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PubMed2003

Unique tertiary and neighbor interactions determine conservation patterns of Cis Watson-Crick A/G base-pairs.

X-ray, phylogenetic and quantum chemical analysis of molecular interactions and conservation patterns of cis Watson-Crick (W.C.) A/G base-pairs in 16S rRNA, 23S rRNA and other molecules was carried out. In these base-pairs, the A and G nucleotides interact with their W.C. edges with glycosidic bonds oriented cis relative to each other. The base-pair is stabilised by two hydrogen bonds, the C1'-C1' distance is enlarged and the G(N2) amino group is left unpaired. Quantum chemical calculations show that, in the absence of other interactions, the unpaired amino group is substantially non-planar due to its partial sp(3) pyramidalization, while the whole base-pair is internally propeller twisted and very flexible. The unique molecular properties of the cis W.C. A/G base-pairs make them distinct from other base-pairs. They occur mostly at the ends of canonical helices, where they serve as interfaces between the helix and other motifs. The cis W.C. A/G base-pairs play crucial roles in natural RNA structures with salient sequence conservation patterns. The key contribution to conservation is provided by the unpaired G(N2) amino group that is involved in a wide range of tertiary and neighbor contacts in the crystal structures. Many of them are oriented out of the plane of the guanine base and utilize the partial sp(3) pyramidalization of the G(N2). There is a lack of A/G to G/A covariation, which, except for the G(N2) position, would be entirely isosteric. On the contrary, there is a rather frequent occurrence of G/A to G/U covariation, as the G/U wobble base-pair has an unpaired amino group in the same position as the cis W.C. G/A base-pair. The cis W.C. A/G base-pairs are not conserved when there is no tertiary or neighbor interaction. Obtaining the proper picture of the interactions and phylogenetic patterns of the cis W.C. A/G base-pairs requires a detailed analysis of the relation between the molecular structures and the energetics of interactions at a level of single H-bonds and contacts.

Base Pairing↗

PubMed2002

Crystal structure of NC1 domains. Structural basis for type IV collagen assembly in basement membranes.

Type IV collagen, which is present in all metazoan, exists as a family of six homologous alpha(IV) chains, alpha1-alpha6, in mammals. The six chains assemble into three different triple helical protomers and self-associate as three distinct networks. The network underlies all epithelia as a component of basement membranes, which play important roles in cell adhesion, growth, differentiation, tissue repair and molecular ultrafiltration. The specificity of both protomer and network assembly is governed by amino acid sequences of the C-terminal noncollagenous (NC1) domain of each chain. In this study, the structural basis for protomer and network assembly was investigated by determining the crystal structure of the ubiquitous [(alpha1)(2).alpha2](2) NC1 hexamer of bovine lens capsule basement membrane at 2.0 A resolution. The NC1 monomer folds into a novel tertiary structure. The (alpha1)(2).alpha2 trimer is organized through the unique three-dimensional domain swapping interactions. The differences in the primary sequences of the hypervariable region manifest in different secondary structures, which determine the chain specificity at the monomer-monomer interfaces. The trimer-trimer interface is stabilized by the extensive hydrophobic and hydrophilic interactions without a need for disulfide cross-linking.

Amino Acid Sequence↗

PubMed2001

X-ray diffraction and atomic force microscopy analysis of twinned crystals: rhombohedral canavalin.

The structure of canavalin, the vicilin-class storage protein from jack bean, was refined to 1.7 A resolution in a highly twinned rhombohedral crystal of space group R3 and unit-cell parameters a = b = c = 83.0 A, alpha = beta = gamma = 111.1 degrees. The resulting R and R(free) were 0.176 and 0.245, respectively. The orthorhombic crystal structure (space group C222(1), unit-cell parameters a = 136.5, b = 150.3, c = 133.4 A) was also refined with threefold non-crystallographic symmetry restraints. R and R(free) were 0.181 and 0.226, respectively, for 2.6 A resolution data. No significant difference in the protein structure was seen between these two crystal forms, nor between these two and the hexagonal and cubic crystal forms reported elsewhere [Ko et al. (1993), Acta Cryst. D49, 478-489; Ko et al. (1993), Plant Physiol. 101, 729-744]. A phosphate ion was identified in the lumen of the C-terminal beta-barrel. Lattice interactions showed that the trimeric molecule could be well accommodated in both 'top-up' and 'bottom-up' orientations in a rhombohedral unit cell of the R3 crystal and explained the presence of a high twin fraction. The large inter-trimer stacking interface of the C222(1) crystal may account for its relative stability. Atomic force microscopy (AFM) investigations of the growth of three crystal forms of canavalin indicate the rhombohedral form to be unique. Unlike the other two crystal forms, it contains at least an order of magnitude more screw dislocations and stacking faults than any other macromolecular crystal yet studied, and it alone grows principally by generation of steps from the screw dislocations. The unusually high occurrence of the screw dislocations and stacking faults is attributed to mechanical stress produced by the alternate molecular orientations in the rhombohedral crystals and their organization into discrete domains or blocks. At boundaries of alternate domains, lattice strain is relieved by the formation of the screw dislocations.

Crystallization↗

PubMed2005

The T cell receptor: critical role of the membrane environment in receptor assembly and function.

Recent studies have demonstrated that cell membranes provide a unique environment for protein-protein and protein-lipid interactions that are critical for the assembly and function of the T cell receptor (TCR)-CD3 complex. Highly specific polar interactions among transmembrane (TM) domains that are uniquely favorable in the lipid environment organize the association of the three signaling dimers with the TCR. Each of these three assembly steps depends on the formation of a three-helix interface between one basic and two acidic residues in the membrane environment. The same polar TM residues that drive assembly also play a central role in quality control and export by directing the retention and degradation of free subunits and partial complexes, while membrane proximal cytoplasmic signals control recycling and degradation of surface receptors. Recent studies also suggest that interactions between the membrane and the cytoplasmic domains of CD3 proteins may be important for receptor triggering.

Amino Acid Motifs↗

PubMed2005

Experimental and simulative dissociation of dimeric Cu,Zn superoxide dismutase doubly mutated at the intersubunit surface.

The equilibrium properties of dimeric Photobacterium leiognathi Cu,Zn superoxide dismutase mutant bearing two negative charges in the amino acid clusters at the association interface has been studied, experimentally and computationally, and compared to those of the native enzyme. Pressure-dependent dissociation is observed for the mutant, as observed by the fluorescence shift of the unique tryptophan residue located at the intersubunit surface. The spectral shift occurs slowly, reaching a plateau after 15-20 min, and is fully reversible. Measurement of the degree of dissociation allows us to calculate the standard volume variation upon association and the dissociation constant at atmospheric pressure. On the other hand the native protein is undissociable at any pressure. In the simulative approach, the dissociation free energy has been calculated through the blue moon calculation method for the case of a multidimensional reaction coordinate, corrected for the rotational contribution within the semiclassical approximation for a free rigid-body rotor. The scheme permits to define a definite path for the rupture of the dimer and to calculate the effective force involved in the process. The calculated free energy difference is close to the experimental one, and the value obtained for the mutant is well below that obtained for the native protein, indicating that the theoretical reaction scheme is able to reproduce the experimental trend. Moreover, we find that, when the separation distance increases, the protein structure of the monomer is stable in line with the fast recovery of the original fluorescence properties after decompression, which excludes the presence of partly unfolded intermediates during the dimer-monomer transition.

Amino Acids↗

PubMed2003

Lamellarin D: a novel potent inhibitor of topoisomerase I.

We report the identification and characterization of a novel potent inhibitor of DNA topoisomerase I: lamellarin D (LAM-D), initially isolated from a marine mollusk, Lamellaria sp., and subsequently identified from various ascidians. This alkaloid, which displays potent cytotoxic activities against multidrug-resistant tumor cell lines and is highly cytotoxic to prostate cancer cells, bears a 6H-[1]benzopyrano[4',3':4,5]pyrrolo[2,1-a]isoquinolin-one pentacyclic planar chromophore, whereas its synthetic 5,6-dehydro analogue, LAM-501, has a significantly tilted structure. DNA binding measurements by absorbance, fluorescence, and electric linear dichroism spectroscopy show that LAM-D is a weak DNA binder that intercalates between bp of the double helix. In contrast, the nonplanar analogue LAM-501 did not bind to DNA and failed to inhibit topoisomerase I. DNA intercalation may be required for the stabilization of topoisomerase I-DNA complexes by LAM-D. In the DNA relaxation assay, LAM-D strongly promoted the conversion of supercoiled DNA into nicked DNA in the presence of topoisomerase I. The marine product was approximately 5 times less efficient than camptothecin (CPT) at stabilizing topoisomerase I-DNA complexes, but interestingly, the two drugs exhibited slightly distinct sequence specificity profiles. Topoisomerase I-mediated DNA cleavage in the presence of LAM-D occurred at some sites common to CPT, but a few specific sites identified with CPT but not with LAM-D or conversely unique sites cleaved by LAM-D but not by CPT were detected. The distinct specificity profiles suggest that LAM-D and CPT interact differently with the topoisomerase I-DNA interface. A molecular modeling analysis provided structural information on the orientation of LAM-D within the topoisomerase I-DNA covalent complex. The marine alkaloid did not induce DNA cleavage by topoisomerase II. Immunoblotting experiments revealed that endogenous topoisomerase I was efficiently trapped on DNA by LAM-D in P388 and CEM leukemia cells. P388/CPT5 and CEM/C2 cell lines, both resistant to CPT and expressing a mutated top1 gene, were cross-resistant to LAM-D. Collectively, the results identify LAM-D as a novel lead candidate for the development of topoisomerase I-targeted antitumor agents.

Animals↗

PubMed1985

Environmental modulation of the expression of differentiation and malignancy in six human squamous cell carcinoma cell lines.

Cell lines from six human squamous cell carcinomas exhibiting different degrees of differentiation and malignancy were studied under in vitro and in vivo growth conditions. The stability of phenotypic traits of these carcinoma cells and their sensitivity to environmental influences were analyzed to further elucidate the interdependency of differentiation and malignancy expressed under experimental conditions. In conventional (submerged) cultures the cell lines exhibited unique growth patterns with an individual but generally poor expression of differentiation (stratification). In a new organotypical culture assay where the cells grew on lifted collagen gels at the air-medium interface, three-dimensional structures were formed exhibiting organizational features and degrees of differentiation similar to those of the respective tumors. Both in tumors formed after s.c. injection of cells and in transplants (performed with silicone chambers on the dorsal muscle fascia) in nude mice, an enhancement of the individually distinct pattern of differentiation was observed. While anchorage independent growth was an unreliable marker for malignancy, all six lines were tumorigenic after s.c. injection into nude mice. However, the tumor yield (20 to 100%) and latency period (2 to 12 weeks) varied considerably. In contrast all lines exhibited (within 1 to 2 weeks) invasive growth in 100% of animals after transplantation onto the dorsal muscle fascia. All tumors (squamous carcinomas) and invading cells were identified as epithelial and as human by specific antibodies. The two new test systems, the organotypical culture assay in vitro and the transplantation assay in vivo, proved to be reliable and sensitive models also for human squamous carcinoma cells to analyze their differentiative and malignant potential. In comparing the individually maintained degrees of differentiation and malignancy in the different test systems, it was apparent that, opposite to the prevailing opinion, cell lines with the highest differentiation potential were at least as malignant as were the least differentiated ones.

Animals↗

PubMed1994

Photoaffinity labeling of the adenine binding sites of two Dolichos biflorus lectins.

Two differentially expressed lectins from the legume Dolichos biflorus, the seed lectin and a stem and leaf lectin (DB58), were photoaffinity-labeled at their adenine binding sites using the probe [2-3H]8-azidoadenine. Both heteromeric subunits I and II of the seed lectin and alpha and beta of DB58 were specifically labeled. This result, combined with the adenine binding site stoichiometries of two identical sites/seed lectin tetramer or one site/DB58 dimer, indicates that the adenine binding site resides at a heterologous subunit interface. Three radiolabeled peaks from seed lectin and one from DB58 were isolated from chymotryptic digests of the labeled lectins by reverse phase chromatography at pH 7.0. From these four peaks, six unique peptide sequences were determined. When aligned with the concanavalin A sequence, four of these peptides map to three loops in the metal binding domain of concanavalin A. The remaining two sequences represent carboxyl-terminal peptides unique to the D. biflorus lectins which may extend to the putative binding site from adjacent, heterologous subunits. It thus appears that the adenine binding sites of these D. biflorus lectins are within the metal binding domain and adjacent to the carbohydrate binding site.

Adenine↗

PubMed2006

Interface parameters of impact-induced mild traumatic brain injury.

Commonly considered a continuum of injuries, diffuse brain injury (DBI) ranges from mild concussion to severe diffuse axonal injury. The lower end of the spectrum is generally referred to as mild traumatic brain injury (MTBI). More severe forms of DBI have garnered extensive experimentation while these milder cases are considerably less explored. Recently, a new device was designed to generate DBI in the rodent using impact-induced angular acceleration. This device is modifiable so the entire spectrum of DBI can be investigated. Severity of DBI is critically dependent on magnitude of angular acceleration. A small animal surrogate like a rodent has a relatively small brain mass. This constraint poses a unique problem because the angular acceleration necessary for DBI is inversely related to brain mass. Prior experimentation estimated an angular acceleration of approximately 350 krad/s2 is necessary for the induction of mild traumatic brain injury (MTBI) in the rodent. To induce these magnitudes of angular acceleration in a repeatable manner, the impacting interface must be critically analyzed. This investigation uses a mathematical model based on parameters of a previously developed experimental model to assess the impacting interface such that angular accelerations are sufficient to produce MTBI in the rodent.

Acceleration↗

PubMed1997

The unique heme-heme interactions of the homodimeric Scapharca inaequivalvis hemoglobin as probed in the protein reconstituted with unnatural 2,4 heme derivatives.

In the homodimeric hemoglobin from Scapharca, HbI, functional communication between the two heme groups is based on their direct structural linkage across the subunit interface through the heme propionates. The heme-protein interactions have been altered in deutero- and meso-HbI by substituting the vinyl groups at positions 2 and 4 of protoheme with hydrogen and ethyl groups, respectively. In meso-HbI the introduction of the ethyl groups in the heme pocket induces significant alterations in the conformation of the heme peripheral substituents, including the propionates, and in the structure of bound CO, as revealed by the resonance Raman spectra. The functional counterpart of these structural changes is the loss of cooperativity in carbon monoxide binding and in the rate of oxygen dissociation. Oxygen pulse and flash photolysis experiments indicate that meso-HbI is locked in the liganded conformation. It is postulated that the ethyl groups, which occupy a larger volume than vinyl ones, impair the ligand-linked movement of the heme relative to its pocket and in turn the expression of cooperativity. In deutero-HbI structural alterations have not been monitored. Functionally, cooperativity in the CO binding kinetics is increased as if hydrogen atoms at positions 2 and 4 permitted more marked movements of the heme than in the native protein.

Animals↗

PubMed1998

Structural basis of inhibitor selectivity in MAP kinases.

BACKGROUND: The mitogen-activated protein (MAP) kinases are important signaling molecules that participate in diverse cellular events and are potential targets for intervention in inflammation, cancer, and other diseases. The MAP kinase p38 is responsive to environmental stresses and is involved in the production of cytokines during inflammation. In contrast, the activation of the MAP kinase ERK2 (extracellular-signal-regulated kinase 2) leads to cellular differentiation or proliferation. The anti-inflammatory agent pyridinylimidazole and its analogs (SB [SmithKline Beecham] compounds) are highly potent and selective inhibitors of p38, but not of the closely-related ERK2, or other serine/threonine kinases. Although these compounds are known to bind to the ATP-binding site, the origin of the inhibitory specificity toward p38 is not clear. RESULTS: We report the structural basis for the exceptional selectivity of these SB compounds for p38 over ERK2, as determined by comparative crystallography. In addition, structural data on the origin of olomoucine (a better inhibitor of ERK2) selectivity are presented. The crystal structures of four SB compounds in complex with p38 and of one SB compound and olomoucine in complex with ERK2 are presented here. The SB inhibitors bind in an extended pocket in the active site and are complementary to the open domain structure of the low-activity form of p38. The relatively closed domain structure of ERK2 is able to accommodate the smaller olomoucine. CONCLUSIONS: The unique kinase-inhibitor interactions observed in these complexes originate from amino-acid replacements in the active site and replacements distant from the active site that affect the size of the domain interface. This structural information should facilitate the design of better MAP-kinase inhibitors for the treatment of inflammation and other diseases.

Adenosine Triphosphate↗

PubMed2000

S-layer-supported lipid membranes.

Many prokaryotic organisms (archaea and bacteria) are covered by a regularly ordered surface layer (S-layer) as the outermost cell wall component. S-layers are built up of a single protein or glycoprotein species and represent the simplest biological membrane developed during evolution. Pores in S-layers are of regular size and morphology, and functional groups on the protein lattice are aligned in well-defined positions and orientations. Due to the high degree of structural regularity S-layers represent unique systems for studying the structure, morphogenesis, and function of layered supramolecular assemblies. Isolated S-layer subunits of numerous organisms are able to assemble into monomolecular arrays either in suspension, at air/water interfaces, on planar mono- and bilayer lipid films, on liposomes and on solid supports (e.g. silicon wafers). Detailed studies on composite S-layer/lipid structures have been performed with Langmuir films, freestanding bilayer lipid membranes, solid supported lipid membranes, and liposomes. Lipid molecules in planar films and liposomes interact via their head groups with defined domains on the S-layer lattice. Electrostatic interactions are the most prevalent forces. The hydrophobic chains of the lipid monolayers are almost unaffected by the attachment of the S-layer and no impact on the hydrophobic thickness of the membranes has been observed. Upon crystallization of a coherent S-layer lattice on planar and vesicular lipid membranes, an increase in molecular order is observed, which is reflected in a decrease of the membrane tension and an enhanced mobility of probe molecules within an S-layer-supported bilayer. Thus, the terminology 'semifluid membrane' has been introduced for describing S-layer-supported lipid membranes. The most important feature of composite S-layer/lipid membranes is an enhanced stability in comparison to unsupported membranes.

Bacterial Proteins↗

PubMed2006

Optimized peptide separation and identification for mass spectrometry based proteomics via free-flow electrophoresis.

Multidimensional LC-MS based shotgun proteomics experiments at the peptide level have traditionally been carried out by ion exchange in the first dimension and reversed-phase liquid chromatography in the second. Recently, it has been shown that isoelectric focusing (IEF) is an interesting alternative approach to ion exchange separation of peptides in the first dimension. Here we present an improved protocol for peptide separation by continuous free-flow electrophoresis (FFE) as the first dimension in a two-dimensional peptide separation work flow. By the use of a flat pI gradient and a mannitol and urea based separation media we were able to perform high-throughput proteome analysis with improved interfacing between FFE and RPLC-MS/MS. The developed protocol was applied to a cytosolic fraction from Schneider S2 cells from Drosophila melanogaster, resulting in the identification of more than 10,000 unique peptides with high probability. To improve the accuracy of the peptide identification following FFE-IEF we incorporated the pI information as an additional parameter into a statistical model for discrimination between correct and incorrect peptide assignments to MS/MS spectra.

Animals↗

PubMed1997

Crystal structure of the RAG1 dimerization domain reveals multiple zinc-binding motifs including a novel zinc binuclear cluster.

The crystal structure of the dimerization domain of the V(D)J recombination-activating protein, RAG1, was solved using zinc anomalous scattering. The structure reveals an unusual combination of multi-class zinc-binding motifs, including a zinc RING finger and a C2H2 zinc finger, that together from a single structural domain. The domain also contains a unique zinc binuclear cluster in place of a normally mononuclear zinc site in the RING finger. Together, four zinc ions help organize the entire domain, including the two helices that form the dimer interface.

Binding Sites↗

PubMedDate not supplied

A disposable blood pressure transducer system.

A unique disposable invasive hemodynamic blood pressure transducer system has been developed. The system consists of a disposable piezoresistive flow-through transducer with twelve-inch pigtail, a reusable extension cable, an electronic interface module and a custom interconnect cable to attach to most monitors. The transducer has Linden Luer fittings and replaces the dome and reusable transducer in monitoring systems. The cost of the transducer is kept low so that it can be disposable through efficient modern, high-volume semiconductor technology and the fact that additional electrical isolation is accomplished in the interface module. Besides providing electrical isolation, the interface module provides a universal output which will accommodate all common AC, DC, and pulsed excitation signals from monitors.

Blood Pressure Determination↗

PubMed2006

Orientation selection in lamellar phases by oscillatory shears.

In order to address the selection mechanism that is responsible for the unique lamellar orientation observed in block copolymers under oscillatory shears, we use a constitutive law for the dissipative part of the stress tensor that respects the uniaxial symmetry of a lamellar phase. An interface separating two domains oriented parallel and perpendicular to the shear is shown to be hydrodynamically unstable, a situation analogous to the thin layer instability of stratified fluids under shears. The resulting secondary flows break the degeneracy between the parallel and perpendicular lamellar orientation, leading to a preferred perpendicular orientation in certain ranges of parameters of the polymer and of the shear.

Journal Article↗

PubMedDate not supplied

Image-based stained glass.

We present a method of restyling an image so that it approximates the visual appearance of a work of stained glass. To this end, we develop a novel approach which involves image warping, segmentation, querying, and colorization along with texture synthesis. In our method, a given input image is first segmented. Each segment is subsequently transformed to match real segments of stained glass queried from a database of image exemplars. By using real sources of stained glass, our method produces high quality results in this nascent area of nonphotorealistic rendering. The generation of the stained glass requires only modest amounts of user interaction. This interaction is facilitated with a unique region-merging tool.

Algorithms↗

PubMed2004

Collagen fibril characteristics at the corneo-scleral boundary and rabbit corneal stromal swelling.

BACKGROUND: The aim of this investigation was to reassess the impact of the scleral rim on the swelling of the mammalian corneal stroma and to investigate the ultrastructural features of the scleral rim and corneal stromal tissues. METHODS: The epithelium and endothelium were removed from corneas excised from three-month-old female rabbits. The resulting preparations consisted of the corneal stroma plus a surrounding scleral rim, excised corneal stroma or a nine-millimetre button of central corneal stroma. These preparations were immersed in a 35 mM bicarbonate-buffered mixed salt solution (equilibrated with five per cent CO2-air, pH 7.54 at 37 degrees Centigrade for nine hours. Some sclero-corneal preparations were fixed for light or transmission electron microscopy. RESULTS: The initial rate of swelling of corneal stromal buttons was greatest at 127 +/- 8 per cent per hour, less for complete stromal preparations (118 +/- 9 per cent per hour) and least for sclero-stromal preparations (76 +/- 12 per cent per hour). The swelling continued over three to nine hours but sclera preparations swelled up to 40 per cent with no further swelling. Light microscopy demonstrated that the sclero-corneal rim tissue limited the swelling of the posterior corneal stroma. TEM sections of the episclera and sclera indicate that most fibril bundles show a radial orientation to the cornea. There are marked anterio-posterior differences in the collagen fibrils of the scleral surround that are distinctly different from previous reports. Average fibril diameters were 62.8 +/- 7.9 nm in the episclera, 122.4 +/- 18.9 nm, 133.5 +/- 51.9 nm and 56.5 +/- 11.2 nm in the anterior, mid- and posterior scleral stroma, compared to an average fibril diameter of 33.5 +/- 3.5 nm for the posterior corneal stroma. CONCLUSIONS: When there is a scleral rim in place, the swelling of the corneal stroma is substantially less than for isolated corneal stroma. The effect can be attributed to the absence of a cut-edge effect for the sclero-corneal stromal preparation but the unique and largely radial arrangement of the collagen fibrils in the scleral rim plays a part in limiting the swelling of the adjacent corneal stroma. The heterogeneous nature of this sclero-corneal interface requires further investigation to define the mechanism of the effect.

Animals↗