP directly effects microtubule assembly.

Animals↗
PubMed2003

Functional assembly of thylakoid deltapH-dependent/Tat protein transport pathway components in vitro.

Assembly of the components of the thylakoid deltapH-dependent/Tat protein transport machinery was analyzed in vitro. Upon incubation with intact chloroplasts, precursors to all three components, Hcf106, cpTatC and Tha4, were imported into the organelle and assembled into characteristic endogenous complexes. In particular, all of the imported cpTatC and approximately two-thirds of the imported Hcf106 functionally assembled into 700 kDa complexes capable of binding Tat pathway precursor proteins. The amounts assembled into thylakoids by this procedure were moderate. However, physiological quantities of mature forms of Tha4 and Hcf106 were integrated into isolated thylakoids and a significant percentage of the Hcf106 so integrated was assembled into the 700 kDa complex. Interestingly, a mutant form of Hcf106 in which an invariant transmembrane glutamate was changed to glutamine integrated into the membrane but did not assemble into the receptor complex. Analysis of energy and known pathway component requirements indicated that Hcf106 and Tha4 integrate by an unassisted or 'spontaneous' mechanism. The functionality of in vitro integrated Tha4 was verified by its ability to restore transport to thylakoid membranes from the maize tha4 mutant, which lacks the Tha4 protein. Development of this functional in vitro assembly assay will facilitate structure-function studies of the thylakoid Tat pathway translocation machinery.

Arginine↗
PubMed2004

Protein assembly of photosystem II and accumulation of subcomplexes in the absence of low molecular mass subunits PsbL and PsbJ.

The protein assembly and stability of photosystem II (PSII) (sub)complexes were studied in mature leaves of four plastid mutants of tobacco (Nicotiana tabacum L), each having one of the psbEFLJ operon genes inactivated. In the absence of psbL, no PSII core dimers or PSII-light harvesting complex (LHCII) supercomplexes were formed, and the assembly of CP43 into PSII core monomers was extremely labile. The assembly of CP43 into PSII core monomers was found to be necessary for the assembly of PsbO on the lumenal side of PSII. The two other oxygen-evolving complex (OEC) proteins, PsbP and PsbQ, were completely lacking in Delta psbL. In the absence of psbJ, both intact PSII core monomers and PSII core dimers harboring the PsbO protein were formed, whereas the LHCII antenna remained detached from the PSII dimers, as demonstrated by 77 K fluorescence measurements and by the lack of PSII-LHCII supercomplexes. The Delta psbJ mutant was characterized by a deficiency of PsbQ and a complete lack of PsbP. Thus, both the PsbL and PsbJ subunits of PSII are essential for proper assembly of the OEC. The absence of psbE and psbF resulted in a complete absence of all central PSII core and OEC proteins. In contrast, very young, vigorously expanding leaves of all psbEFLJ operon mutants accumulated at least traces of D2, CP43 and the OEC proteins PsbO and PsbQ, implying developmental control of the expression of the PSII core and OEC proteins. Despite severe problems in PSII assembly, the thylakoid membrane complexes other than PSII were present and correctly assembled in all psbEFLJ operon mutants.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2002

The gamma-Na+,K+-ATPase subunit assembles selectively with alpha1/beta1-Na+,K+-ATPase but not with the colonic H+,K+-ATPase.

BACKGROUND: The ubiquitous Na+-pump (Na+,K+-ATPase) assembles as a heterodimer of composition alpha/beta in some nephron segments, while in other segments it may exist as a heterotrimer of composition alpha/beta/gamma. The gamma-subunit has been reported to increase the affinity of the Na+-pump for adenosine 5'-triphosphate (ATP), and decrease affinity for both Na+ and K+. The alpha-subunit of the colonic H+,K+-ATPase (cHK) shares 75% sequence similarity with alpha1-Na+,K+-ATPase (alpha1) and assembles with beta1-Na+,K+-ATPase (beta1) in distal colon and renal medulla. Differences in pharmacological properties have been ascribed to when heterologously expressed function has been compared to function in vitro. The purpose of this study was to determine if cHK might associate with the gamma-subunit of the Na+,K+-ATPase (gamma) as a possible explanation for these variations in function. METHODS: An antibody specific for the gamma was used in coimmunoprecipitation experiments to determine if the gamma assembles stably in vitro with cHK and beta1 in rat renal medulla or distal colon. RESULTS: Our results demonstrate that the gamma-subunit assembles specifically with the Na+-pump, but not with cHK. Furthermore, the gamma-subunit assembly was specific for rat kidney and was not observed in distal colon. CONCLUSION: Since the gamma-subunit did not assemble with the cHK/beta1 complex, gamma-subunit assembly cannot explain those variations in ex vivo and in vitro pharmacologic properties ascribed to cHK.

Animals↗
PubMed2004

Self-assembly of peptides into a beta-barrel motif.

We report the results of a study of the self-assembly of four minimalist peptide strands with a native beta-barrel structure. Using a soft-well potential to mimic cellular crowding, molecular dynamics simulations were performed in confining spheres of varying radii. By utilizing a previously introduced scaling factor lambda for the non-native hydrophobic interactions (0<lambda<1), we were able to study models with varying degrees of frustration. Both the thermodynamics and kinetics of a Go-like model (lambda=0) and a highly frustrated model (lambda=0.9) were studied. Additionally, we used an extrapolation technique to investigate the thermodynamics of assembly at intermediate values of lambda. As in our earlier work [J. Chem. Phys. 118, 8106 (2003)] on a connected Go-like model beta-barrel protein, we find that the stability of the assembled protein increases with decreasing sphere size, and that larger confining spheres result in increased assembly times. Additionally, the lambda=0 model seems to undergo distinct phase transitions during the assembly process. In contrast, the more frustrated model (lambda=0.9) appears to undergo a glasslike transition at temperatures comparable to the assembly temperature of the Go model, and that this transition is relatively nonspecific. Our results suggest the assembly process is dependent on both sequence and environment, with implications for the formation of misassembled aggregates.

Computer Simulation↗
PubMed2004

Sequential shape-and-solder-directed self-assembly of functional microsystems.

We demonstrate the fabrication of packaged microsystems that contain active semiconductor devices and passive components by using a directed self-assembly technique. The directed self-assembly is accomplished by combining geometrical shape recognition with site-specific binding involving liquid solder. Microfabricated components with matching complementary shapes, circuits, and liquid solder-coated areas were suspended in ethylene glycol and agitated by using a turbulent liquid flow to initiate the self-assembly. Microsystems were obtained by sequentially adding components of different types. Six hundred AlGaInP/GaAs light-emitting diode segments with a chip size of 200 microm were assembled onto device carriers with a yield of 100% in 2 min. Packaged light-emitting diodes formed with yields exceeding 97% as a result of two self-assembly steps in 4 min. This self-assembly procedure, based on geometrical shape recognition and subsequent binding to form mechanical and electrical connections, provides a high distinguishing power between different components and a route to nonrobotic parallel assembly of electrically functional hybrid microsystems in three dimensions.

Computers↗
PubMed2001

Nanohedra: using symmetry to design self assembling protein cages, layers, crystals, and filaments.

A general strategy is described for designing proteins that self assemble into large symmetrical nanomaterials, including molecular cages, filaments, layers, and porous materials. In this strategy, one molecule of protein A, which naturally forms a self-assembling oligomer, A(n), is fused rigidly to one molecule of protein B, which forms another self-assembling oligomer, B(m). The result is a fusion protein, A-B, which self assembles with other identical copies of itself into a designed nanohedral particle or material, (A-B)(p). The strategy is demonstrated through the design, production, and characterization of two fusion proteins: a 49-kDa protein designed to assemble into a cage approximately 15 nm across, and a 44-kDa protein designed to assemble into long filaments approximately 4 nm wide. The strategy opens a way to create a wide variety of potentially useful protein-based materials, some of which share similar features with natural biological assemblies.

Amino Acid Sequence↗
PubMed2006

Self-assembled single-crystal silicon circuits on plastic.

We demonstrate the use of self-assembly for the integration of freestanding micrometer-scale components, including single-crystal, silicon field-effect transistors (FETs) and diffusion resistors, onto flexible plastic substrates. Preferential self-assembly of multiple microcomponent types onto a common platform is achieved through complementary shape recognition and aided by capillary, fluidic, and gravitational forces. We outline a microfabrication process that yields single-crystal, silicon FETs in a freestanding, powder-like collection for use with self-assembly. Demonstrations of self-assembled FETs on plastic include logic inverters and measured electron mobility of 592 cm2/V-s. Finally, we extend the self-assembly process to substrates each containing 10,000 binding sites and realize 97% self-assembly yield within 25 min for 100-microm-sized elements. High-yield self-assembly of micrometer-scale functional devices as outlined here provides a powerful approach for production of macroelectronic systems.

Journal Article↗
PubMed2006

Atomic structures of peptide self-assembly mimics.

Although the beta-rich self-assemblies are a major structural class for polypeptides and the focus of intense research, little is known about their atomic structures and dynamics due to their insoluble and noncrystalline nature. We developed a protein engineering strategy that captures a self-assembly segment in a water-soluble molecule. A predefined number of self-assembling peptide units are linked, and the beta-sheet ends are capped to prevent aggregation, which yields a mono-dispersed soluble protein. We tested this strategy by using Borrelia outer surface protein (OspA) whose single-layer beta-sheet located between two globular domains consists of two beta-hairpin units and thus can be considered as a prototype of self-assembly. We constructed self-assembly mimics of different sizes and determined their atomic structures using x-ray crystallography and NMR spectroscopy. Highly regular beta-sheet geometries were maintained in these structures, and peptide units had a nearly identical conformation, supporting the concept that a peptide in the regular beta-geometry is primed for self-assembly. However, we found small but significant differences in the relative orientation between adjacent peptide units in terms of beta-sheet twist and bend, suggesting their inherent flexibility. Modeling shows how this conformational diversity, when propagated over a large number of peptide units, can lead to a substantial degree of nanoscale polymorphism of self-assemblies.

Amino Acid Sequence↗
PubMed2006

AMP-activated protein kinase regulates the assembly of epithelial tight junctions.

AMP activated protein kinase (AMPK), a sensor of cellular energy status in all eukaryotic cells, is activated by LKB1-dependent phosphorylation. Recent studies indicate that activated LKB1 induces polarity in epithelial cells and that this polarization is accompanied by the formation of tight junction structures. We wished to determine whether AMPK also contributes to the assembly of tight junctions in the epithelial cell polarization process. We found that AMPK is activated during calcium-induced tight junction assembly. Activation of AMPK by 5-aminoimidazole-4-carboxamide ribonucleoside facilitates tight junction assembly under conditions of normal extracellular Ca2+ concentrations and initiates tight junction assembly in the absence of Ca2+ as revealed by the relocation of zonula occludens 1, the establishment of transepithelial electrical resistance, and the paracellular flux assay. Expression of a dominant negative AMPK construct inhibits tight junction assembly in MDCK cells, and this defect in tight junction assembly can be partially ameliorated by rapamycin. These results suggest that AMPK plays a role in the regulation of tight junction assembly.

AMP-Activated Protein Kinases↗
PubMed1982

Assembly map of the large subunit (50S) of Escherichia coli ribosomes.

Stoichiometric amounts of ribosomal proteins and RNA derived from the 50S subunit reconstitute to fully active particles under the conditions of a two-step incubation procedure. After the first incubation, all components are found in a particle that is activated in the second incubation [Dohme, F. & Nierhaus, K. H. (1976) J. Mol. Biol. 107, 585-599]. Here we describe the assembly dependences of the ribosomal components in the first incubation. Assembly dependence is the requirements of one protein that, before it binds, another must be first built into the ribosome. After incubation of 23S RNA and the proteins under observation, the mixture was subjected to sucrose gradient analysis. The RNA-protein complex was precipitated with trichloroacetic acid and the proteins were identified by NaDodSO4 gel electrophoresis. The assembly dependences of 26 proteins could be elucidated. In a second series of experiments, the incorporation of 3H-labeled 5S RNA in the 23S-protein complex was analyzed. It was found that L5, L15, and L18 are absolutely required for 5S RNA incorporation. In addition, two of the three proteins L2, L3, and L4 are needed, in excellent agreement with the protein dependences. The data are summarized in an assembly map. Comparison with other data shows a structural domain at the 5' end of 23S RNA around protein L20 combining all proteins essential in the early assembly. All the proteins essential for the reconstitution of the peptidyltransferase protein form a skeleton of strong assembly dependences. Finally, L proteins whose genes are present in large transcriptional units on the chromosome depend on each other during assembly.

Escherichia coli↗