Plant growth in elevated CO2 alters mitochondrial number and chloroplast fine structure

With increasing interest in the effects of elevated atmospheric CO2 on plant growth and the global carbon balance, there is a need for greater understanding of how plants respond to variations in atmospheric partial pressure of CO2. Our research shows that elevated CO2 produces significant fine structural changes in major cellular organelles that appear to be an important component of the metabolic responses of plants to this global change. Nine species (representing seven plant families) in several experimental facilities with different CO2-dosing technologies were examined. Growth in elevated CO2 increased numbers of mitochondria per unit cell area by 1.3–2.4 times the number in control plants grown in lower CO2 and produced a statistically significant increase in the amount of chloroplast stroma (nonappressed) thylakoid membranes compared with those in lower CO2 treatments. There was no observable change in size of the mitochondria. However, in contrast to the CO2 effect on mitochondrial number, elevated CO2 promoted a decrease in the rate of mass-based dark respiration. These changes may reflect a major shift in plant metabolism and energy balance that may help to explain enhanced plant productivity in response to elevated atmospheric CO2 concentrations.

Tertiary structure of an amyloid immunoglobulin light chain protein: a proposed model for amyloid fibril formation.

An immunoglobulin light chain protein was isolated from the urine of an individual (BRE) with systemic amyloidosis. Complete amino acid sequence of the variable region of the light chain (VL) protein established it as a kappa I, which when compared with other kappa I amyloid associated proteins had unique residues, including Ile-34, Leu-40, and Tyr-71. To study the tertiary structure, BRE VL was expressed in Escherichia coli by using a PCR product amplified from the patient BRE's bone marrow DNA. The PCR product was ligated into pCZ11, a thermal-inducible replication vector. Recombinant BRE VL was isolated, purified to homogeneity, and crystallized by using ammonium sulfate as the precipitant. Two crystal forms were obtained. In crystal form I the BRE VL kappa domain crystallizes as a dimer with unit cell constants isomorphous to previously published kappa protein structures. Comparison with a nonamyloid VL kappa domain from patient REI, identified significant differences in position of residues in the hypervariable segments plus variations in framework region (FR) segments 40-46 (FR2) and 66-67 (FR3). In addition, positional differences can be seen along the two types of local diads, corresponding to the monomer-monomer and dimer-dimer interfaces. From the packing diagram, a model for the amyloid light chain (AL) fibril is proposed based on a pseudohexagonal spiral structure with a rise of approximately the width of two dimers per 360 degree turn. This spiral structure could be consistent with the dimensions of amyloid fibrils as determined by electron microscopy.

Third-generation synchrotron x-ray diffraction of 6-μm crystal of raite, ≈Na3Mn3Ti0.25Si8O20(OH)2⋅10H2O, opens up new chemistry and physics of low-temperature minerals

The crystal structure of raite was solved and refined from data collected at Beamline Insertion Device 13 at the European Synchrotron Radiation Facility, using a 3 × 3 × 65 μm single crystal. The refined lattice constants of the monoclinic unit cell are a = 15.1(1) Å; b = 17.6(1) Å; c = 5.290(4) Å; β = 100.5(2)°; space group C2/m. The structure, including all reflections, refined to a final R = 0.07. Raite occurs in hyperalkaline rocks from the Kola peninsula, Russia. The structure consists of alternating layers of a hexagonal chicken-wire pattern of 6-membered SiO4 rings. Tetrahedral apices of a chain of Si six-rings, parallel to the c-axis, alternate in pointing up and down. Two six-ring Si layers are connected by edge-sharing octahedral bands of Na+ and Mn3+ also parallel to c. The band consists of the alternation of finite Mn–Mn and Na–Mn–Na chains. As a consequence of the misfit between octahedral and tetrahedral elements, regions of the Si–O layers are arched and form one-dimensional channels bounded by 12 Si tetrahedra and 2 Na octahedra. The channels along the short c-axis in raite are filled by isolated Na(OH,H2O)6 octahedra. The distorted octahedrally coordinated Ti4+ also resides in the channel and provides the weak linkage of these isolated Na octahedra and the mixed octahedral tetrahedral framework. Raite is structurally related to intersilite, palygorskite, sepiolite, and amphibole.

Lipidic cubic phases: A novel concept for the crystallization of membrane proteins

Understanding the mechanisms of action of membrane proteins requires the elucidation of their structures to high resolution. The critical step in accomplishing this by x-ray crystallography is the routine availability of well-ordered three-dimensional crystals. We have devised a novel, rational approach to meet this goal using quasisolid lipidic cubic phases. This membrane system, consisting of lipid, water, and protein in appropriate proportions, forms a structured, transparent, and complex three-dimensional lipidic array, which is pervaded by an intercommunicating aqueous channel system. Such matrices provide nucleation sites (“seeding”) and support growth by lateral diffusion of protein molecules in the membrane (“feeding”). Bacteriorhodopsin crystals were obtained from bicontinuous cubic phases, but not from micellar systems, implying a critical role of the continuity of the diffusion space (the bilayer) on crystal growth. Hexagonal bacteriorhodopsin crystals diffracted to 3.7 Å resolution, with a space group P63, and unit cell dimensions of a = b = 62 Å, c = 108 Å; α = β = 90° and γ = 120°.

An amyloid-forming peptide from the yeast prion Sup35 reveals a dehydrated β-sheet structure for amyloid

X-ray diffraction and other biophysical tools reveal features of the atomic structure of an amyloid-like crystal. Sup35, a prion-like protein in yeast, forms fibrillar amyloid assemblies intrinsic to its prion function. We have identified a polar peptide from the N-terminal prion-determining domain of Sup35 that exhibits the amyloid properties of full-length Sup35, including cooperative kinetics of aggregation, fibril formation, binding of the dye Congo red, and the characteristic cross-β x-ray diffraction pattern. Microcrystals of this peptide also share the principal properties of the fibrillar amyloid, including a highly stable, β-sheet-rich structure and the binding of Congo red. The x-ray powder pattern of the microcrystals, extending to 0.9-Å resolution, yields the unit cell dimensions of the well-ordered structure. These dimensions restrict possible atomic models of this amyloid-like structure and demonstrate that it forms packed, parallel-stranded β-sheets. The unusually high density of the crystals shows that the packed β-sheets are dehydrated, despite the polar character of the side chains. These results suggest that amyloid is a highly intermolecularly bonded, dehydrated array of densely packed β-sheets. This dry β-sheet could form as Sup35 partially unfolds to expose the peptide, permitting it to hydrogen-bond to the same peptide of other Sup35 molecules. The implication is that amyloid-forming units may be short segments of proteins, exposed for interactions by partial unfolding.

Photosystem II single crystals studied by EPR spectroscopy at 94 GHz: The tyrosine radical Y\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{D}^{{\bullet}}}}\end{equation*}\end{document}

Electron paramagnetic resonance (EPR) spectroscopy at 94 GHz is used to study the dark-stable tyrosine radical Y\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{D}^{{\bullet}}}}\end{equation*}\end{document} in single crystals of photosystem II core complexes (cc) isolated from the thermophilic cyanobacterium Synechococcus elongatus. These complexes contain at least 17 subunits, including the water-oxidizing complex (WOC), and 32 chlorophyll a molecules/PS II; they are active in light-induced electron transfer and water oxidation. The crystals belong to the orthorhombic space group P212121, with four PS II dimers per unit cell. High-frequency EPR is used for enhancing the sensitivity of experiments performed on small single crystals as well as for increasing the spectral resolution of the g tensor components and of the different crystal sites. Magnitude and orientation of the g tensor of Y\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{D}^{{\bullet}}}}\end{equation*}\end{document} and related information on several proton hyperfine tensors are deduced from analysis of angular-dependent EPR spectra. The precise orientation of tyrosine Y\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{D}^{{\bullet}}}}\end{equation*}\end{document} in PS II is obtained as a first step in the EPR characterization of paramagnetic species in these single crystals.

Theoretical prediction of a crystal structure.

The diffusion equation method of global minimization is applied to compute the crystal structure of S6, with no a priori knowledge about the system. The experimental lattice parameters and positions and orientations of the molecules in the unit cell are predicted correctly.

Preferential self-association of basic fibroblast growth factor is stabilized by heparin during receptor dimerization and activation.

Central to signaling by fibroblast growth factors (FGFs) is the oligomeric interaction of the growth factor and its high-affinity cell surface receptor, which is mediated by heparin-like polysaccharides. It has been proposed that the binding of heparin-like polysaccharides to FGF induces a conformational change in FGF, resulting in the formation of FGF dimers or oligomers, and this biologically active form is 'presented' to the FGF receptor for signal transduction. In this study, we show that monomeric basic FGF (FGF-2) preferentially self-associates and forms FGF-2 dimers and higher-order oligomers. As a consequence, FGF-2 monomers are oriented for binding to heparin-like polysaccharides. We also show that heparin-like polysaccharides can readily bind to self-associated FGF-2 without causing a conformational change in FGF-2 or disrupting the FGF-2 self-association, but that the bound polysaccharides only additionally stabilize the FGF-2 self-association. The preferential self-association corresponds to FGF-2 translations along two of the unit cell axes of the FGF-2 crystal structures. These two axes represent the two possible heparin binding directions, whereas the receptor binding sites are oriented along the third axis. Thus, we propose that preferential FGF-2 self-association, further stabilized by heparin, like "beads on a string," mediates FGF-2-induced receptor dimerization and activation. The observed FGF-2 self-association, modulated by heparin, not only provides a mechanism of growth factor activation but also represents a regulatory mechanism governing FGF-2 biological activity.

Crystallization and preliminary x-ray studies of NADPH-cytochrome P450 reductase.

NADPH-cytochrome P450 reductase (CPR; NADPH:ferrihemoprotein reductase, EC 1.6.2.4) catalyzes the transfer of electrons to all known microsomal cytochromes P450. CPR is unique in that it is one of only two mammalian enzymes known to contain both flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), the other being the various isoforms of nitric oxide synthase. Similarities in amino acid sequence and in functional domain arrangement with other key flavoproteins, including nitric oxide synthase, make CPR an excellent prototype for studies of interactions between two flavin cofactors. We have obtained diffraction-quality crystals of rat liver CPR, expressed in Escherichia coli and solubilized by limited proteolysis with trypsin. The crystals were grown in Hepes buffer (pH 7.0), containing polyethylene glycol 4500 and NaCl. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions a = 103.3 A, b = 116.1 A, and c = 120.4 A. If we assume that there are two molecules of the 72-kDa CPR polypeptide per asymmetric unit, the calculated value of Vm is 2.54 A3/Da.

Assembly of a catalytic unit for RNA microhelix aminoacylation using nonspecific RNA binding domains

An assembly of a catalytic unit for aminoacylation of an RNA microhelix is demonstrated here. This assembly may recapitulate a step in the historical development of tRNA synthetases. The class-defining domain of a tRNA synthetase is closely related to the primordial enzyme that catalyzed synthesis of aminoacyl adenylate. RNA binding elements are imagined to have been added so that early RNA substrates could be docked proximal to the activated amino acid. RNA microhelices that recapitulate the acceptor stem of modern tRNAs are potential examples of early substrates. In this work, we examined a fragment of Escherichia coli alanyl-tRNA synthetase, which catalyzes aminoacyl adenylate formation but is virtually inactive for catalysis of RNA microhelix aminoacylation. Fusion to the fragment of either of two unrelated nonspecific RNA binding domains activated microhelix aminoacylation. Although the fusion proteins lacked the RNA sequence specificity of the natural enzyme, their activity was within 1–2 kcal⋅mol−1 of a truncated alanyl-tRNA synthetase that has aminoacylation activity sufficient to sustain cell growth. These results show that, starting with an activity for adenylate synthesis, barriers are relatively low for building catalytic units for aminoacylation of RNA helices.

Lack of cell cycle regulation of telomerase activity in human cells

Conflicting reports have appeared concerning the cell cycle regulation of telomerase activity and its possible repression during quiescence and cell differentiation. We have reexamined these issues in an attempt to uncover the basis for the discrepancies. Variations in extracted telomerase activity during the cell cycle are not observed in cells sorted on the basis of DNA content. Variations are observed in cells synchronized using some biochemical cell cycle inhibitors, but only with those agents where cellular toxicity is evident. A progressive decline in telomerase activity is observed in cells whose growth rate is reduced from seven to eight population doublings per week to one to two doublings per week. Telomerase is largely absent in cells that truly exit the cell cycle and do not divide over the 7-day period. Although it is not necessary for all cell types to regulate telomerase in the same way, we conclude that in the immortal cultured cell lines examined, extracted telomerase activity does not change significantly during progression through the stages of the cell cycle. Telomerase activity generally correlates with growth rate and is repressed in cells that exit the cell cycle and become quiescent.

Telomeres in the mouse have large inter-chromosomal variations in the number of T2AG3 repeats

The ultra-long telomeres that have been observed in mice are not in accordance with the concept that critical telomere shortening is related to aging and immortalization. Here, we have used quantitative fluorescence in situ hybridization to estimate (T2AG3)n lengths of individual telomeres in various mouse strains. Telomere lengths were very heterogeneous, but specific chromosomes of bone marrow cells and skin fibroblasts from individual mice had similar telomere lengths. We estimate that the shortest telomeres are around 10 kb in length, indicating that each mouse cell has a few telomeres with (T2AG3)n lengths within the range of human telomeres. These short telomeres may be critical in limiting the replicative potential of murine cells.

An autonomous folding unit mediates the assembly of two-stranded coiled coils

Subunit oligomerization of many proteins is mediated by coiled-coil domains. Although the basic features contributing to the thermodynamic stability of coiled coils are well understood, the mechanistic details of their assembly have not yet been dissected. Here we report a 13-residue sequence pattern that occurs with limited sequence variations in many two-stranded coiled coils and that is absolutely required for the assembly of the Dictyostelium discoideum actin-bundling protein cortexillin I and the yeast transcriptional activator GCN4. The functional relationship between coiled-coil “trigger” sequences was manifested by replacing the intrinsic trigger motif of GCN4 with the related sequence from cortexillin I. We demonstrate that these trigger sequences represent autonomous helical folding units that, in contrast to arbitrarily chosen heptad repeats, can mediate coiled-coil formation. Aside from being of general interest for protein folding, trigger motifs should be of particular importance in the protein de novo design.

Comprehensive Identification of Cell Cycle–regulated Genes of the Yeast Saccharomyces cerevisiae by Microarray HybridizationD⃞

We sought to create a comprehensive catalog of yeast genes whose transcript levels vary periodically within the cell cycle. To this end, we used DNA microarrays and samples from yeast cultures synchronized by three independent methods: α factor arrest, elutriation, and arrest of a cdc15 temperature-sensitive mutant. Using periodicity and correlation algorithms, we identified 800 genes that meet an objective minimum criterion for cell cycle regulation. In separate experiments, designed to examine the effects of inducing either the G1 cyclin Cln3p or the B-type cyclin Clb2p, we found that the mRNA levels of more than half of these 800 genes respond to one or both of these cyclins. Furthermore, we analyzed our set of cell cycle–regulated genes for known and new promoter elements and show that several known elements (or variations thereof) contain information predictive of cell cycle regulation. A full description and complete data sets are available at http://cellcycle-www.stanford.edu