Molecular Architecture of Plant Thylakoids under Physiological and Light Stress Conditions: A Study of Lipid–Light-Harvesting Complex II Model Membranes

In this study, we analyzed multibilayer lipid-protein membranes composed of the photosynthetic light-harvesting complex II (LHCII; isolated from spinach [Spinacia oleracea]) and the plant lipids monogalcatosyldiacylglycerol and digalactosyldiacylglycerol. Two types of pigment-protein complexes were analyzed: those isolated from dark-adapted leaves (LHCII) and those from leaves preilluminated with high-intensity light (LHCII-HL). The LHCII-HL complexes were found to be partially phosphorylated and contained zeaxanthin. The results of the x-ray diffraction, infrared imaging microscopy, confocal laser scanning microscopy, and transmission electron microscopy revealed that lipid-LHCII membranes assemble into planar multibilayers, in contrast with the lipid-LHCII-HL membranes, which form less ordered structures. In both systems, the protein formed supramolecular structures. In the case of LHCII-HL, these structures spanned the multibilayer membranes and were perpendicular to the membrane plane, whereas in LHCII, the structures were lamellar and within the plane of the membranes. Lamellar aggregates of LHCII-HL have been shown, by fluorescence lifetime imaging microscopy, to be particularly active in excitation energy quenching. Both types of structures were stabilized by intermolecular hydrogen bonds. We conclude that the formation of trans-layer, rivet-like structures of LHCII is an important determinant underlying the spontaneous formation and stabilization of the thylakoid grana structures, since the lamellar aggregates are well suited to dissipate excess energy upon overexcitation.     

"Open" structures of MurD: domain movements and structural similarities with folylpolyglutamate synthetase

UDP-N-acetylmuramoyl-l-alanine:d-glutamate (MurD) ligase catalyses the addition of d-glutamate to the nucleotide precursor UDP-N-acetylmuramoyl-l-alanine (UMA). The crystal structures of Escherichia coli in the substrate-free form and MurD complexed with UMA have been determined at 2.4 A and 1.88 A resolution, respectively. The MurD structure comprises three domains each of a topology reminiscent of nucleotide-binding folds. In the two structures the C-terminal domain undergoes a large rigid-body rotation away from the N-terminal and central domains. These two "open" structures were compared with the four published "closed" structures of MurD. In addition the comparison reveals which regions are affected by the binding of UMA, ATP and d-Glu. Also we compare and discuss two structurally characterized enzymes which belong to the same ligase superfamily: MurD and folylpolyglutamate synthetase (FGS). The analysis allows the identification of key residues involved in the reaction mechanism of FGS. The determination of the two "open" conformation structures represents a new step towards the complete elucidation of the enzymatic mechanism of the MurD ligase.     

Combinatorics of locally optimal RNA secondary structures

It is a classical result of Stein and Waterman that the asymptotic number of RNA secondary structures is $1.104366 \cdot n^{-3/2} \cdot 2.618034^n$ . Motivated by the kinetics of RNA secondary structure formation, we are interested in determining the asymptotic number of secondary structures that are locally optimal, with respect to a particular energy model. In the Nussinov energy model, where each base pair contributes $-1$ towards the energy of the structure, locally optimal structures are exactly the saturated structures, for which we have previously shown that asymptotically, there are $1.07427\cdot n^{-3/2} \cdot 2.35467^n$ many saturated structures for a sequence of length $n$ . In this paper, we consider the base stacking energy model, a mild variant of the Nussinov model, where each stacked base pair contributes $-1$ toward the energy of the structure. Locally optimal structures with respect to the base stacking energy model are exactly those secondary structures, whose stems cannot be extended. Such structures were first considered by Evers and Giegerich, who described a dynamic programming algorithm to enumerate all locally optimal structures. In this paper, we apply methods from enumerative combinatorics to compute the asymptotic number of such structures. Additionally, we consider analogous combinatorial problems for secondary structures with annotated single-stranded, stacking nucleotides (dangles).     

Crystal structures of complexes of PcrA DNA helicase with a DNA substrate indicate an inchworm mechanism

We have determined two different structures of PcrA DNA helicase complexed with the same single strand tailed DNA duplex, providing snapshots of different steps on the catalytic pathway. One of the structures is of a complex with a nonhydrolyzable analog of ATP and is thus a "substrate" complex. The other structure contains a bound sulphate ion that sits in a position equivalent to that occupied by the phosphate ion produced after ATP hydrolysis, thereby mimicking a "product" complex. In both complexes, the protein is monomeric. Large and distinct conformational changes occur on binding DNA and the nucleotide cofactor. Taken together, these structures provide evidence against an "active rolling" model for helicase action but are instead consistent with an "inchworm" mechanism.     

Determinants of DNA quadruplex structural type: sequence and potassium binding

There are DNA sequences which adopt the same quadruplex structural type in the presence of sodium as in the presence of sodium and potassium. There are also sequences that appear to have a requirement for the presence of potassium for the adoption of a particular quadruplex structural type. Information about the basis for these potassium effects has been obtained by examining the structures of a set of DNAs with differing numbers of loop residues and different lengths of runs of dG residues in the presence of sodium alone and in the presence of potassium and sodium. On the basis of the results, obtained primarily via solution-state NMR, it appears that very small loops favor parallel stranded quartet structures which do not require the presence of potassium. DNAs with loops of two to four residues and runs of two dG residues can form quadruplex structures of the "edge" or "chair" type in the presence of potassium but not in the presence of sodium alone. When all of the loops contain four residues, a "crossover" or "basket" type structure can be formed in the presence of sodium as well as in the presence of sodium and potassium. Structures with runs of three or four dG residues and with loops from two to four residues can form basket or crossover type structures in the absence of potassium. The presence of a purine in a loop can block both potassium binding and formation of chair type structures. Modeling of the interactions of cations with these quadruplex structures indicates that the potassium ions required for chair type structures interact with a terminal quartet and residues in the adjacent loop.     

Sidedness of membrane structures in Rhodopseudomonas sphaeroides. Electrochemical titration of the spectrum changes of carotenoid in spheroplasts, spheroplast membrane vesicles and chromatophores.

The shift of the carotenoid absorption spectrum induced by illumination and valinomycin-K+ addition was investigated in membrane structures with different characteristics and opposite sidednesses isolated from Rhodopseudomonas sphaeroides. Right-side-out membrane structures were prepared by isotonic lysozyme-EDTA treatment of the cells (spheroplasts) and by hypotonic treatment of spheroplasts (spheroplast membrane vesicles). Inside-out membrane structures ("chromatophores") were obtained by treating spheroplast membrane vesicles by French press or sonication. The membrane structures with either sidedness showed the same light-induced change of the "red shift" type. However, the absorbance change by K+ addition in the presence of valinomycin in the right-side-out membrane structures were opposite to that in the inverted vesicles, "blue shift" in the former and "red shift" in the latter. The carotenoid absorbance change was linear to membrane potential, calculated from the concentration of KCl added, with a reference on the cytoplasmic side, through positive and negative ranges.     

Morphology and development of mirror-image doublets of Stylonychia mytilus

This paper describes the cortical anatomy and development of mirror-image doublets of Stylonychia mytilus, analyzed using the protargol technique. The reversed, or "left-handed" (LH) component of these doublets is a mirror image of the normal or "right handed" (RH) component with regard to the arrangement of cortical structures. The mirror-image patterning is imperfect, however, as the individual ciliary structures of the LH component all are of normal internal asymmetry, and the orientation of membranelles is inverted. Certain structures that would be expected to form near the line of symmetry are absent. During cell division and cortical reorganization, ciliary primordia arise and become arranged in a mirror-image pattern that is more perfect than that exhibited by the mature structures. Deviations from a mirror-image pattern appear at late stages when organelle sets differentiate within ciliary primordia: for example, the membranelle set differentiates within the oral primordium of the LH component in a sequence that is an inversion rather than a mirror image of the corresponding sequence of the RH component. This mixed control of oral development by different cortical "informational systems" accounts for some of the characteristic abnormalities of the mature oral structures of the LH component.     

Crystal structures of the complexes between vancomycin and cell-wall precursor analogs

The crystal structures of three vancomycin complexes with two vancomycin-sensitive cell-wall precursor analogs (diacetyl-Lys-D-Ala-D-Ala and acetyl-D-Ala-D-Ala) and a vancomycin-resistant cell-wall precursor analog (diacetyl-Lys-D-Ala-D-lactate) were determined at atomic resolutions of 1.80 A, 1.07 A, and 0.93 A, respectively. These structures not only reconfirm the "back-to-back" dimerization of vancomycin monomers and the ligand-binding scheme proposed by previous experiments but also show important structural features of strategies for the generation of new glycopeptide antibiotics. These structural features involve a water-mediated antibiotic-ligand interaction and supramolecular structures such as "side-by-side" arranged dimer-to-dimer structures, in addition to ligand-mediated and "face-to-face" arranged dimer-to-dimer structures. In the diacetyl-Lys-D-Ala-D-lactate complex, the interatomic O...O distance between the carbonyl oxygen of the fourth residue of the antibiotic backbone and the ester oxygen of the D-lactate moiety of the ligand is clearly longer than the corresponding N-H...O hydrogen-bonding distance observed in the two other complexes due to electrostatic repulsion. In addition, two neighboring hydrogen bonds are concomitantly lengthened. These observations provide, at least in part, a molecular basis for the reduced antibacterial activity of vancomycin toward vancomycin-resistant bacteria with cell-wall precursors terminating in -D-Ala-D-lactate.     

The Three-Dimensional Solution Structure of the Src Homology Domain-2 of the Growth Factor Receptor-Bound Protein-2

A set of high-resolution three-dimensional solution structures of the Src homology region-2 (SH2) domain of the growth factor receptor-bound protein-2 was determined using heteronuclear NMR spectroscopy. The NMR data used in this study were collected on a stable monomeric protein solution that was free of protein aggregates and proteolysis. The solution structure was determined based upon a total of 1439 constraints, which included 1326 nuclear Overhauser effect distance constraints, 70 hydrogen bond constraints, and 43 dihedral angle constraints. Distance geometry-simulated annealing calculations followed by energy minimization yielded a family of 18 structures that converged to a root-mean-square deviation of 1.09 Å for all backbone atoms and 0.40 Å for the backbone atoms of the central -sheet. The core structure of the SH2 domain contains an antiparallel -sheet flanked by two parallel -helices displaying an overall architecture that is similar to other known SH2 domain structures. This family of NMR structures is compared to the X-ray structure and to another family of NMR solution structures determined under different solution conditions.     

Mucin-producing bronchioloalveolar-cell carcinoma. With special reference to a characteristic structure revealed by phosphotungstic acid-hematoxylin staining

In four cases of bronchioloalveolar-cell carcinoma of the mucin-producing type, the cells were characterized electron microscopically by cored microvilli seen on the free surface of the tumor cells, structures that corresponded well to the phosphotungstic acid-hematoxylin (PTAH) stained structures seen by light microscopy in histologic specimens. The exfoliative cytology specimens contained corresponding PTAH-stained structures on the cell surfaces, namely, a prominent cell membrane with a "beaded" or "peg-like" configuration and, in places, a "feathery" nature, findings quite dissimilar to the surfaces of other well-differentiated adenocarcinoma cells. The cytologic specimens also exhibited characteristic nuclear indentations and tightly connected cell groupings. These results indicate the existence of a type of bronchioloalveolar-cell carcinoma not derived from type II alveolar epithelial cells or Clara cells.     

Models of the three-dimensional structures of echidna, horse, and pigeon lysozymes: Calcium-binding lysozymes and their relationship with α-lactalbumins

Similarities in amino acid sequences, three-dimensional structures, and the exon-intron patterns of their genes have indicated thatc-type lysozymes and-lactalbumins are homologous proteins, i.e., descended by divergent evolution from a common ancestor. Like the-lactalbumins, echidna milk, horse milk, and pigeon eggwhite lysozymes all bind Ca(II). Models of their three-dimensional structures, based on their amino acid sequences and the known crystal structures of domestic hen eggwhite and human lysozymes and baboon and human-lactalbumins, have been built. The several structures have been compared and their relationships discussed.     

Is the nexus necessary for cell-to-cell coupling of smooth muscle?

Summary Electronmicroscopic study of electrically coupled smooth muscles was undertaken to determine the distribution of nexuses in various types of smooth muscle. The study revealed that while nexal structures were commonplace in some types of smooth muscle, they were very rare or absent in others, even though in some cases these cells were only a few nanometers distant from one another. The persistence in thin section of these structures in the main circular muscle of dog intestine after poor fixation, fixation under strain, cell shrinkage, and metabolic damage of various sorts seems to rule out the thesis that they are labile. The absence of nexuses in longitudinal muscle of dog intestine examined both by thin section and by freeze fracture suggests that in this tissue they are absent or very rarein vivo and cannot account for electrical coupling.Nexuses were discernible in thin sections of main circular muscle after a variety of experimental conditions of fixation. Metabolic inhibition orin vitro permanganate fixation partially destroyed nexal contacts. These procedures induced tissue, membrane apposition and an accompanying increase in the number of structures which resemble nexuses at low magnification (nexus-like structures). Nexus-like structures occurred in all smooth muscle fixed byin vitro permanganate associated with apposition of membranes and poor preservation of basement membrane. A technique ofin vitro permanganate fixation was developed which prevented tissue swelling; consequently nexus-like structures were absent in tissues so treated. The suggestion is made that some structures described in the literature as nexuses, following permanganate fixation, may represent nexus-like structures.The balance of evidence suggests that nexuses need not be present for electrical coupling of some smooth muscle cells, in which other types of cell-to-cell contacts must be invoked.     

Dynamics of Vacuoles and H+-Pyrophosphatase Visualized by Monomeric Green Fluorescent Protein in Arabidopsis: Artifactual Bulbs and Native Intravacuolar Spherical Structures

We prepared Arabidopsis thaliana lines expressing a functional green fluorescent protein (GFP)-linked vacuolar H⁺-pyrophosphatase (H+-PPase) under the control of its own promoter to investigate morphological dynamics of vacuoles and tissue-specific expression of H+-PPase. The lines obtained had spherical structures in vacuoles with strong fluorescence, which are referred to as bulbs. Quantitative analyses revealed that the occurrence of the bulbs correlated with the amount of GFP. Next, we prepared a construct of H+-PPase linked with a nondimerizing GFP (mGFP); we detected no bulbs. These results indicate that the membranes adhere face-to-face by antiparallel dimerization of GFP, resulting in the formation of bulbs. In plants expressing H⁺-PPase-mGFP, intravacuolar spherical structures with double membranes, which differed from bulbs in fluorescence intensity and intermembrane spacing, were still observed in peripheral endosperm, pistil epidermis and hypocotyls. Four-dimensional imaging revealed the dynamics of formation, transformation, and disappearance of intravacuolar spherical structures and transvacuolar strands in living cells. Visualization of H⁺-PPase-mGFP revealed intensive accumulation of the enzyme, not only in dividing and elongating cells but also in mesophyll, phloem, and nectary cells, which may have high sugar content. Dynamic morphological changes including transformation of vacuolar structures between transvacuolar strands, intravacuolar sheet-like structures, and intravacuolar spherical structures were also revealed.     

Comparison of protein solution structures refined by molecular dynamics simulation in vacuum,with a generalized Born model,and with explicit water

The inclusion of explicit solvent water in molecular dynamics refinement of NMR structures ought to provide the most physically meaningful accounting for the effects of solvent on structure, but is computationally expensive. In order to evaluate the validity of commonly used vacuum refinements and of recently developed continuum solvent model methods, we have used three different methods to refine a set of NMR solution structures of a medium sized protein, Escherichia coliglutaredoxin 2, from starting structures calculated using the program DYANA. The three different refinement protocols used molecular dynamics simulated annealing with the program AMBER in vacuum (VAC), including a generalized Born (GB) solvent model, and a full calculation including explicit solvent water (WAT). The structures obtained using the three methods of refinements were very similar, a reflection of their generally well-determined nature. However, the structures refined with the generalized Born model were more similar to those from explicit water refinement than those refined in vacuum. Significant improvement was seen in the percentage of backbone dihedral angles in the most favored regions of , space and in hydrogen bond pattern for structures refined with the GB and WAT models, compared with the structures refined in vacuum. The explicit water calculation took an average of 200 h of CPU time per structure on an SGI cluster, compared to 15–90 h for the GB calculation (depending on the parameters used) and 2 h for the vacuum calculation. The generalized Born solvent model proved to be an excellent compromise between the vacuum and explicit water refinements, giving results comparable to those of the explicit water calculation. Some improvement for and angle distribution and hydrogen bond pattern can also be achieved by energy minimizing the vacuum structures with the GB model, which takes a much shorter time than MD simulations with the GB model.     

Fractal geometry and root system structures of heterogeneous plant communities

Above-ground plant growth is widely known in terms of structural diversity. Likewise, the below-ground growth presents a mosaic of heterogeneous structures of differing complexity. In this study, root system structures of heterogeneous plant communities were recorded as integral systems by using the trench profile method. Fractal dimensions of the root images were calculated from image files by the box-counting method. This method allows the structural complexity of such associations to be compared between plant communities, with regard to their potentials for soil resource acquisition and utilization. Distinct and partly significant differences are found (fractal dimension between 1.46±0.09 and 1.71±0.05) in the below-ground structural complexity of plant communities, belonging to different biotope types. The size of the heterogeneous plant community to be examined has an crucial influence on the fractal dimension of the root system structures. The structural heterogeneity becomes particularly evident (fractal dimensions between 1.32 and 1.77) when analysing many small units of a complex root system association. In larger plant communities, a broad variety of below-ground structures is recorded in its entirety, integrating the specific features of single sub-structures. In that way, extreme fractal dimensions are lost and the diversity decreases. Therefore, the analysis of larger units of root system associations provides a general knowledge of the complexity of root system structures for heterogeneous plant communities.     

The difference between protein structures obtained by x-ray analysis and nuclear magnetic resonance

We have analyzed the pairs of protein structures obtained by X-ray diffraction analysis and nuclear magnetic resonance (X-ray and NMR structures) that display no major differences when superimposed on one another (61 pairs). Analyzing atom-to-atom contacts (contact distances 2–8 Å), it has been found that the NMR structures (compared to the X-ray structures) have more contacts at distances below 3.5 Å and above 5.5 Å. In the case of residue-to-residue contacts, the NMR structures have more contacts at distances below 3 Å and between 4.5 and 6.5 Å. At other distances analyzed, the X-ray structures have more contacts. The difference in the numbers of atom-to-atom and residue-to-residue contacts is greater for buried residues inaccessible to water than for surface residues. Another important difference is related to the number of hydrogen bonds in the main chain: this number is greater in the X-ray structures. The coefficient of correlation between the numbers of hydrogen bonds identified in the structures obtained by both methods is only 32%. If a complete set of NMR models of protein structure is considered, the total number of hydrogen bonds proves to be 1.2 times greater than in the X-ray structures, whereas the correlation coefficient increases to only 65%. We have also demon-strated that -helices in the NMR structures are more distorted (compared to the ideal -helix) than those in the X-ray structures.Translated from Molekulyarnaya Biologiya, Vol. 39, No. 1, 2005, pp. 129–138.Original Russian Text Copyright © 2005 by Melnik, Garbuzynskiy, Lobanov, Galzitskaya.     

XPD structure reveals its secrets

The XPD protein is central to our understanding of the relationship between NER deficiencies and human disorders. Three recent papers report the crystal structures of XPD from archaea. Apart from anticipated helicase domains the structures reveal a 4FeS cluster and novel "Arch" domain. The structures help our understanding of genotype-phenotype relationships in the XPD gene.     

Holliday intermediates and reaction by-products in FLP protein-promoted site-specific recombination.

Holliday structures are formed and resolved by FLP protein during site-specific recombination. These structures have been isolated and are visualized in both native and partially denatured states by electron microscopy. No single-strand breaks are found within the junction, indicating that the structure results from a reciprocal exchange of strands. These structures have properties consistent with being reaction intermediates. Double-strand cleavage products and "Y structures" are also detected and appear to be by-products of the reaction. The Y structures are three-armed branched molecules with a covalently closed junction located at the FLP recombination target site. Models are discussed, suggesting that both of these novel structures are made by aberrant cleavages during formation and resolution of the Holliday intermediate.     

The Mechanism of Fractal-Like Structure Formation by Bacterial Populations

Three types of population growth and development of chemotaxic motile bacteria Escherichia coli on semi-solid nutrient media are investigated: a) stable development – circular symmetrical waves; b) bursts; c) fractal-like self-organization. Experimental investigation of the burst formation is presented. The microscopic analysis of growing, fractal-like structures is carried out, and a mechanism for such structure formation is suggested. It is supposed that fractal-like bacterial structures growth is based on the principle of successively forming multiple micro-bursts. A mathematical model has been suggested to reproduce the experimental results. The structures obtained by numerical modeling of population growth in the parameter space substrate concentration - bacterial movement rate reproduce the corresponding experimental structures in the space nutrient concentration in the media – the density of the media.