Alpha-helical, but not beta-sheet, propensity of proline is determined by peptide environment.

Proline is established as a potent breaker of both alpha-helical and beta-sheet structures in soluble (globular) proteins. Thus, the frequent occurrence of the Pro residue in the putative transmembrane helices of integral membrane proteins, particularly transport proteins, presents a structural dilemma. We propose that this phenomenon results from the fact that the structural propensity of a given amino acid may be altered to conform to changes imposed by molecular environment. To test this hypothesis on proline, we synthesized model peptides of generic sequence H2N-(Ser-LyS)2-Ala- Leu-Z-Ala-Leu-Z-Trp-Ala-Leu-Z-(Lys-Ser)3-OH (Z = Ala and/or Pro). Peptide conformations were analyzed by circular dichroism spectroscopy in aqueous buffer, SDS, lysophosphatidylglycerol micelles, and organic solvents (methanol, trifluoroethanol, and 2-propanol). The helical propensity of Pro was found to be greatly enhanced in the membrane-mimetic environments of both lipid micelles and organic solvents. Proline was found to stabilize the alpha-helical conformation relative to Ala at elevated temperatures in 2-propanol, an observation that argues against the doctrine that Pro is the most potent alpha-helix breaker as established in aqueous media. Parallel studies in deoxycholate micelles of the temperature-induced conformational transitions of the single-spanning membrane bacteriophage IKe major coat protein, in which the Pro-containing wild type was compared with Pro30 --> Ala mutant, Pro was found to protect the helix, but disrupt the beta-sheet structure as effectively as it does to model peptides in water. The intrinsic capacity of Pro to disrupt beta-sheets was further reflected in a survey of porins where Pro was found to be selectively excluded from the core of membrane-spanning beta-sheet barrels. The overall data provide a rationale for predicting and understanding the structural consequences when Pro occurs in the context of a membrane.

Structure and conformational changes of DNA topoisomerase II visualized by electron microscopy.

Type II DNA topoisomerases, which create a transient gate in duplex DNA and transfer a second duplex DNA through this gate, are essential for topological transformations of DNA in prokaryotic and eukaryotic cells and are of interest not only from a mechanistic perspective but also because they are targets of agents for anticancer and antimicrobial chemotherapy. Here we describe the structure of the molecule of human topoisomerase II [DNA topoisomerase (ATP-hydrolyzing), EC 5.99.1.3] as seen by scanning transmission electron microscopy. A globular approximately 90-angstrom diameter core is connected by linkers to two approximately 50-angstrom domains, which were shown by comparison with genetically truncated Saccharomyces cerevisiae topoisomerase II to contain the N-terminal region of the approximately 170-kDa subunits and that are seen in different orientations. When the ATP-binding site is occupied by a nonhydrolyzable ATP analog, a quite different structure is seen that results from a major conformational change and consists of two domains approximately 90 angstrom and approximately 60 angstrom in diameter connected by a linker, and in which the N-terminal domains have interacted. About two-thirds of the molecules show an approximately 25 A tunnel in the apical part of the large domain, and the remainder contain an internal cavity approximately 30 A wide in the large domain close to the linker region. We propose that structural rearrangements lead to this displacement of an internal tunnel. The tunnel is likely to represent the channel through which one DNA duplex, after capture in the clamp formed by the N-terminal domains, is transferred across the interface between the enzyme's subunits. These images are consistent with biochemical observations and provide a structural basis for understanding the reaction of topoisomerase II.

Human plectin: organization of the gene, sequence analysis, and chromosome localization (8q24).

Plectin, a 500-kDa intermediate filament binding protein, has been proposed to provide mechanical strength to cells and tissues by acting as a cross-linking element of the cytoskeleton. To set the basis for future studies on gene regulation, tissue-specific expression, and pathological conditions involving this protein, we have cloned the human plectin gene, determined its coding sequence, and established its genomic organization. The coding sequence contains 32 exons that extend over 32 kb of the human genome. Most of the introns reside within a region encoding the globular N-terminal domain of the molecule, whereas the entire central rod domain and the entire C-terminal globular domain were found to be encoded by single exons of remarkable length, >3 kb and >6 kb, respectively. Overall, the organization of the human plectin gene was strikingly similar to that of human bullous pemphigoid antigen 1 (BPAG1), confirming that both proteins belong to the same gene family. Comparison of the deduced protein sequences for human and rat plectin revealed that they were 93% identical. By using fluorescence in situ hybridization, we have mapped the plectin gene to the long arm of chromosome 8 within the telomeric region. This gene locus (8q24) has previously been implicated in the human blistering skin disease epidermolysis bullosa simplex Ogna. Detailed knowledge of the structure of the plectin gene and its chromosome localization will aid in the elucidation of whether this or any other pathological conditions are linked to alterations in the plectin gene.

The neck region of the myosin motor domain acts as a lever arm to generate movement.

The myosin head consists of a globular catalytic domain that binds actin and hydrolyzes ATP and a neck domain that consists of essential and regulatory light chains bound to a long alpha-helical portion of the heavy chain. The swinging neck-level model assumes that a swinging motion of the neck relative to the catalytic domain is the origin of movement. This model predicts that the step size, and consequently the sliding velocity, are linearly related to the length of the neck. We have tested this point by characterizing a series of mutant Dictyostelium myosins that have different neck lengths. The 2xELCBS mutant has an extra binding site for essential light chain. The delta RLCBS mutant myosin has an internal deletion that removes the regulatory light chain binding site. The delta BLCBS mutant lacks both light chain binding sites. Wild-type myosin and these mutant myosins were subjected to the sliding filament in vitro motility assay. As expected, mutants with shorter necks move slower than wild-type myosin in vitro. Most significantly, a mutant with a longer neck moves faster than the wild type, and the sliding velocities of these myosins are linearly related to the neck length, as predicted by the swinging neck-lever model. A simple extrapolation to zero speed predicts that the fulcrum point is in the vicinity of the SH1-SH2 region in the catalytic domain.

Enhanced conformational sampling in Monte Carlo simulations of proteins: application to a constrained peptide.

A Monte Carlo simulation method for globular proteins, called extended-scaled-collective-variable (ESCV) Monte Carlo, is proposed. This method combines two Monte Carlo algorithms known as entropy-sampling and scaled-collective-variable algorithms. Entropy-sampling Monte Carlo is able to sample a large configurational space even in a disordered system that has a large number of potential barriers. In contrast, scaled-collective-variable Monte Carlo provides an efficient sampling for a system whose dynamics is highly cooperative. Because a globular protein is a disordered system whose dynamics is characterized by collective motions, a combination of these two algorithms could provide an optimal Monte Carlo simulation for a globular protein. As a test case, we have carried out an ESCV Monte Carlo simulation for a cell adhesive Arg-Gly-Asp-containing peptide, Lys-Arg-Cys-Arg-Gly-Asp-Cys-Met-Asp, and determined the conformational distribution at 300 K. The peptide contains a disulfide bridge between the two cysteine residues. This bond mimics the strong geometrical constraints that result from a protein's globular nature and give rise to highly cooperative dynamics. Computation results show that the ESCV Monte Carlo was not trapped at any local minimum and that the canonical distribution was correctly determined.

Significance of bound water to local chain conformations in protein crystals.

We examine how the polypeptide chain in protein crystal structures exploits the multivalent hydrogen-bonding potential of bound water molecules. This shows that multiple interactions with a single water molecule tend to occur locally along the chain. A distinctive internal-coordinate representation of the local water-binding segments reveals several consensus conformations. The fractional water occupancy of each was found by comparison of the total number of conformations in the database regardless of the presence or absence of bound water. The water molecule appears particularly frequently in type II beta-turn geometries and an N-terminal helix feature. This work constitutes a first step into assessing not only the generality but also the significance of specific water binding in globular proteins.

Submillisecond folding of monomeric lambda repressor.

The folding kinetics of a truncated form of the N-terminal domain of phage lambda repressor [lambda 6-85] has been investigated by using the technique of dynamic NMR. lambda 6-85 has been shown previously to fold in a purely two-state fashion. This allows the determination of folding and unfolding rates from simulation of the exchange-broadened aromatic resonances of Tyr-22. The folding kinetics were determined over a range of 1.35 to 3.14 M urea. The urea dependence of both folding and unfolding rate constants is exponential, suggesting that the rate-determining step is invariant at the urea concentrations studied. The folding and unfolding rates extrapolated to 0 M urea at 37 degrees C are 3600 +/- 400 s-1 and 27 +/- 6 s-1, respectively. The observed lambda 6-85 folding rate constant exceeds that of other fast-folding globular proteins by a factor of 14-54. The urea dependence of the folding and unfolding rate constants suggests that the transition state of the rate-determining step is considerably more exposed to solvent than previously studied protein-folding transition states. The surprising rapidity of lambda 6-85 folding and unfolding may be the consequence of its all-helical secondary structure. These kinetic results clearly demonstrate that all of the fundamental events of protein folding can occur on the submillisecond time scale.

Pex1, a pollen-specific gene with an extensin-like domain.

We report here the identification of a pollen-specific gene from Zea mays that contains multiple Ser-(Pro)n repeats, the motif found in the cell wall-associated extensins. Sequence analysis reveals that the encoded protein has a putative globular domain at the N terminus and an extensin-like domain at the C terminus. The Pex1 (pollen extensin-like) gene is expressed exclusively in pollen, not in vegetative or female tissues, and is not induced in leaves upon wounding. We propose that the encoded protein may have a role in reproduction, either as a structural element deposited in the pollen tube wall during its rapid growth or as a sexual recognition molecule that interacts with partner molecules in the pistil.

Avaliação do processamento por atrito linear em chapas da liga de titânio Ti-6Al-4V.

Esta tese tem por objetivo a aplicação do processamento por atrito linear na liga de titânio Ti-6Al-4V. Derivado da solda por atrito linear, é um processo recente desenvolvido na década de 90 para união de alumínio. Sua aplicação em outros tipos de materiais como aços e ligas de alto desempenho, em especial o titânio, tem interessado a industria. A metodologia utilizada nesta tese para avaliar o processamento por atrito linear, consistiu na execução de ensaios mecânicos de tração em condições mistas em chapas da liga de titânio Ti-6Al-4V. A máquina utilizada para o processamento das chapas foi um centro de usinagem CNC convencional, adaptado com dispositivos especiais. Além dos ensaios de tração em condições mistas, foram executadas medições de microdurezas nas regiões atingidas pelo processo, avaliação das microestruturas resultantes e medições de tensão residual para uma caracterização mais ampla do processo. As microestruturas na região processada são caracterizadas por uma estrutura totalmente transformada. As temperaturas de pico na região processada excederam a temperatura -transus durante o processamento e a transformação da fase + ocorreu durante a fase de resfriamento. A transformação da fase para resultou na formação de agulhas de fase nos contornos e pelo interior dos grãos da fase . Pequenas regiões com estrutura equiaxial de grãos ( globular) foram observados na zona de processamento. A abordagem dos resultados quantitativos foi feita de forma estatística, visando identificar os parâmetros de maior interação com os resultados observados. Foi identificado nesta tese que a rotação da ferramenta apresentou a maior influência nos resultados de tensão residual, microdureza e tensão de escoamento. Uma importante contribuição à modelagem da tensão de escoamento para materiais anisotrópicos é proposta, baseado em um critério de escoamento ortotrópico. Equações complementares baseadas nos testes mistos de tração e cisalhamento são propostas para modificar o modelo ortotrópico. O intuito deste modelo é indicar em que condições o material tem seu regime de escoamento atingido, podendo servir de base para simulações práticas de peças em condições similares.

New galactic star clusters discovered in the VVV survey. Candidates projected on the inner disk and bulge

Context. VISTA Variables in the Vía Láctea (VVV) is one of six ESO Public Surveys using the 4 meter Visible and Infrared Survey Telescope for Astronomy (VISTA). The VVV survey covers the Milky Way bulge and an adjacent section of the disk, and one of the principal objectives is to search for new star clusters within previously unreachable obscured parts of the Galaxy. Aims. The primary motivation behind this work is to discover and analyze obscured star clusters in the direction of the inner Galactic disk and bulge. Methods. Regions of the inner disk and bulge covered by the VVV survey were visually inspected using composite JHKS color images to select new cluster candidates on the basis of apparent overdensities. DR1, DR2, CASU, and point spread function photometry of 10 × 10 arcmin fields centered on each candidate cluster were used to construct color–magnitude and color–color diagrams. Follow-up spectroscopy of the brightest members of several cluster candidates was obtained in order to clarify their nature. Results. We report the discovery of 58 new infrared cluster candidates. Fundamental parameters such as age, distance, and metallicity were determined for 20 of the most populous clusters.

The Gaia-ESO Survey: The analysis of high-resolution UVES spectra of FGK-type stars

Context. The ongoing Gaia-ESO Public Spectroscopic Survey is using FLAMES at the VLT to obtain high-quality medium-resolution Giraffe spectra for about 105 stars and high-resolution UVES spectra for about 5000 stars. With UVES, the Survey has already observed 1447 FGK-type stars. Aims. These UVES spectra are analyzed in parallel by several state-of-the-art methodologies. Our aim is to present how these analyses were implemented, to discuss their results, and to describe how a final recommended parameter scale is defined. We also discuss the precision (method-to-method dispersion) and accuracy (biases with respect to the reference values) of the final parameters. These results are part of the Gaia-ESO second internal release and will be part of its first public release of advanced data products. Methods. The final parameter scale is tied to the scale defined by the Gaia benchmark stars, a set of stars with fundamental atmospheric parameters. In addition, a set of open and globular clusters is used to evaluate the physical soundness of the results. Each of the implemented methodologies is judged against the benchmark stars to define weights in three different regions of the parameter space. The final recommended results are the weighted medians of those from the individual methods. Results. The recommended results successfully reproduce the atmospheric parameters of the benchmark stars and the expected Teff-log  g relation of the calibrating clusters. Atmospheric parameters and abundances have been determined for 1301 FGK-type stars observed with UVES. The median of the method-to-method dispersion of the atmospheric parameters is 55 K for Teff, 0.13 dex for log  g and 0.07 dex for [Fe/H]. Systematic biases are estimated to be between 50−100 K for Teff, 0.10−0.25 dex for log  g and 0.05−0.10 dex for [Fe/H]. Abundances for 24 elements were derived: C, N, O, Na, Mg, Al, Si, Ca, Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Y, Zr, Mo, Ba, Nd, and Eu. The typical method-to-method dispersion of the abundances varies between 0.10 and 0.20 dex. Conclusions. The Gaia-ESO sample of high-resolution spectra of FGK-type stars will be among the largest of its kind analyzed in a homogeneous way. The extensive list of elemental abundances derived in these stars will enable significant advances in the areas of stellar evolution and Milky Way formation and evolution.

Three-Dimensional Structure and Disposition of the Air Conducting and Gas Exchange Conduits of the Avian Lung: The Domestic Duck (Cairina moschata)

The anatomy of the domestic duck lung was studied macroscopically, by casting and by light, transmission, and scanning electron microscopy. The lung had four categories of secondary bronchi (SB), namely, the medioventral (MV, 4-5), laterodorsal (LD, 6-10), lateroventral (LV, 2-4), and posterior secondary bronchi (PO, 36-44). The neopulmonic parabronchi formed an intricate feltwork on the ventral third of the lung and inosculated those from the other SB. The lung parenchyma was organized into cylindrical parabronchi separated by thin septa containing blood vessels. Atria were shallow and well-fortified by epithelial ridges reinforced by smooth muscle bundles and gave rise to 2-6 elongate infundibulae. Air capillaries arose either directly from the atria or from infundibulae and were tubular or globular in shape with thin interconnecting branches. The newly described spatial disposition of the conducting air conduits closely resembles that of the chicken. This remarkable similarity between the categories, numbers, and 3D arrangement of the SB in the duck and chicken points to a convergence in function-oriented design. To illuminate airflow dynamics in the avian lung, precise directions of airflow in the various categories of SB and parabronchi need to be characterized.

(Table 1) Crystal sizes of measured samples

Due to experimental difficulties grain size distributions of gas hydrate crystallites are largely unknown in natural samples. For the first time, we were able to determine grain size distributions of six natural gas hydrates for samples retrieved from the Gulf of Mexico and from Hydrate Ridge offshore Oregon from varying depths. High-energy synchrotron radiation provides high photon fluxes as well as high penetration depth and thus allows for investigation of bulk sediment samples. The gas hydrate crystallites appear to be (log-) normally distributed in the natural samples and to be of roughly globular shape. The mean grain sizes are in the range from 300-600 µm with a tendency for bigger grains to occur in greater depth, possibly indicating a difference in the formation age. Laboratory produced methane hydrate, starting from ice and aged for 3 weeks, shows half a log-normal curve with a mean value of ~40 µm. This one order-of-magnitude smaller grain sizes suggests that care must be taken when transposing grain-size sensitive (petro-)physical data from laboratory-made gas hydrates to natural settings.

Organic carbon and amino acid concentration of Karelian shungite rocks from the lake Onego area, Russia

The study of amino acids in the Precambrian shungite rocks of Karelia showed that their contents vary within 25-89 µg/g depending on proportions between shungite and mineral components. It was established that the amino acids exhibit an excess of L-enantiomers. In the shungite rocks, they form organomineral complexes with silica and aluminosilicates, being built in the globular structure of shungite matter. There are several sources of amino acids in shungites: secondary synthesis, microbial pollution, and original amino acids of organic matter in shungite rocks.

(Table 1) Distribution of Lower Cretaceous calcareous nannofossils at DSDP Hole 93-603B

Upper Berriasian to lower Aptian calcareous nannofossil assemblages have been studied from a siliciclastic deep-sea fan complex and a subjacent limestone sequence drilled beneath the lower continental rise in the western North American Basin, 270 miles (435 km) off Cape Hatteras, North Carolina (USA). Sharp lithologic facies changes and reworking by turbidites complicate the biostratigraphic interpretation, but provide an excellent opportunity to better distinguish "nearshore" from open-ocean nannofossil species, and to investigate the introduction of neritic taxa into the deep-see environment, a phenomenon that appears to have been widespread within the circum-North Atlantic during Neocomian times. Well-preserved assemblages in dark, carbonaceous claystones were probably displaced from the oxygen minimum zone along the upper slope or outer shelf. Neritic, continental margin species prevalent in this facies include the holococcolith Zebrashapka vanhintei n. gen., n. sp., Lithraphidites alatus magnus n. spp., Pickelhaube furtiva n. gen., and a host of nannoconids and micrantholiths. A qualitative evaluation of widely used guide fossils suggests that the triad of proposed markers for the base of Roth's Zone NC3 make their first appearances in the following (ascending) order: Diadorhombus rectus, TUbodiscus verenae, Calcicalathina oblongata. Of these, we chose the nominative species for the zone, T. verenae, to mark its base and to approximate the Berriasian/Valangian boundary. Cyclagelosphaera deflandrei is strongly affected by diagenesis and is therefore not a reliable index species for the base of Zone NC4 near the Valanginian/Hauterivian boundary (the last occurrence of T. verenae is also not suitable there). In addition, Lithraphidites bollii, a form apparently confined to the low latitudes of the Tethyan region, was absent at the more temperate Site 603 and not available as a subzonal marker for the upper Hautervian-lower Barremian (mid-NC4 and mid-NC5, respectively). Cruciellipsis cuvillieri, however, provides a reliable datum just below the Hauterivian/Barremian boundary (base of NC5), despite the potential for reworking in this section. Nannoconids tend to be reworked in this section, and do not provide trustworthy forms to mark the Barremian/Aptian boundary (base of NC6). Hayesites irregularis n. comb, probably does provide a useful first appearance datum within the lower Aptian, if it is not confused with a more birefringent and globular form, Rucinolithus terebrodentarius n. sp. Rhagodiscus angustus is mimicked by a similar form (Zeughrabdotusl pseudoangustus n. sp.), which apparently ranges down to the Hauterivian, thus Lithastrinus floralis provides a more useful first appearance datum for the base of the middle-upper Aptian Rhagodiscus angustus Zone (NC7). Aside from the new taxa mentioned above, the following are also described: Cretarhabdusl delicatus n. sp. and Cyclagelosphaera jiangii n. sp.