Nuclear magnetic resonance studies of the dynamic aspects of molecular structure and interaction in biological systems.
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Aspartate Carbamoyltransferase↗
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Perspectives for neural regeneration with changes in macromolecular metabolism.
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Animals↗
[Signal transducing systems: G protein mediated signaling systems and NDP kinase].
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Adenylyl Cyclases↗
Metabolic requirements for hormone-induced resistance to antibody-complement mediated killing of tumor cells.
Line-1 guinea pig hepatoma cells are susceptible to killing by anti-Forssman IgM antibody plus guinea pig complement (GPC). When these tumor cells are incubated with insulin, epinephrine, hydrocortisone, or prednisolone, the cells show a marked reduction in their susceptibility to antibody-C-killing. If the ability of the cells to synthesize DNA, RNA, and protein is impaired by pretreatment with metabolic inhibitors, x-irradiation, or culture in nutrient-deficient media, the hormones are no longer effective in rendering the cells resistant to killing. If only DNA synthesis is impaired, but not RNA and protein synthesis, the hormones are effective. The inability of cells inhibited in their macromolecular synthesis to be rendered resistant to killing after hormone treatment is not due to an inability of the cells to bind hormone.
Amino Acids↗
A systems biology analysis of the Drosophila phagosome.
Phagocytes have a critical function in remodelling tissues during embryogenesis and thereafter are central effectors of immune defence. During phagocytosis, particles are internalized into 'phagosomes', organelles from which immune processes such as microbial destruction and antigen presentation are initiated. Certain pathogens have evolved mechanisms to evade the immune system and persist undetected within phagocytes, and it is therefore evident that a detailed knowledge of this process is essential to an understanding of many aspects of innate and adaptive immunity. However, despite the crucial role of phagosomes in immunity, their components and organization are not fully defined. Here we present a systems biology analysis of phagosomes isolated from cells derived from the genetically tractable model organism Drosophila melanogaster and address the complex dynamic interactions between proteins within this organelle and their involvement in particle engulfment. Proteomic analysis identified 617 proteins potentially associated with Drosophila phagosomes; these were organized by protein-protein interactions to generate the 'phagosome interactome', a detailed protein-protein interaction network of this subcellular compartment. These networks predicted both the architecture of the phagosome and putative biomodules. The contribution of each protein and complex to bacterial internalization was tested by RNA-mediated interference and identified known components of the phagocytic machinery. In addition, the prediction and validation of regulators of phagocytosis such as the 'exocyst', a macromolecular complex required for exocytosis but not previously implicated in phagocytosis, validates this strategy. In generating this 'systems-based model', we show the power of applying this approach to the study of complex cellular processes and organelles and expect that this detailed model of the phagosome will provide a new framework for studying host-pathogen interactions and innate immunity.
Animals↗
Evaluation of nonideality from gel chromatographic partition coefficients. A technique with greater versatility than equilibrium dialysis.
Frontal gel chromatography has been used to measure partition coefficients which enable a quantitative evaluation of the thermodynamic nonideality of small solutes generated by the presence of high concentrations of macromolecular solutes. Equivalence of results obtained by the present method and by equilibrium dialysis is demonstrated in a comparison of results for dextran sulfate-NaCl and dextran-sorbitol systems. Interaction coefficients obtained for dextran-sorbitol and protein-polyethylene glycol 4000 systems yields results which are in reasonable agreement with those predicted on the statistical-mechanical basis of excluded volume. Because of its greater versatility in regard to the range of systems that may be studied, the frontal gel chromatographic procedure is likely to be of particular value for the quantitative characterization of thermodynamic nonideality arising from excluded volume effects in concentrated mixtures of macromolecular solutes.
Biopolymers↗
Teaching electron diffraction and imaging of macromolecules.
Electron microscopic analysis can be used to determine the three-dimensional structures of macromolecules at resolutions ranging between 3 and 30 A. It differs from nuclear magnetic resonance spectroscopy or x-ray crystallography in that it allows an object's Coulomb potential functions to be determined directly from images and can be used to study relatively complex macromolecular assemblies in a crystalline or noncrystalline state. Electron imaging already has provided valuable structural information about various biological systems, including membrane proteins, protein-nucleic acid complexes, contractile and motile protein assemblies, viruses, and transport complexes for ions or macromolecules. This article, organized as a series of lectures, presents the biophysical principles of three-dimensional analysis of objects possessing different symmetries.
Biophysics↗
Evaluation of protein docking predictions using Hex 3.1 in CAPRI rounds 1 and 2.
This article describes and reviews our efforts using Hex 3.1 to predict the docking modes of the seven target protein-protein complexes presented in the CAPRI (Critical Assessment of Predicted Interactions) blind docking trial. For each target, the structure of at least one of the docking partners was given in its unbound form, and several of the targets involved large multimeric structures (e.g., Lactobacillus HPr kinase, hemagglutinin, bovine rotavirus VP6). Here we describe several enhancements to our original spherical polar Fourier docking correlation algorithm. For example, a novel surface sphere smothering algorithm is introduced to generate multiple local coordinate systems around the surface of a large receptor molecule, which may be used to define a small number of initial ligand-docking orientations distributed over the receptor surface. High-resolution spherical polar docking correlations are performed over the resulting receptor surface patches, and candidate docking solutions are refined by using a novel soft molecular mechanics energy minimization procedure. Overall, this approach identified two good solutions at rank 5 or less for two of the seven CAPRI complexes. Subsequent analysis of our results shows that Hex 3.1 is able to place good solutions within a list of <or=20 for four of the seven targets. This finding shows that useful in silico protein-protein docking predictions can now be made with increasing confidence, even for very large macromolecular complexes.
Algorithms↗
Macromolecular differentiation of Golgi stacks in root tips of Arabidopsis and Nicotiana seedlings as visualized in high pressure frozen and freeze-substituted samples.
The plant root tip represents a fascinating model system for studying changes in Golgi stack architecture associated with the developmental progression of meristematic cells to gravity sensing columella cells, and finally to "young" and "old", polysaccharide-slime secreting peripheral cells. To this end we have used high pressure freezing in conjunction with freeze-substitution techniques to follow developmental changes in the macromolecular organization of Golgi stacks in root tips of Arabidopsis and Nicotiana. Due to the much improved structural preservation of all cells under investigation, our electron micrographs reveal both several novel structural features common to all Golgi stacks, as well as characteristic differences in morphology between Golgi stacks of different cell types. Common to all Golgi stacks are clear and discrete differences in staining patterns and width of cis, medial and trans cisternae. Cis cisternae have the widest lumina (approximately 30 nm) and are the least stained. Medial cisternae are narrower (approximately 20 nm) and filled with more darkly staining products. Most trans cisternae possess a completely collapsed lumen in their central domain, giving rise to a 4-6 nm wide dark line in cross-sectional views. Numerous vesicles associated with the cisternal margins carry a non-clathrin type of coat. A trans Golgi network with clathrin coated vesicles is associated with all Golgi stacks except those of old peripheral cells. It is easily distinguished from trans cisternae by its blebbing morphology and staining pattern. The zone of ribosome exclusion includes both the Golgi stack and the trans Golgi network. Intercisternal elements are located exclusively between trans cisternae of columella and peripheral cells, but not meristematic cells. In older peripheral cells only trans cisternae exhibit slime-related staining. Golgi stacks possessing intercisternal elements also contain parallel rows of freeze-fracture particles in their trans cisternal membranes. We propose that intercisternal elements serve as anchors of enzyme complexes involved in the synthesis of polysaccharide slime molecules to prevent the complexes from being dragged into the forming secretory vesicles by the very large slime molecules. In addition, we draw attention to the similarities in composition and apparent site of synthesis of xyloglucans and slime molecules.
Arabidopsis↗
NMDA receptor subunits: diversity, development and disease.
N-methyl-D-aspartate receptors (NMDARs) are present at many excitatory glutamate synapses in the central nervous system and display unique properties that depend on their subunit composition. Biophysical, pharmacological and molecular methods have been used to determine the key features conferred by the various NMDAR subunits, and have helped to establish which NMDAR subtypes are present at particular synapses. Recent studies are beginning to address the functional significance of NMDAR diversity under normal and pathological conditions.
Alternative Splicing↗
Introducing a biosensor based technology for real-time biospecific interaction analysis.
This report describes a system for real-time biospecific interaction analysis, using biosensor technology based on the optical phenomenon surface plasmon resonance. The biospecific interface is a sensor chip consisting of a thin gold film deposited on a glass support and covered with a hydrogel matrix. One component of the interaction being studied is attached covalently to the hydrogel, and other interactants are passed over the chip in solution. The interaction is followed in real time in terms of changes in the mass concentration of biomolecules at the sensor surface. Surface concentrations down to 10 pg/mm2 can be measured. The technique does not require molecular labels such as isotopes or spectroscopic markers, and purification of interacting components can often be avoided. Repeated analyses can be performed on the same sensor chip. With this system, the same general procedure can be used for a wide range of different applications, including concentration determination, kinetic measurements and multi-site binding studies. The sensitivity of the technique can be adjusted by choice of reagents and experimental procedure: determination of specific proteins in serum down to 20 ng/ml and macromolecular association constants from 10(7) M-1 up to 4 x 10(11) M-1 are documentated examples. No other single analytical system has the same versatility and general applicability to biospecific interaction analysis. The system is developed and marketed by Pharmacia Biosensor AB, Sweden.
Biosensing Techniques↗
Messenger RNA on the move: implications for cell polarity.
RNA sorting is an evolutionarily conserved mechanism for establishing asymmetries within a given cell concerning the macromolecular equipment of defined domains. mRNAs as well as non-coding transcripts are delivered to specific subcellular compartments in diverse organisms including developmental systems of Drosophila, Xenopus, ascidiens, zebrafish and echinoderms and in differentiated cells from yeast to mammals. The composition of the RNA localization machinery is complex. Both sequence- or structural motifs within RNA molecules to be transported (cis-acting elements) and various proteins (trans-acting factors) contribute to the localization procedure. Most often these macromolecular ribonucleoprotein complexes exhibit a granular appearance, and granule localization depends on intact microfilaments or microtubules. When delivered to their ultimate destinations mRNAs are anchored to await translational activation at the appropriate timepoint. Beyond doubt, RNA localization plays a pivotal role in embryonic development, where mRNA mislocations cause severe body pattern defects. In terminally differentiated vertebrate cells RNA transport and local on-site translation presumably have an impact on various cellular functions such as cell motility, myelinization of nerve cell axons and nerve cell communications in the central and peripheral nervous system.
Animals↗
Efficacy of macromolecular crowding in forcing proteins to fold.
The intrinsically unstructured protein, reduced and carboxyamidated RNase T1 (TCAM) was used to determine the degree to which macromolecular crowding agents increase the equilibrium constant for folding. TCAM is not catalytically active in an aqueous assay system alone, but becomes catalytically active on addition of 400 mg/ml dextran 70. The activity observed accounts for approximately 16% of the total available TCAM in solution. We interpret this result to mean that 16% of the TCAM becomes folded protein in the presence of the 400 mg/ml dextran 70, and this translates into an approximately five-fold increase in the equilibrium constant for folding. Sarcosine-induced folding of TCAM was performed in the presence of 0, 100, 200 and 300 mg/ml dextran 70, and apparent deltaG(o)(N-D) values determined from the linear extrapolation method provide an estimated 22% folded TCAM formed in the limit of zero sarcosine concentration and in presence of 400 mg/ml dextran 70. The increase in TCAM folding equilibrium constant using this method of determination is approximately 7.5-fold. Overall, the results indicate that macromolecular crowding agents are only modestly effective in promoting folding of this intrinsically unstructured protein.
Catalysis↗
Carboxymethyl benzylamide sulfonate dextrans (CMDBS), a family of biospecific polymers endowed with numerous biological properties: a review.
The functionalized dextrans termed carboxymethyl benzylamide sulfonate dextran (CMDBS) represent a family encompassing a wide range of polymers. These soluble macromolecular compounds, which are substituted with specific chemical functional groups, are designed to interact with living systems. By analogy with glycosaminoglycan heparin, a natural highly charged anionic polysaccharide that exerts a variety of biological effects, we postulated that CMDBS compounds also possess binding sites capable of specific interactions with biological constituents, depending on the overall composition of the polymer. The synthesis and heparin-like properties of these CMDBS have been extensively investigated. Thus, it appears that dextran derivatives can mimic the action of heparin in regard to its interactions with antithrombin and serine proteases involved in blood coagulation. Other derivatives interact with various components of the immune system or with adhesive proteins such as fibronectin in modulating the proliferation of Staphylococcus aureus. Because they are able to stimulate wound healing in various in vivo models, these polysaccharides may also constitute a family of tissue repair agents because of their protecting and potentiating effects with heparin binding growth factors. Moreover, dextran derivatives in contact with cells such as endothelial cells, smooth muscle cells, or tumoral cells can affect both cell proliferation and metabolism. It appears that these bioactive polymers are also efficient tools to investigate the precise mechanism of action of individual biological activities by contrasting their mode of action to that of heparin. In addition to their numerous biological properties and biospecificity, functionalized dextrans are relatively simple to manufacture and exempt of donor contaminant, which make them attractive in a variety of clinical applications.
Animals↗
Role of oxygen radicals in tumor promotion.
Tumor promoters provoke the elaboration of oxygen radicals by direct chemical generation and through the indirect activation or alteration of cellular sources including membrane oxidases, peroxisomes, and electron transport chains in mitochondria and endoplasmic reticulum. Although direct measurement of amplified oxygen radical production in response to tumor promoters in target tissues remains problematic, studies with scavengers of reactive oxygen species demonstrate inhibition of biochemical and biological sequelae of tumor promoter exposure and provide strong presumptive evidence for oxygen radical involvement in this late stage of carcinogenesis. The critical macromolecular targets for these oxygen radicals remain undefined; however, they may include lipids, DNA, DNA repair systems, and other enzymes.
Animals↗
Indanoyl amino acid conjugates: tunable elicitors of plant secondary metabolism.
The unassuming nature of plants belies their viciously effective defensive strategies in the face of herbivore attack. Under the direction of, among others, octadecanoid hormones, plants respond by producing phytoalexins, bitter and toxic alkaloids, protease inhibitors, and even volatile compounds that call predatory insects to the herbivores. A rational design of 4-oxoindanoyl amino acid conjugates based on the phytotoxin, coronatine, as a structural guide resulted in a series of highly active compounds which turn on defensive systems in much the same way as octadecanoid hormones. The developments in the syntheses of indanoyl amino acid conjugates have created easy access to substantial amounts of a variety of such compounds. When these compounds were tested in biological systems, they showed abilities to induce defensive responses that surpassed octadecanoid hormones. In addition, small changes in the structures of these compounds resulted in large differences in the particular defensive systems that were activated. Indanoyl amino acid conjugates are promising tools in photoaffinity approaches towards the macromolecular targets of octadecanoids and their subcellular localization. Owing to the strong activation of plant defense or their efficient induction of fruit abscission which facilitates mechanical harvest, the compounds are promising candidates for future application in agriculture.
Amino Acids↗
Characterization of a tight molecular complex between integrin alpha 6 beta 4 and laminin-5 extracellular matrix.
In many adult epithelia, e.g., epidermis or intestine, adhesion of epithelial cells to basement membrane requires the integrin alpha6 beta4 and laminin-5 (Ln-5). In the absence of one or the other, extensive blistering and exfoliation occur. While alpha6 beta4 was reported to be a receptor for Ln-5, this interaction is poorly understood. We characterize complexes between alpha6 beta4 and Ln-5 in cell-free preparations of extracellular matrix (ECM) from the epithelial cell line, 804G. By microsequencing, Ln-5 and alpha6 beta4 were the major proteins in this ECM and were likely engaged in receptor/ligand complexes because, by immunofluorescence, alpha6 beta4 was colocalized with Ln-5 both in cell monolayers and in cell-free ECM preparations, but they disappeared after preincubation of the monolayers with alpha6 beta4 or Ln-5 function-blocking antibodies. The alpha6 beta4/Ln-5 complexes were resistant to dissociation by extreme pH, urea, chaotropes, eDTA, non-ionic detergents, and b-mercaptoethanol. They were only dissociated by strong anionic detergents, e.g., 1% SDS, suggesting receptor/ligand interactions based on high affinity or avidity. We propose that these alpha6 beta4/Ln-5 complexes may provide links between plasma membrane and basement membrane that resist mechanical stress and support epithelial integrity.
Amino Acid Sequence↗
Proteolytic and conformational control of virus capsid maturation: the bacteriophage HK97 system.
Bacteriophage capsid assembly pathways provide excellent model systems to study large-scale conformational changes and other mechanisms that regulate the formation of macromolecular complexes. These capsids are formed from proheads: relatively fragile precursor particles which mature by undergoing extensive remodeling. Phage HK97 employs novel features in its strategy for building capsids, including assembly without a scaffolding protein, and the formation of a network of covalent cross-links between neighboring subunits in the mature virion. In addition, proteolytic cleavage of the capsid protein from 42 kDa to 31 kDa is essential for maturation. To investigate the structural bases for proteolysis and cross-linking, we have used cryo-electron micrographs to reconstruct the three-dimensional structures of purified particles from four discrete stages in the assembly pathway: Prohead I, Prohead II, Head I and Head II. Prohead I has icosahedral T = 7 packing of blister-shaped pentamers and hexamers. The pentamers are 5-fold symmetric, but the hexamers exhibit an unusual departure from 6-fold symmetry, as if two trimers had undergone a shear dislocation of about 25 A. Proteolytic conversion to Prohead II leaves the outer surface largely unchanged, but a major loss of density from the inner surface is observed, which we infer to represent the excision of the amino-terminal domains of the capsid protein. Upon expansion to the Head I state, the capsid becomes markedly larger, thinner walled, and more polyhedral: moreover, the capsomer shapes change radically; especially notable is the disappearance of the large hexon dislocation. No differences between Head I and the covalently cross-linked Head II could be observed at the current resolution of about 25 A, from which we infer that it is the conformational rearrangements effected by expansion that create the micro-environments needed for the autocatalytic formation of the isodipeptide bonds found in the mature virions ("pseudo-active sites").
Amino Acid Sequence↗