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PubMed2001

Assembly of cytochrome c oxidase: what can we learn from patients with cytochrome c oxidase deficiency?

Cytochrome c oxidase is an intricate metalloprotein that transfers electrons from cytochrome c to oxygen in the last step of the mitochondrial respiratory chain. It uses the free energy of this reaction to sustain a transmembrane electrochemical gradient of protons. Site-directed mutagenesis studies of bacterial terminal oxidases and the recent availability of refined crystal structures of the enzyme are rapidly expanding the understanding of the coupling mechanism between electron transfer and proton translocation. In contrast, relatively little is known about the assembly pathway of cytochrome c oxidase. Studies in yeast have indicated that assembly is dependent on numerous proteins in addition to the structural subunits and prosthetic groups. Human homologues of a number of these assembly factors have been identified and some are now known to be involved in disease. To dissect the assembly pathway of cytochrome c oxidase, we are characterizing tissues and cell cultures derived from patients with genetically defined cytochrome c oxidase deficiency, using biochemical, biophysical and immunological techniques. These studies have allowed us to identify some of the steps of the assembly process.

Animals↗
PubMed2005

Do protein-lipid interactions determine the recognition of transmembrane helices at the ER translocon?

Membrane-protein integration, folding and assembly processes in vivo depend on complex targeting, translocation, chaperoning, and sorting machineries that somehow read the 'molecular code' built into the nascent polypeptide, ultimately producing a properly folded protein integrated into the correct target membrane. Although the main molecular constituents and the basic mechanistic principles of many of these machines are known in outline, the codes remain poorly defined and there is little quantitative information on how protein sequence affects the final structure of membrane proteins. By carefully designing model protein constructs, we have derived the first true biological hydrophobicity scale and have been able to get a first impression of how the position of a given type of residue within a transmembrane segment affects its ability to promote membrane insertion.

Computer Simulation↗
PubMed1996

Histone-like protein HU as a specific transcriptional regulator: co-factor role in repression of gal transcription by GAL repressor.

BACKGROUND: Transcription initiation from the two overlapping promoters of the gal operon in Escherichia coli is negatively regulated by binding of Gal repressor (GalR) to bipartite operators, which encompass the promoters. Coordinated repression of the two promoters requires GalR binding to both operators. In a purified system, GalR, nevertheless, fails to show the coordinated repression, predicting the participation of an additional factor(s) in the regulation in vivo. RESULTS: We have purified a protein that restored the expected GalR-mediated repression for the in vitro system and have identified this factor to be the bacterial histone-like protein HU. In vitro transcription assays in the presence of GalR and HU show that, just as in vivo, the coordinated repression of the two gal promoters requires GalR binding to both operators and is sensitive to the inducer, D-galactose. The GalR and HU dependent repression also requires supercoiled DNA template and prevents open complex formation. CONCLUSION: We propose that HU, acting as a co-factor, brings about the GalR-mediated repression by forming a distinct nucleoprotein complex of higher order structure. Although how HU participates in the assembly process is unknown, there may be a cooperative effect in the formation of the repression complex.

Bacterial Proteins↗
PubMed1999

Fission yeast APC/cyclosome subunits, Cut20/Apc4 and Cut23/Apc8, in regulating metaphase-anaphase progression and cellular stress responses.

BACKGROUND: The 20S cyclosome/APC complex promotes metaphase-anaphase transition by ubiquitinating its specific substrates such as mitotic cyclins and anaphase inhibitor Cut2/Pds1/securin. The complex has been shown to contain more than 10 proteins in budding yeast and frog. In fission yeast, however, only five (Cut4, Cut9, Nuc2, Apc10, Hcn1) have been identified. RESULTS: More than five hundred temperature-sensitive mutants were screened for identifying those defective in mitotic anaphase. Fifty-five showed the cut (cell untimely torn) phenotype or metaphase-arrest phenotypes, 27 of them locating at new loci. Their extracts were run in sucrose gradient centrifugation, and four showed alterations in the sedimentation profiles. The gene products of cut20+ and cut23+ were thus identified. Phenotypes of cut20-100 mutant highly resemble cut4-533 in many ways: they are hypersensitive to canavanine and CdCl2, and suppressed by PKA-inactivating regulators, cAMP-dependent phosphodiesterase and PKA regulatory subunits. Cut20 interacts closely with Cut4 in the assembly process of cyclosome. But cut20 mutant differs from cut4, as a novel gene stw1+ suppresses cut20 mutant but not cut4. cut23-194 mutant cells are sterile and blocked at metaphase, but does not show sensitivity to the stress and cAMP. TPR repeat-containing Cut23 may not be the stable component of APC/cyclosome, and its level significantly fluctuates during cell cycle. Cut23 may be ubiquitinated and degraded in a cell cycle dependent fashion. CONCLUSIONS: We identified two new subunits of fission yeast cyclosome/APC complex. Our observations indicate that cyclosome components are divided into several subgroups with distinctly different roles.

Adenomatous Polyposis Coli Protein↗
PubMed1998

Oligomeric structure of the repressor of the bacteriophage Mu early operon.

The regulation of the lytic and lysogenic development in the life cycle of bacteriophage Mu is regulated in part by its repressor, c, which binds to three operator sites, O1, O2 and O3, overlapping two divergent promoters. The oligomeric structure of this repressor protein was investigated by hydrodynamic and biochemical methods. Size-exclusion chromatography, analytical ultracentrifugation, dynamic light scattering, crosslinking and direct electron microscopy observations suggest that c exists primarily as a hexamer with a molecular mass of 120-140 kDa at low concentrations, i.e. in the 10-microM range. This molecule undergoes a self-assembly process leading to dodecamers and higher order species as the concentration is further increased in a manner depending on the nature of the solvent. Our results also suggest that these species have an elongated structure, and a possible arrangement of the subunits within the hexamer is proposed. The implication of this unusual quaternary structure for a repressor in its interaction with the operator sites O1 and O2 remains to be elucidated.

Bacteriophage mu↗
PubMed2003

Reversible dissociation of collagen in tissues.

The turbidity of most biologic tissues hinders the use of lasers for diagnostic and therapeutic purposes. Hyperosmotic agents such as glycerol have been used to alter the optical scattering properties of turbid tissues. The mechanism of this effect, "optical clearing", however, remains incompletely understood. Multiphoton microscopy utilizing second harmonic generation can be used to monitor collagen structural changes in the presence of glycerol. This study suggests that the use of glycerol for tissue "optical clearing" screens noncovalent intermolecular interactions between low-order collagen structures, resulting in fiber disassembly. Dissociation of collagen fibers in native and engineered tissues in the presence of glycerol and reassociation with the application of saline are imaged dynamically. Collagen fiber reassembly is governed by the displacement of glycerol by saline in native and engineered skin. These results reveal the assembly process of high-order collagen structures and propose a molecular mechanism for the increase in tissue transparency observed after glycerol application.

Animals↗
PubMed2006

Nanofunctionalisation for the treatment of peripheral nervous system injuries.

A construct based on the electrostatic layer-by-layer self assembly technique has been fabricated, to be used as a tailored device to encourage nerve regeneration. A multilayered nanocoating composed of three precursor bilayers of cationic poly(dimethyldiallylammonium) chloride (PDDA) and anionic poly(styrenesulfonate) (PSS), followed by bilayers of poly-D-lysine (PDL) and antibody specific to transforming growth factor 1 (anti-TGF-1), has been deposited on HYAFF 11. The assembly process has been monitored by quartz crystal microbalance (QCM) for its characterisation and then it has been used on HYAFF 11. Structural studies of the resulting multilayers confirmed stepwise deposition of anti-TGF-1, with an average layer thickness of 2.2+/-0.2 nm and an average surface density of 0.36+/-0.03 mug cm(-2). Scanning electron microscopy has been used to characterise multilayer uniformity. Finally, the immunological activity of the multilayered structure has been assessed. The results show that anti-TGF-1 can be included in its active form in a predetermined multilayered structure onto HYAFF 11 with quantitative control of layer thickness and weight, providing a high tool with great potential in tissue engineering.

Animals↗
PubMed2004

Biomolecule-directed assembly of nanoscale building blocks studied via lattice Monte Carlo simulation.

We perform lattice Monte Carlo simulations to study the self-assembly of functionalized inorganic nanoscale building blocks using recognitive biomolecule linkers. We develop a minimal coarse-grained lattice model for the nanoscale building block (NBB) and the recognitive linkers. Using this model, we explore the influence of the size ratio of linker length to NBB diameter on the assembly process and the structural properties of the resulting aggregates, including the spatial distribution of NBBs and aggregate topology. We find the constant-kernel Smoluchowski theory of diffusion-limited cluster-cluster aggregation describes the aggregation kinetics for certain size ratios.

Algorithms↗
PubMed2005

Following the aggregation of amyloid-forming peptides by computer simulations.

There is experimental evidence suggesting that the toxicity of neurodegenerative diseases such as Alzheimer's disease may result from the soluble intermediate oligomers. It is therefore important to characterize extensively the early steps of oligomer formation at atomic level. As these structures are metastable and short lived, experimental data are difficult to obtain and they must be complemented with numerical simulations. In this work, we use the activation-relaxation technique coupled with a coarse-grained energy model to study in detail the mechanisms of aggregation of four lys-phe-phe-glu (KFFE) peptides. This is the shortest peptide known to form amyloid fibrils in vitro. Our simulations indicate that four KFFE peptides adopt a variety of oligomeric states (tetramers, trimers, and dimers) with various orientations of the chains in rapid equilibrium. This conformational distribution is consistent with all-atom molecular-dynamics simulations in explicit solvent and is sequence dependent; as seen experimentally, the lys-pro-gly-glu (KPGE) peptides adopt disordered structures in solution. Our unbiased simulations also indicate that the assembly process is much more complex than previously thought and point to intermediate structures which likely are kinetic traps for longer chains.

Amyloid beta-Peptides↗
PubMed2005

Simulated annealing study of morphological transitions of diblock copolymers in solution.

The simulated annealing method was applied to study the self-assembling process of diblock copolymers in selective solvents for one block. The simulation results illustrated that the morphologies of the copolymer aggregates strongly depend on the interactions between the core-forming blocks and the solvents and on the length of the corona-forming blocks. Multiple morphological transitions were observed in one system. The transition sequence (disordered state-spherical micelles-short rodlike micelles-long rodlike micelles-onionlike aggregates) was observed for copolymers with increasing core-solvent interaction. Similar transitions were observed with the decrease of the length of the corona-forming blocks. The mechanisms of these transitions are investigated. The simulation results are compared with experiments and other simulations.

Journal Article↗
PubMed2006

A molecular dynamics simulation study of buckyballs in water: atomistic versus coarse-grained models of C60.

A simple coarse-grained water-C(60) model potential is proposed to study the solvation behavior of C(60) in water. Extensive molecular dynamics simulations are performed to compare the hydration behavior of the coarse-grained C(60) with that of its atomistic counterpart. In the coarse-grained description, we model the C(60) as a spherical solute and its interaction with water is represented by a simple two-body central potential as obtained from a coarse graining of the interactions of a water molecule with all the atoms of the C(60). On the other hand, the atomistic model takes into account a discrete nature of all the atoms of C(60) explicitly. Molecular dynamics simulations are carried out in an isothermal-isobaric ensemble at normal temperature and pressure. Various spatial and orientational correlations of the water around the two model C(60)s are compared. The coarse-grained model is shown to reproduce the hydration behavior of the C(60) quite well. The simplicity and the computational economy of the coarse-grained model will allow for simulations of self-assembly processes of a much larger system over a longer period of time.

Journal Article↗
PubMed2002

Entropically driven self-assembly of multichannel rosette nanotubes.

Rosette nanotubes are a new class of organic nanotubes obtained through the hierarchical self-assembly of low molecular weight synthetic modules in water. Here we demonstrate that these materials can serve as scaffolds for the supramolecular synthesis of multichannel nanotubular architectures and report on the discovery of their entropy-driven self-assembly process.

Journal Article↗
PubMed2004

Capillarity-driven assembly of two-dimensional cellular carbon nanotube foams.

Capillary forces arising during the evaporation of liquids from dense carbon nanotube arrays are used to reassemble the nanotubes into two-dimensional contiguous cellular foams. The stable nanotube foams can be elastically deformed, transferred to other substrates, or floated out to produce free-standing macroscopic fabrics. The lightweight cellular foams made of condensed nanotubes could have applications as shock-absorbent structural reinforcements and elastic membranes. The ability to control the length scale, orientation, and shape of the cellular structures and the simplicity of the assembly process make this a particularly attractive system for studying pattern formation in ordered media.

Manufactured Materials↗
PubMed2006

Principles of self-assembly of helical pores from dendritic dipeptides.

The self-assembly of the dendritic dipeptides (4-3,4-3,5)nG2-CH2-Boc-L-Tyr-L-Ala-OMe and their achiral dendritic alcohol (4-3,4-3,5)nG2-CH2OH precursors, both with n = 1-16, where n represents the number of methylenic units in the alkyl groups of the dendron, are reported. All chiral dendritic dipeptides and achiral dendritic alcohols self-assemble into helical porous columns that are stable in both solution and solid state. The pore diameter (D(pore)) of the columns self-assembled from dendritic dipeptides is approximately 10 A larger than that of structures assembled from dendritic alcohols. The increase of the D(pore) at the transition from dendritic alcohol to dendritic dipeptide is accompanied by a decreased solid angle of the building block. This trend is in agreement with previous pore size-solid angle dependences observed with different protective groups of the dipeptide and primary structures of the dendron. However, within the series of dendritic alcohols and dendritic dipeptides with various n, the D(pore) increases when the solid angle increases. The results of these investigations together with those of previous studies on the role of dipeptide stereochemistry and protective groups on this self-assembly process provide the molecular principles required to program the construction of supramolecular helical pores with diameter controlled at the A level from a single dendritic dipeptide architecture. These principles are expected to be valid for libraries of dendritic dipeptides based on dendrons and dipeptides with various primary structures.

Circular Dichroism↗
PubMed2002

Anion-templated self-assembly of tetrahedral cage complexes of cobalt(II) with bridging ligands containing two bidentate pyrazolyl-pyridine binding sites.

The bridging ligands L(1) and L(2) contain two N,N-bidentate pyrazolyl-pyridine units linked to a central aromatic spacer unit (1,2-phenyl or 2,3-naphthyl, respectively). Reaction with Ni(II) salts and treatment with the anions tetrafluoroborate or perchlorate result in formation of dinuclear complexes having a 2:3 metal:ligand ratio, with one bridging and two terminal tetradentate ligands. In contrast, reaction of L(1) and L(2) with Co(II) salts, followed by treatment with tetrafluoroborate or perchlorate, results in assembly of cage complexes having a 4:6 metal:ligand ratio; these complexes have a metal ion at each corner of an approximate tetrahedron, and a bis-bidentate bridging ligand spanning each edge. The central cavity is occupied by a tetrahedral counterion that forms multiple hydrogen-bonding interactions with the methylene protons of the bridging ligands. The anionic guest fits tightly into the central cavity of the cage to which it is ideally complementary in terms of shape, size, and charge. Solution NMR experiments show that the central anion acts as a template for cage formation, with a mixture of Co(II) and the appropriate bridging ligand alone giving no assembly into a cage until the tetrahedral anion is added, at which point cage assembly is fast and quantitative. The difference between the structures of the complexes with Ni(II) and Co(II) illustrate how the uncoordinated anions can exert a profound influence on the course of the assembly process.

Anions↗
PubMed2006

Tail-interacting protein TIP47 is a connector between Gag and Env and is required for Env incorporation into HIV-1 virions.

The presence of the envelope glycoprotein Env in HIV-1 virions is essential for infectivity. To date, the molecular mechanism by which Env is packaged into virions has been largely unknown. Here, we show that TIP47 (tail-interacting protein of 47 kDa), which has been shown to interact with Env, also binds the MA (matrix) domain of HIV-1 Gag protein and that these three proteins form a ternary complex. Mutations in Gag that abrogate interaction with TIP47 inhibit Env incorporation and virion infectivity as well as colocalization between Gag and Env. We also show that TIP47 silencing impairs Env incorporation and infectivity and abolishes coimmunoprecipitation of Gag with Env. In contrast, overexpression of TIP47 increases Env packaging. Last, we demonstrate that TIP47 can interact simultaneously with Env and Gag. Taken together, our results show that TIP47 is a cellular cofactor that plays an essential role in Env incorporation, allowing the encounter and the physical association between HIV-1 Gag and Env proteins during the viral assembly process.

Amino Acid Sequence↗
PubMed2002

Positioning protein molecules on surfaces: a nanoengineering approach to supramolecular chemistry.

We discuss a nanoengineering approach for supramolecular chemistry and self assembly. The collective properties and biofunctionalities of molecular ensembles depend not only on individual molecular building blocks but also on organization at the molecular or nanoscopic level. Complementary to "bottom-up" approaches, which construct supramolecular ensembles by the design and synthesis of functionalized small molecular units or large molecular motifs, nanofabrication explores whether individual units, such as small molecular ligands, or large molecules, such as proteins, can be positioned with nanometer precision. The separation and local environment can be engineered to control subsequent intermolecular interactions. Feature sizes as small as 2 x 4 nm(2) (32 alkanethiol molecules) are produced. Proteins may be aligned along a 10-nm-wide line or within two-dimensional islands of desired geometry. These high-resolution engineering and imaging studies provide new and molecular-level insight into supramolecular chemistry and self-assembly processes in bioscience that are otherwise unobtainable, e.g., the influence of size, separation, orientation, and local environment of reaction sites. This nanofabrication methodology also offers a new strategy in construction of two- and three-dimensional supramolecular structures for cell, virus, and bacterial adhesion, as well as biomaterial and biodevice engineering.

Animals↗
PubMed2001

The crystal structure of a heptameric archaeal Sm protein: Implications for the eukaryotic snRNP core.

Sm proteins form the core of small nuclear ribonucleoprotein particles (snRNPs), making them key components of several mRNA-processing assemblies, including the spliceosome. We report the 1.75-A crystal structure of SmAP, an Sm-like archaeal protein that forms a heptameric ring perforated by a cationic pore. In addition to providing direct evidence for such an assembly in eukaryotic snRNPs, this structure (i) shows that SmAP homodimers are structurally similar to human Sm heterodimers, (ii) supports a gene duplication model of Sm protein evolution, and (iii) offers a model of SmAP bound to single-stranded RNA (ssRNA) that explains Sm binding-site specificity. The pronounced electrostatic asymmetry of the SmAP surface imparts directionality to putative SmAP-RNA interactions.

Amino Acid Sequence↗