Wood samples of 111 Vaccinieae specimens (Vaccinioideae, Ericaceae s.l.) representing 98 species and 26 genera are investigated with light microscopy and scanning electron microscopy. The wood... Show moreWood samples of 111 Vaccinieae specimens (Vaccinioideae, Ericaceae s.l.) representing 98 species and 26 genera are investigated with light microscopy and scanning electron microscopy. The wood structure of Vaccinieae delivers taxonomically important characters that can be used to define some subclades within the tribe. The wood of the large polyphyletic genus Vaccinium strongly resembles non-vaccinioid members of the family, which are characterized by bordered vessel-ray pits and relatively narrow (2- to 4-seriate) and low multiseriate rays (often less than 1000 m) with exclusively or mainly procumbent body ray cells. The East Malesian clade, Meso-American/Caribbean clade, and the Andean clade show a combination of wood anatomical features that is lacking in other representatives of the family. These features include scalariform vessel-ray pits with strongly reduced borders, a high portion of upright body ray cells, wide (4- to 14-seriate) and high multiseriate rays (often more than 3000 m), and prismatic crystals in chambered ray cells (although absent in Symphysia racemosa). The presence of secretory ducts in the primary xylem and in the pith tissue may represent a synapomorphy for the Andean clade. Furthermore, the presence of undivided axial parenchyma cells, usually ranging from 500 to 900 m, seems to be unique in the subfamily. Show less
An ensemble approach for force networks in static granular packings is developed. The framework is based on the separation of packing and force scales, together with an a priori flat measure in the... Show moreAn ensemble approach for force networks in static granular packings is developed. The framework is based on the separation of packing and force scales, together with an a priori flat measure in the force phase space under the constraints that the contact forces are repulsive and balance on every particle. In this paper we will give a general formulation of this force network ensemble, and derive the general expression for the force distribution P(f). For small regular packings these probability densities are obtained in closed form, while for larger packings we present a systematic numerical analysis. Since technically the problem can be written as a noninvertible matrix problem (where the matrix is determined by the contact geometry), we study what happens if we perturb the packing matrix or replace it by a random matrix. The resulting P(f)’s differ significantly from those of normal packings, which touches upon the deep question of how network statistics is related to the underlying network structure. Overall, the ensemble formulation opens up a different perspective on force networks that is analytically accessible, and which may find applications beyond granular matter. Show less
A comparative study of the thermal stability of wild type poplar plastocyanin and of a mutant form containing a disulfide bridge between residues 21 and 25 was performed using differential scanning... Show moreA comparative study of the thermal stability of wild type poplar plastocyanin and of a mutant form containing a disulfide bridge between residues 21 and 25 was performed using differential scanning calorimetry and optical spectroscopic techniques.For wild type plastocyanin the transition temperature, determined from the calorimetric profiles, is 62.7 degreesC at the scan rate of 60 degreesC/h, whereas for the mutant it is reduced to 58.0 degreesC. In both cases, the endothermic peak is followed by an exothermic one at higher temperatures.The unfolding process monitored by optical absorption at 596 nm also reveals a reduced thermal stability of the mutated plastocyanin compared to the wild type protein, with transition temperatures of 54.8 and 58.0 degreesC, respectively. For both proteins, the denaturation process was found to be irreversible and dependent on the scan rate preventing the thermodynamic analysis of the unfolding process.In parallel, small conformational changes between wild type and mutant plastocyanin emerge from fluorescence spectroscopy measurements. Here, a difference in the interaction of the two proteins between the microenvironment surrounding the fluorophores and the solvent was proposed.The destabilization observed in the disulfide containing mutant of plastocyanin suggests that the double mutation, Ile2lCys and Glu25Cys, introduces strain into the protein which offsets the stabilizing effect expected from the formation of a covalent crosslink. (C) 2004 Elsevier B.V. All rights reserved. Show less
Molecular dynamics (MD) simulations have been performed on quercetin 2,3 dioxygenase (2,3QD) to study the mobility and flexibility of the substrate cavity. 2,3QD is the only firmly established Cu... Show moreMolecular dynamics (MD) simulations have been performed on quercetin 2,3 dioxygenase (2,3QD) to study the mobility and flexibility of the substrate cavity. 2,3QD is the only firmly established Cu-containing dioxygenase known so far. It catalyses the breakage of the O-heterocycle of flavonols. The substrates occupy a shallow and overall hydrophobic cavity proximal to the metal centre of the homo-dimeric enzyme. The linker connecting the C-terminal and N-terminal domains in the monomer is partly disordered in the crystal structure and part of it forms a flexible lid at the entrance of the substrate cavity. This loop has been tentatively assigned a role in the enzyme mechanism: it helps lock the substrate into place. The dynamics of this loop has been investigated by MD simulation. The initial coordinates were taken from the crystal structure of 2,3QD in the presence of the substrate kaempferol (KMP). After equilibration and simulation over 7.2 ns the substrate was removed and another equilibration and simulation of 7.2 ns was performed. The results show that the structures of the free enzyme as well as of the enzyme-substrate complex are stable in MD simulation. The linker shows strongly enhanced mobility in the loop region that is close to the entrance to the substrate cavity (residues 154-169). Movement of the loop takes place on a timescale of 5-10 ns. To confirm the conclusions about the loop dynamics drawn from the 7.2 ns simulation, the simulation was extended with another 8 ns. When substrate binds into the cavity the loop orders remarkably, although mobility is retained by residues 155-158. Some regions of the loop (residues 154-160 and 164-176) move over a considerable distance and approach the substrate closely, reinforcing the idea that they lock the substrate in the substrate cavity The enthalpic component of the interaction of the loop with the protein and the KMP appears to favour the locking of the substrate. Two water molecules were found immobilised in the cavity, one of which exhibited rotation on the picosecond timescale. When the substrate is removed, the empty cavity fills up with water within 200 ps. (C) 2004 Elsevier Ltd. All rights reserved. Show less
Obtaining chemical shift anisotropy (CSA) principal values from large biomolecular systems is often a laborious process preparing many singly isotopically labeled samples and performing multiple... Show moreObtaining chemical shift anisotropy (CSA) principal values from large biomolecular systems is often a laborious process preparing many singly isotopically labeled samples and performing multiple independent CSA measurements. We present CSA tensor principal values measured in the biomolecular building blocks tyrosine(.)HCl, histidine(.)HCl, and all-E-retinal, both isotopically labeled and unlabeled forms at 17.6 T. The measured tensor values are identical for most carbon sites despite significant dipolar couplings between the spins. Quantum mechanical simulations of all arbitrary three spin system were used to evaluate the accuracy of direct CSA measurement as a function of applied magnetic field strength and molecular parameters. It was found that for a CSA asymmetry of 0.2 or more, an accurate measure of the CSA parameters is obtained when the CSA anisotropy is more than six times the largest dipolar coupling in frequency units. If the CSA asymmetry is more than 0.5, this requirement is relaxed, and accurate results are obtained if the anisotropy is more than three times the dipolar coupling. While these limits are insufficient for measurement of CSA's for alpha-carbons and aliphatic sidechain sites in proteins at current field strengths. they open the way for routine systematic CSA measurements of sites with relatively large CSA tensor values in extensively isotopically labeled biomolecules in widely available magnetic fields. (C) 2004 Published by Elsevier Inc. Show less
We have studied the morphological, conformational, and electron-transfer (ET) function of the metalloprotein azurin in the solid state, by a combination of physical investigation methods, namely... Show moreWe have studied the morphological, conformational, and electron-transfer (ET) function of the metalloprotein azurin in the solid state, by a combination of physical investigation methods, namely atomic force microscopy, intrinsic fluorescence spectroscopy, and scanning tunneling microscopy. We demonstrate that a “solid state protein film” maintains its nativelike conformation and ET function, even after removal of the aqueous solvent. Show less
Partly biosynthetic site-directed isotopically C-13 enriched photosynthetic light-harvesting 2(LH2) complexes have been prepared from Rhodopseudomonas acidophila strain 10050 by using chemically... Show morePartly biosynthetic site-directed isotopically C-13 enriched photosynthetic light-harvesting 2(LH2) complexes have been prepared from Rhodopseudomonas acidophila strain 10050 by using chemically labeled [1,2,3,4-C-13], [1,4-C-13] and [2,3-C-13] succinic acid as a precursor in the growth medium. Two-dimensional proton driven spin diffusion (PDSD) solid state NMR correlation spectroscopy has been used to trace each individual 13C isotope from the labeled succinic acid precursor to its destination into the protein and into the embedded major light-absorbing bacteriochlorophyll cofactors. For both the residues of the protein and for the cofactors distinct labeling patterns have been deduced, for protein complexes prepared from [1,4-C-13]-succinic acid or [2,3-C-13]-succinic labeled media. All residues, except isoleucine and leucine, have been labeled almost homogeneously by the succinic acid precursor. Carbonyl carbons in the protein backbone were labeled by [1,4-C-13]-succinic acid, while the Calpha and Cbeta carbons of the residues were labeled by [2,3-C-13]-succinic acid. Leucine and isoleucine residues were labeled using a uniformly labeled amino acid mixture in the medium. The pattern labeling yields an increase of the resolution and less spectral crowding. The partial labeling technique in combination with conventional solid state NMR methods at ultra high magnetic fields provides an attractive route to resolve chemical shifts for alpha-helical transmembrane protein structures. Show less
Nowadays, the development of experimental procedures for the determination of the secondary structure of RNA molecules is taking advantage of the novel single-molecule probing and imaging... Show moreNowadays, the development of experimental procedures for the determination of the secondary structure of RNA molecules is taking advantage of the novel single-molecule probing and imaging techniques. We report a method for the mapping of the secondary structure of RNA molecules spread on a flat surface by means of the atomic force microscope. Globular domains comprising groups of RNA secondary and tertiary structure elements separated by unstructured domains can be discerned in the micrographs and their position along the molecule contour can be measured directly on unstained specimens. We have analyzed the morphology of a population of single molecules of 3' fragments of the Turnip Yellow Mosaic Virus RNA shorter than 1 kb in different temperature and electrolytic conditions. We found a satisfying agreement of the shape of the imaged structures with previously available evidence. The method we have developed can be used to map also different types of RNA molecules and has the advantage of showing the distribution of the single molecule conformations within the population. (C) 2005 Wiley-Liss, Inc. Show less
Fusion of biological membranes is mediated by distinct integral membrane proteins, e.g., soluble N-ethylmaleimide-sensitive factor attachment protein receptors and viral fusion proteins. Previous... Show moreFusion of biological membranes is mediated by distinct integral membrane proteins, e.g., soluble N-ethylmaleimide-sensitive factor attachment protein receptors and viral fusion proteins. Previous work has indicated that the transmembrane segments (TMSs) of such integral membrane proteins play an important role in fusion. Furthermore, peptide mimics of the transmembrane part can drive the fusion of liposomes, and evidence had been obtained that fusogenicity depends on their conformational flexibility. To test this hypothesis, we present a series of unnatural TMSs that were designed de novo based on the structural properties of hydrophobic residues. We find that the fusogenicity of these peptides depends on the ratio of alpha-helix-promoting Leu and beta-sheet-promoting Val residues and is enhanced by helix-destabilizing Pro and Gly residues within their hydrophobic cores. The ability of these peptides to refold from an alpha-helical state to a beta-sheet conformation and backwards was determined under different conditions. Membrane fusogenic peptides with mixed Leu/ Val sequences tend to switch more readily between different conformations than a nonfusogenic peptide with an oligo-Leu core. We propose that structural flexibility of these TMSs is a prerequisite of fusogenicity. Show less
Boder, I. de; Matysik, J.; Erkelens, C.; Sasaki, S.; Miyatake, T.; Yagai, S.; ... ; Groot, H.J.M. de 2004
Magic angle spinning NMR spectroscopy has been used to investigate the self-organization of bacteriochlorophylls in chlorosomal light-harvesting antennae. Two model cadmium chlorins were studied... Show moreMagic angle spinning NMR spectroscopy has been used to investigate the self-organization of bacteriochlorophylls in chlorosomal light-harvesting antennae. Two model cadmium chlorins were studied that were uniformly C-13 and N-15 enriched in the ring moieties. The chlorin models differ from the natural BChl c in the central metal and the 3-, 12-, 17-, and 20-side chains. One model system has the farnesyl tail replaced by a methyl, whereas the other has a stearyl tail. The Cd-113 MAS NMR signals indicate a five-coordination of the Cd metal. In particular, the combined NMR data show a (HOCd)-Cd-... coordination, very similar to the (HOMg)-Mg-... coordination in the natural system. Anomalously large H-1 ring-current shifts of up to 10 ppm reveal a dense orderly stacking of the molecules in planar layers, for which a correlation length of at least 24 A was defined from long-range ring-current shift calculations. In addition, our model structures confirm and validate the essential role of the [3(1)R] and [3(1)S] stereoisomers in the formation of the chlorosomal antennae, as tubular structures are not formed without this chirality. The 3D arrangement of the layers is revealed by intermolecular C-13-C-13 correlations obtained from CP3 CHHC experiments. With the tail truncated to methyl, a microcrystalline solid is formed with favorable interactions between the planar sheets in a head-to-tail orientation. The stearyl tails lead to a considerably disordered aggregate consisting of both syn and anti layers similar to the chlorosomes, as indicated by a doubling of the N-D signal. These results reveal a balance between relatively strong local interactions and contributions to the free energy of the system associated with a longer length scale. This leads to a robust chlorosome structure, stable against thermodynamic noise, and allows for fine-tuning of the structure. Show less
Het is inmiddels al enige tijd geleden, maar daarom niet minder interessant om aandacht aan te besteden: het LCB 2004 Grenzeloos Verniewen? Vernieuwen is (zoals de titel al zegt) grenzeloos en... Show moreHet is inmiddels al enige tijd geleden, maar daarom niet minder interessant om aandacht aan te besteden: het LCB 2004 Grenzeloos Verniewen? Vernieuwen is (zoals de titel al zegt) grenzeloos en immer actueel. Ondanks de ruimte tijdspanne tussen het uitkomen van deze BB en de datum waarop het congres gehouden werd (18 en 19 mei 2004), wil de redactie u een klein verslag niet onthouden. Niet in de laatste plaats omdat Leiden het congres dit jaar weer eens mocht organiseren. Show less
