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PubMed1984

Molecular weight dependency of heparin inhibition of microtubule assembly in vitro.

Low molar ratios of heparin inhibited in vitro assembly of bovine brain microtubule proteins and disassembled preformed microtubules. Addition of purified microtubule-associated proteins counteracted the assembly inhibition by heparin. Our results suggest that the polyanion heparin affects microtubule assembly by binding to the microtubule-associated proteins. This complex can not support nucleation or stabilize the microtubule structure although it still can associate with the tubulin polymer. In the presence of heparin, the critical concentration needed for microtubule assembly was increased. Furthermore, the absolute assembly difference induced by heparin, the delta A350, was only dependent on the concentration and the molecular weight of heparin, not of the total microtubule protein concentration, or the addition of microtubule-associated proteins. Commercial, standard heparin (Mr 6000-25 000) had an I50 of about 0.1/tubulin dimer. The heparin fraction(s) with a high molecular weight had a stronger effect than those with lower molecular weight. Substoichiometric amounts of taxol completely relieved the inhibition of assembly by heparin, although aberrant forms were present. These microtubules had a reduced amount of coassembled microtubule-associated proteins, and furthermore contained heparin.

Alkaloids↗
PubMed1995

Assembly of mitochondrial ATP synthase in cultured human cells: implications for mitochondrial diseases.

To study the assembly of mitochondrial F1F0 ATP synthase, cultured human cells were labeled with [35S]methionine in pulse-chase experiments. Next, two-dimensional electrophoresis and fluorography were used to analyze the assembly pattern. Two assembly intermediates could be demonstrated. First the F1 part appeared to be assembled, and next an intermediate product that contained F1 and subunit c. This product probably also contained subunits b, F6 and OSCP, but not the mitochondrially encoded subunits a and A6L. Both intermediate complexes accumulated when mitochondrial protein synthesis was inhibited, suggesting that mitochondrially encoded subunits are indispensable for the formation of a fully assembled ATP synthase complex, but not for the formation of the intermediate complexes. The results and methods described in this study offer an approach to study the effects of mutations in subunits of mitochondrial ATP synthase on the assembly of this complex. This might be of value for a better understanding of deficiencies of ATP synthase activity in mitochrondrial diseases.

Anti-Bacterial Agents↗
PubMed2006

Promotion of tubulin assembly by poorly soluble taxol analogs.

Promotion or inhibition of tubulin assembly into microtubules is the standard in vitro assay for evaluating potential antimicrotubule agents. Many agents to be tested are poorly soluble in aqueous solution and require a cosolvent such as dimethyl sulfoxide (DMSO). However, DMSO itself can promote tubulin assembly, and its inclusion in assays for compounds that induce tubulin assembly complicates interpretation of the results. Substituting GDP for GTP in the exchangeable nucleotide binding site of tubulin produces a less active form of the protein, tubulin-GDP. Here it is shown that tubulin-GDP can be assembled into normal microtubules in DMSO concentrations up to 15% (v/v), and polymerization assays performed under these conditions can be compared with assays run under more standard conditions. Assays for measuring the effective concentration of a ligand for promotion of tubulin assembly (EC(50)), measuring the concentration for inhibition of tubulin assembly (IC(50)) by a colchicine site ligand, and measuring tubulin critical concentrations in the presence of poorly soluble taxol derivatives are illustrated.

Biopolymers↗
PubMed2004

Conformational transition state is responsible for assembly of microtubule-binding domain of tau protein.

In the brains of Alzheimer's disease patients, the tau protein dissociates from the axonal microtubule and abnormally aggregates to form a paired helical filament (PHF). One of the priorities in Alzheimer research is to clarify the mechanism of PHF formation. Although several reports on the regulation of tau assembly have been published, it is not yet clear whether in vivo PHFs are composed of beta-structures or alpha-helices. Since the four-repeat microtubule-binding domain (4RMBD) of the tau protein has been considered to play an essential role in PHF formation, its heparin-induced assembly propensity was investigated by the thioflavin fluorescence method to clarify what conformation is most preferred for the assembly. We analyzed the assembly propensity of 4RMBD in Tris-HCl buffer with different trifluoroethanol (TFE) contents, because TFE reversibly induces the transition of the random structure to the alpha-helical structure in an aqueous solution. Consequently, it was observed that the 4RMBD assembly is most significantly favored to proceed in the 10-30% TFE solution, the concentration of which corresponds to the activated transition state of 4RMBD from a random structure to an alpha-helical structure, as determined from the circular dichroism (CD) spectral changes. Since such an assembly does not occur in a buffer containing TFE of < 10% or > 40%, the intermediate conformation between the random and alpha-helical structures could be most responsible for the PHF formation of 4RMBD. This is the first report to clarify that the non-native alpha-helical intermediate in transition from random coil is directly associated with filament formation at the start of PHF formation.

Amino Acid Sequence↗
PubMed2004

Modification of fibrous poly(L-lactic acid) scaffolds with self-assembling triblock molecules.

Molecular self-assembly offers an effective method to modify the surface properties of common biomaterials by presenting biologically relevant chemistry in a controlled, ordered fashion. This work reports on self-assembling triblock molecules containing rigid cholesteryl segments followed by flexible oligomers of L-(lactic acid) and second generation L-lysine dendrons. Second harmonic generation and small angle X-ray scattering indicate these molecules self-assemble into multilayer polar structures when cast from ethyl acetate solutions and segregate into polar polydomains when annealed. These self-assembled layers significantly improve water wettability when coated onto poly(L-lactic acid) fibers. Scaffolds formed from fibers modified by self-assembly enhance adhesion of 3T3 mouse calvaria cells and produce greater population growth rates. These results demonstrate the use of self-assembly to present biologically relevant chemistry on surfaces of biomaterials. Applications of this technology include the modification of substrates for cell culture, tissue engineering, and cell transplantation.

Acetates↗
PubMed2005

Immobilization of rhodopsin on a self-assembled multilayer and its specific detection by electrochemical impedance spectroscopy.

Rhodopsin, the G protein-coupled receptor (GPCR) which mediates the sense of vision, was prepared from calf eyes and used as receptor enriched membrane fraction. In this study it was immobilized onto gold electrode by two different techniques: Langmuir-Blodgett (LB) and a strategy based on a self-assembled multilayer. We demonstrated that Langmuir and LB films of rhodopsin are not stable. Thus, in this study a new protein multilayer was prepared on gold electrode by building up layer-by-layer a self-assembled multilayer. It is composed of a mixed self-assembled monolayer formed by MHDA and biotinyl-PE, followed by a biotin-avidin system which allows binding of biotinylated antibody specific to rhodopsin. The immobilization of rhodopsin in membrane fraction, by the specific antibody bound previously on self-assembled multilayer, was monitored with electrochemical impedance spectroscopy (EIS). In addition, the specificity and sensitivity of this self-assembled multilayer system to the presence of rhodopsin were investigated. No effect was observed when the system was in contact with olfactory receptor I7 in membrane fraction used for control measurements. All these results demonstrate that rhodopsin can be immobilized efficiently, specifically, quantitatively and stably on gold electrode through the self-assembled multilayer.

Adsorption↗
PubMed2005

Open tubular capillary electrochromatography using capillaries coated with films of alkanethiol-self-assembled gold nanoparticle layers.

We have used alkanethiol self-assembly and dithiol layer-by-layer (LBL) self-assembly processes to prepare an Au nanoparticle (NP)-coated open tubular capillary electrochromatography (OTCEC) column for the separation of three neutral steroid drugs (testosterone, progesterone, and testosterone propionate). The CEC column was fabricated through LBL self-assembly of Au NPs on a 3-aminopropyltrimethoxysilane (APTMS)-modified fused-silica capillary and subsequent surface functionalization of the Au NPs through self-assembly of alkanethiols. We investigated the electrochromatographic properties of the resulting Au NP-coated CEC column using a "reversed phase" test mixture of three steroid drugs. We found that the key factors affecting the separation performance were the number of Au NP layers, the length of the carbon-atom chain of the alkanethiol self-assembled on the Au NPs, the percentage of organic modifier, and the pH of the running electrolyte. This study reveals that the self-assembly of alkanethiols and dithiols onto Au NPs provides stationary phases for CEC separation that are easy to prepare and whose retention behavior is highly controllable and reproducible. We believe that our findings will contribute to further studies of the application of nanotechnology to separation science.

Acetonitriles↗
PubMed2006

The characteristics of elastic fiber assembled with recombinant tropoelastin isoform.

OBJECTIVE: It is known that elastin mRNA is transcribed from a single gene. The variety of tropoelastin isoforms results from multiple alternative splicing of the primary transcript. The purpose of this study was to investigate the characteristics of elastic fiber assembled with tropoelastin isoform, which is full-length human tropoelastin (HTE), exon 26A missing tropoelastin (Delta26A), and exon 32 missing tropoelastin (Delta32). DESIGN AND METHODS: We demonstrated the process of elastic fiber assembly and the existence of elastic fiber resistant to pancreatic elastase with HTE, Delta26A, or Delta32 fiber using an in vitro model of elastic fiber assembly. These elastic fibers were evaluated by immunofluorescent staining, the quantitative analysis of cross-linked amino acids, and semi-quantitative analysis of matrix-associated tropoelastin. RESULTS: There were no big differences getting into the matrix among these tropoelastins in immunofluorescence microscopy and semi-quantitative analysis. In the comparison with the HTE, the Delta26A and the Delta32 significantly increased and decreased, respectively, the formation of cross-linking amino acids and the binding to scaffold proteins. Furthermore, it was found that it is difficult to degrade the Delta26A assembly with pancreatic elastase as compared with HTE or Delta32 assembly. CONCLUSION: The elastic fiber assembled with the tropoelastin isoforms was characterized using an in vitro model. The present study provides important information regarding the pathology of human diseases including emphysema and atherosclerosis.

Alternative Splicing↗
PubMed2004

The rate of bipolar spindle assembly depends on the microtubule-gliding velocity of the mitotic kinesin Eg5.

During early embryonic cycles, the time required for mitotic spindle assembly must match the autonomous cell cycle oscillations because a lack of coordination between these two processes will result in chromosome segregation errors. Members of the widely conserved BimC kinesin family are essential for spindle formation in all eukaryotes, and complete loss of BimC function results in monopolar spindles that have two spindle poles that are not separated. However, the precise roles of BimC motor activity in the spindle assembly process are not known. To examine the contribution of BimC kinesin's motor activity to spindle assembly, we generated and characterized mutants of Eg5, a vertebrate BimC kinesin, with reduced in vitro microtubule-gliding velocities. In Xenopus egg extracts, we replaced endogenous Eg5 with recombinant wild-type or mutant motor proteins. By using centrosome-dependent and centrosome-independent spindle assembly assays, we found that mechanisms that determine spindle size and shape were robust to approximately 6-fold reductions in Eg5 motility. However, the spindle assembly process was slower when Eg5 motor function was impaired. This role of Eg5 was independent of its contribution to centrosome separation. We provide evidence that Eg5 is a rate-limiting component of the cellular machinery that drives spindle assembly in vertebrates.

Animals↗
PubMed2006

Dynamics of the formin for3p in actin cable assembly.

BACKGROUND: Formins are a conserved family of actin nucleators responsible for the assembly of diverse actin structures such as cytokinetic rings and filopodia. In the fission yeast Schizosaccharomyces pombe, the formin for3p is necessary for the formation of actin cables, which are bundles of short parallel actin filaments that regulate cell polarity. These filaments are largely organized with their barbed ends facing the cell tip, where for3p is thought to function in their assembly. RESULTS: Here, using a functional for3p-3GFP fusion expressed at endogenous levels, we find that for3p localizes to small dots that appear transiently at cell tips and then move away on actin cables at a rate of 0.3 microm/s. These movements were dependent on the continuous assembly of actin in cables, on the ability of for3p to bind actin within its FH2 domain, and on profilin and bud6p, two formin binding proteins that promote formin activity. Bud6p transiently colocalizes with for3p at the cell tip and stays behind at the cell tip when for3p detaches. CONCLUSIONS: These findings suggest a new model for actin cable assembly: a for3p particle is activated and promotes the assembly of a short actin filament at the cell tip for only seconds. For3p and the actin filament may then be released from the cell tip and carried passively into the cell interior by retrograde flow of actin filaments in the cable. These studies reveal a complex and dynamic cycle of formin regulation and actin cable assembly in vivo.

Actin Cytoskeleton↗
PubMed2004

Assembly of nicotinic alpha7 subunits in Xenopus oocytes is partially blocked at the tetramer level.

The assembly of nicotinic alpha1beta1gammadelta, alpha3beta4, and alpha7 receptors and 5-hydroxytryptamine 3A (5HT3A) receptors was comparatively evaluated in Xenopus oocytes by blue native PAGE analysis. While alpha1betagammadelta subunits, alpha3beta4 subunits, and 5HT3A subunits combined efficiently to pentamers, alpha7 subunits existed in various assembly states including trimers, tetramers, pentamers, and aggregates. Only alpha7 subunits that completed the assembly process to homopentamers acquired complex-type carbohydrates and appeared at the cell surface. We conclude that Xenopus oocytes have a limited capacity to guide the assembly of alpha7 subunits, but not 5HT3A subunits to homopentamers. Accordingly, ER retention of imperfectly assembled alpha7 subunits rather than inefficient routing of fully assembled alpha7 receptors to the cell surface limits surface expression levels of alpha7 nicotinic acetylcholine receptors.

Animals↗
PubMed2005

Biogenesis of cytochrome oxidase-sophisticated assembly lines in the mitochondrial inner membrane.

Biogenesis of the cytochrome oxidase complex in the mitochondrial inner membrane depends on the concerted action of a variety of proteins. Recent studies shed light on this biological assembly process revealing an astonishingly complex procedure by which the different subunits of the enzymes are put together and the required cofactors are supplied. In this review we present a hypothetical model for the assembly process of cytochrome oxidase based on the current knowledge of the functions of specific assembly factors. According to this model the two largest subunits of the complex are first equipped with their respective cofactors on independent assembly lines. Prior to their assembly with the residual subunits that complete the whole complex, these two subcomplexes remain bound to substrate-specific chaperones. We propose that these chaperones, Mss51 for subunit 1 and Cox20 for subunit 2, control the coordinate assembly process to prevent potentially harmful redox reactions of unassembled or misassembled subunits.

Amino Acid Sequence↗
PubMed2006

Albumin binds self-assembling dyes as specific polymolecular ligands.

Self-assembling dyes with a structure related to Congo red (e.g. Evans blue) form polymolecular complexes with albumin. The dyes, which are lacking a self-assembling property (Trypan blue, ANS) bind as single molecules. The supramolecular character of dye ligands bound to albumin was demonstrated by indicating the complexation of dye molecules outnumbering the binding sites in albumin and by measuring the hydrodynamic radius of albumin which is growing upon complexation of self-assembling dye in contrast to dyes lacking this property. The self-assembled character of Congo red was also proved using it as a carrier introducing to albumin the intercalated nonbonding foreign compounds. Supramolecular, ordered character of the dye in the complex with albumin was also revealed by finding that self-assembling dyes become chiral upon complexation. Congo red complexation makes albumin less resistant to low pH as concluded from the facilitated N-F transition, observed in studies based on the measurement of hydrodynamic radius. This particular interference with protein stability and the specific changes in digestion resulted from binding of Congo red suggest that the self-assembled dye penetrates the central crevice of albumin.

Animals↗
PubMed2004

Crystal structures of the Rhodococcus proteasome with and without its pro-peptides: implications for the role of the pro-peptide in proteasome assembly.

To understand the role of the pro-peptide in proteasome assembly, we have determined structures of the Rhodococcus proteasome and a mutant form that prevents the autocatalytic removal of its pro-peptides. The structures reveal that the pro-peptide acts as an assembly-promoting factor by linking its own beta-subunit to two adjacent alpha-subunits, thereby providing a molecular explanation for the observed kinetics of proteasome assembly. The Rhodococcus proteasome has been found to have a substantially smaller contact region between alpha-subunits compared to those regions in the proteasomes of Thermoplasma, yeast, and mammalian cells, suggesting that a smaller contact area between alpha-subunits is likely the structural basis for the Rhodococcus alpha-subunits not assembling into alpha-rings when expressed alone. Analysis of all available beta-subunit structures shows that the contact area between beta-subunits within a beta-ring is not sufficient for beta-ring self-assembly without the additional contact provided by the alpha-ring. This appears to be a fail-safe mechanism ensuring that the active sites on the beta-subunits are activated only after proteasome assembly is complete.

Amino Acid Sequence↗
PubMed2005

The role of arginine-rich motif and beta-annulus in the assembly and stability of Sesbania mosaic virus capsids.

Sesbania mosaic virus (SeMV) capsids are stabilized by protein-protein, protein-RNA and calcium-mediated protein-protein interactions. The N-terminal random domain of SeMV coat protein (CP) controls RNA encapsidation and size of the capsids and has two important motifs, the arginine-rich motif (ARM) and the beta-annulus structure. Here, mutational analysis of the arginine residues present in the ARM to glutamic acid was carried out. Mutation of all the arginine residues in the ARM almost completely abolished RNA encapsidation, although the assembly of T=3 capsids was not affected. A minimum of three arginine residues was found to be essential for RNA encapsidation. The mutant capsids devoid of RNA were less stable to thermal denaturation when compared to wild-type capsids. The results suggest that capsid assembly is entirely mediated by CP-dependent protein-protein inter-subunit interactions and encapsidation of genomic RNA enhances the stability of the capsids. Because of the unique structural ordering of beta-annulus segment at the icosahedral 3-folds, it has been suggested as the switch that determines the pentameric and hexameric clustering of CP subunits essential for T=3 capsid assembly. Surprisingly, mutation of a conserved proline within the segment that forms the beta-annulus to alanine, or deletion of residues 48-53 involved in hydrogen bonding interactions with residues 54-58 of the 3-fold related subunit or deletion of all the residues (48-59) involved in the formation of beta-annulus did not affect capsid assembly. These results suggest that the switch for assembly into T=3 capsids is not the beta-annulus. The ordered beta-annulus observed in the structures of many viruses could be a consequence of assembly to optimize intersubunit interactions.

Amino Acid Sequence↗
PubMed2006

Impact of disease mutations on the desmin filament assembly process.

It has been documented that mutations in the human desmin gene lead to a severe type of myofibrillar myopathy, termed more specifically desminopathy, which affects cardiac and skeletal as well as smooth muscle. We showed recently that 14 recombinant versions of these disease-causing desmin variants, all involving single amino acid substitutions in the alpha-helical rod domain, interfere with in vitro filament formation at distinct stages of the assembly process. We now provide mechanistic details of how these mutations affect the filament assembly process by employing analytical ultracentrifugation, time-lapse electron microscopy of negatively stained and glycerol-sprayed/low-angle rotary metal-shadowed samples, quantitative scanning transmission electron microscopy, and viscometric studies. In particular, the soluble assembly intermediates of two of the mutated proteins exhibit unusually high s-values, compatible with octamers and other higher-order complexes. Moreover, several of the six filament-forming mutant variants deviated considerably from wild-type desmin with respect to their filament diameters and mass-per-length values. In the heteropolymeric situation with wild-type desmin, four of the mutant variants caused a pronounced "hyper-assembly", when assayed by viscometry. This indicates that the various mutations may cause abortion of filament formation by the mutant protein at distinct stages, and that some of them interfere severely with the assembly of wild-type desmin. Taken together, our findings provide novel insights into the basic intermediate filament assembly mechanisms and offer clues as to how amino acid changes within the desmin rod domain may interfere with the normal structural organization of the muscle cytoskeleton, eventually leading to desminopathy.

Amino Acid Substitution↗
PubMed2006

Visualizing the assembly of human Rad51 filaments on double-stranded DNA.

Rad51 is the core component of the eukaryotic homologous recombination machinery and assembles into extended nucleoprotein filaments on DNA. To study the dynamic behavior of Rad51 we have developed a single-molecule assay that relies on a combination of hydrodynamic force and microscale diffusion barriers to align individual DNA molecules on the surface of a microfluidic sample chamber that is coated with a lipid bilayer. When visualized with total internal reflection fluorescence microscopy (TIRFM), these "molecular curtains" allow for the direct visualization of hundreds of individual DNA molecules. Using this approach, we have analyzed the binding of human Rad51 to single molecules of double-stranded DNA under a variety of different reaction conditions by monitoring the extension of the fluorescently labeled DNA, which coincides with assembly of the nucleoprotein filament. We have also generated several mutants in conserved regions of Rad51 implicated in DNA binding, and tested them for their ability to assemble into extended filaments. We show that proteins with mutations within the DNA-binding surface located on the N-terminal domain still retain the ability to form extended nucleoprotein filaments. Mutations in the L1 loop, which projects towards the central axis of the filament, completely abolish assembly of extended filaments. In contrast, most mutations within or near the L2 DNA-binding loop, which is also located near the central axis of the filament, do not affect the ability of the protein to assemble into extended filaments on double-stranded (ds)DNA. Taken together, these results demonstrate that the L1-loop plays a crucial role in the assembly of extended nucleoprotein filaments on dsDNA, but the N-terminal domain and the L2 DNA-binding loop have significantly less impact on this process. The results presented here also provide an important initial framework for beginning to study the biochemical behaviors of Rad51 nucleoprotein filaments using our novel experimental system.

Adenosine Triphosphatases↗
PubMed2005

Predicting oligomeric assemblies: N-mers a primer.

Multi-protein complexes play key roles in many biological processes. However, since the structures of these assemblies are hard to resolve experimentally, the detailed mechanism of how they work cooperatively in the cell has remained elusive. Similarly, recent advances on in silico prediction of protein-protein interactions have so far avoided this difficult problem. In this paper, we present a general algorithm to predict molecular assemblies of homo-oligomers. Given the number of N-mers and the 3D structure of one monomer, the method samples all the possible symmetries that N-mers can be assembled. Based on a scoring function that clusters the low free energy structures at each binding interface, the algorithm predicts the complex structure as well as the symmetry of the protein assembly. The method is quite general and does not involve any free parameters. The algorithm has been implemented as a public server and integrated to the protein-protein complex prediction server ClusPro. Using this application, we validated predictions for trimers, tetramers (discriminating between dimer of dimers and 4-fold symmetry structures), pentamers and hexamers (discriminating between trimer of dimers, dimer of trimers, and 6-fold symmetry structures), for a total of 107 assemblies. For 85% of the multimers, the server predicts the complex structure within an average rms deviation of 2A from the full crystal. For complexes that involve more than one binding interface, the cluster size at each surface provides a strong indication as to which interface forms first. With improving scoring functions and computer power, our multimer docking approach could be used as a framework to address the more general problem of multi-protein assemblies.

Algorithms↗