T-REX: Bare electro-dynamic tape-tether technology experimetn on sounding rocket S520

The project to verify the performance of space tether technology was successfully demonstrated by the launch of the sounding rocket S520 the 25tu. The project is the space demonstration of science and engineering technologies of a bare tape electrodynamic tether (EDT) in the international campaign between Japan, USA, Europe and Australia. Method of "Inverse ORIGAMI (Tape tether folding)" was employed in order to deploy the bare tape EDT in a short period time of the suborbital flight. The deployment of tape tether was tested in a various experimental schemes on ground to show high reliability of tape tether deployment. The rocket was launched on the summer of 2010 and deployed a bare electro-dynamic tape tether with length 132.6 m, which is the world record of the length deployment of tape tether. The verification of tether technology has found a variety kind of science and technology results as the first in the humankind and will lead a large number of applications of space tether technologies

Low initial aspect-ratio direct-drive target designs for shock- or self-ignition in the context of the laser Megajoule

Analysis of low initial aspect ratio direct-drive target designs is carried out by varying the implosion velocity and the fuel mass. Starting from two different spherical targets with a given 300?g-DT mass, optimization of laser pulse and drive power allows to obtain a set of target seeds referenced by their peak implosion velocities and initial aspect ratio (A = 3 and A = 5). Self-ignition is achieved with higher implosion velocity for A = 5-design than for A = 3-design. Then, rescaling is done to extend the set of designs to a huge amount of mass, peak kinetic energies and peak areal densities. Self-ignition kinetic energy threshold Ek is characterized by a dependance of Ek ? v? with ?-values which depart from self-ignition models. Nevertheless, self-ignition energy is seen lower for smaller initial aspect ratio. An analysis of Two-Plasmons Decay threshold and Rayleigh?Taylor instability e-folding is carried out and it is shown that two-plasmon decay threshold is always overpassed for all designs. The hydrodynamic stability analysis is performed by embedded models to deal with linear and non-linear regime. It is found that the A = 5-designs are always at the limit of disruption of the shell.

Investigación y análisis sobre la idoneidad de la práctica del tenis de mesa en la Educación Primaria

Esta investigación surge a raíz de la experiencia profesional del autor, maestro especialista de Educación Física en el C.E.I.P. “Alhambra” de Madrid, cuando de manera progresiva, aprecia que el tenis de mesa puede ser un deporte muy interesante de desarrollar en las sesiones de Educación Física y de promover dentro de los tiempos de recreo. El autor cree que este deporte desarrolla una serie de objetivos motrices, afectivos, cognitivos y sociales que pueden contribuir a la adquisición de las competencias básicas y al desarrollo integral de los alumnos. Es entonces cuando recibe formación sobre el deporte de tenis de mesa y busca los medios necesarios de financiación para que se dote al centro del material necesario. Así la Junta municipal del distrito de Fuencarral-El Pardo instala en el patio del colegio tres mesas de exterior y, con los recursos del colegio y la ayuda de la Asociación de padres y madres (AMPA), se consiguen cinco mesas de interior plegables y todo el material necesario (redes, raquetas, pelotas, etc.). Tras introducir este deporte desde 3º a 6º de Educación Primaria promueve un campeonato en el colegio cuyo índice de participación ronda el 90% del alumnado, estos resultados crean al autor ciertas incertidumbres que son la motivación y punto de partida para realizar esta investigación que analice si la práctica del tenis de mesa puede resultar idónea en la etapa de Educación Primaria. Introducción La legislación actual en materia de educación, Ley Orgánica 2/2006, de 3 de mayo, de Educación (LOE) modificada por la Ley Orgánica 8/2013, de 9 de diciembre, para la mejora de la calidad educativa. (LOMCE), otorga una gran relevancia al deporte en general. "El deporte es una actividad saludable, divertida y formativa que puede tener profundos beneficios no sólo para su salud y su bienestar sino también para el desarrollo personal integral físico, psicológico y psicosocial del niño, además de sobre su desarrollo deportivo" (Pradas, 2009, p. 151), es pues, un momento idóneo para analizar qué deportes se practican en los colegios o por qué se practican unos más que otros. "El tenis de mesa además de ser un deporte para todos, se presenta como un juego atractivo, en donde su práctica resulta muy divertida a cualquier edad, tanto para niños como para adultos, principalmente porque presenta unas reglas de juego simples, no encerrando peligro alguno para la integridad física de sus practicantes durante su juego" (Pradas, 2009, p. 83). Es un deporte que "está abierto a todos, sin distinción de edad o sexo, tanto como deporte de alto nivel como de práctica familiar o social" (Gatien, 1993, p. 16). No obstante, "son escasas las obras sobre tenis de mesa. Pocos libros, tanto de divulgación como de reflexión sobre el tenis de mesa, adornan los estantes de las librerías y las bibliotecas" (Erb, 1999, p.14) y añade “así pues, el medio escolar padece de falta de obras explicativas y pedagógicas referidas a este tema" (Erb, 1999, p.14 ) En particular, se pretenden conseguir cinco objetivos divididos en tres categorías (el centro, el profesorado y el deporte. • A nivel de Centro: - Conocer el porcentaje de colegios que disponen de espacios y materiales adecuados para la práctica del tenis de mesa, así como identificar, de las distintas Direcciones de Área Territoriales (DAT), cuál tiene los colegios mejor dotados tanto en instalaciones como en materiales para desarrollar programas de promoción del tenis de mesa. - Averiguar las posibles causas por las que el tenis de mesa no se practica tanto como otros deportes, analizando los impedimentos que limitan la implantación del tenis de mesa como un deporte habitual en los centros de Educación Primaria. Analizar la opinión del profesorado en cuanto a los materiales y las instalaciones necesarios para el tenis de mesa. • A nivel de profesorado: - Analizar el nivel de conocimiento que tienen los profesionales que imparten la asignatura de Educación Física sobre el tenis de mesa, así como sus necesidades para incluir unidades didácticas de tenis de mesa en sus programaciones didácticas. - Conocer el perfil de profesor ideal que recomienda la utilización del tenis de mesa y averiguar el interés del profesorado por recibir formación específica del tenis de mesa. • A nivel de deporte: - Analizar la opinión de los profesionales sobre la idoneidad del tenis de mesa en la Educación Primaria atendiendo a los objetivos que persigue, a las competencias que desarrolla, a los contenidos, criterios de evaluación y estándares de aprendizaje que se pueden trabajar y a las lesiones que se producen. Metodología La investigación se caracterizó por utilizar una metodología inductiva, al surgir de la experiencia profesional del autor, también fue transversal al analizar la realidad en un momento concreto y de tipo cuantitativa. La población objeto de estudio fue la totalidad de los colegios públicos de la Comunidad de Madrid, siendo los profesores de Educación Física los encargados de facilitar los datos solicitados. Estos datos se obtuvieron utilizando como instrumento de toma de datos el cuestionario auto administrado con preguntas cerradas de opción múltiple previamente validado por un panel de 5 expertos. Las variables indirectas fueron: el género del profesorado, la edad del profesorado, la experiencia profesional y el tipo de destino. El proceso de la toma de datos supuso un lapso de tiempo de 3 meses, desde mayo de 2015 hasta julio de 2015, en este tiempo hubo dos fases de recogida de datos, una online a través del correo electrónico institucional de los colegios públicos de la Comunidad de Madrid y otra “in situ” con cuestionarios de lápiz y papel. En cuanto a los datos que se obtuvieron, sobre una población de 798 colegios, se consiguió una muestra de 276, esto supuso una tasa de respuesta del 34,59%, asumiendo la situación más desfavorable posible (p=q) y un nivel de confianza del 95%, para el total de los 276 cuestionarios cumplimentados, el error máximo fue del ±4,78%. Resultados En cuanto a los resultados obtenidos, se establecieron de acuerdo a tres dimensiones: A nivel de Centro, a nivel de Profesorado y a nivel del Deporte y pretendieron averiguar si se alcanzaron los cinco objetivos planteados. Tras el análisis de los resultados, se apreció que los colegios públicos de la Comunidad de Madrid disponían de las suficientes instalaciones para el tenis de mesa, en cambio, faltaban materiales específicos y formación por parte del profesorado, así como recursos didácticos y un programa de promoción del tenis de mesa. Se apreció un manifiesto interés por parte del profesorado en recibir formación específica de tenis de mesa pues la mayoría recomendaba la utilización del tenis de mesa dentro de la asignatura de Educación Física en Educación Primaria. Por último, los resultados mostraron la cantidad de objetivos motrices, afectivos, cognitivos y sociales que desarrolla el tenis de mesa así como su contribución a la adquisición de las competencias básicas y al objetivo “k” de la Educación Primaria, que indica “Valorar la higiene y la salud, conocer y respetar el cuerpo humano, y utilizar la Educación Física y el deporte como medios para favorecer el desarrollo personal y social”, además, se mostró el bajo índice de lesiones que provoca. Discusión y conclusiones El tenis de mesa es un deporte idóneo para ser practicado y enseñado en la asignatura de Educación Física en la etapa de Educación Primaria debido a la gran cantidad de contenidos que son susceptibles de ser trabajados a través de este deporte y debido a la gran cantidad de valores, individuales y sociales que se pueden fomentar con la práctica del tenis de mesa. Las causas de que hasta ahora, el tenis de mesa no sea un deporte practicado de forma habitual en los colegios públicos de la Comunidad de Madrid a pesar de trabajar muchos contenidos específicos de la asignatura de Educación Física puede deberse a factores externos al deporte del tenis de mesa y susceptibles de ser solucionados con una adecuada inversión en materiales específicos, formación del profesorado y recursos didácticos. Si se dota a los centros de los materiales y recursos didácticos necesarios y dando formación al profesorado, éste introduciría unidades didácticas de tenis de mesa dentro de sus programaciones anuales. La federación española y madrileña de tenis de mesa, deberían desarrollar un programa de promoción dotando de materiales y recursos a los centros, tal y como lo han hecho otras federaciones como la de voleibol, bádminton o de baloncesto, entre otras. ABSTRACT This research arises from the professional experience of the author, specialized teacher of physical education in the CEIP "Alhambra" in Madrid, where progressively, appreciates that table tennis can be a very interesting sport to develop in physical education sessions and promote within the playtimes. The author believes that this sport develops a range of motor, affective, cognitive and social objectives that can contribute to the acquisition of basic skills and the integral development of students. It is then when receives training on the sport of table tennis and seeks ways of funding in order to outfit the center with necessary equipment. The Municipal District of Fuencarral-El Pardo installed three outdoor tables in the schoolyard and with the resources of the school and the support of the Association of Parents (AMPA), five indoor folding tables are achieved as well as all the necessary material (nets, rackets, balls, etc.). After introduce the sport from 3rd to 6th grade of primary education, promotes a championship in the school where the participation rate is around 90% of students, these results create the uncertainties to the author that are the motivation and starting point for this research to analyze whether the practice of table tennis can be ideal at the stage of primary education. Introduction The current legislation on education, Organic Law 2/2006 of 3 May, on Education (LOE) as amended by Organic Law 8/2013, of December 9, to improve educational quality (LOMCE), attaches great importance to the sport in general, "Sport is a healthy, funny and educational activity that can have great benefits not only for their health and well-being but also for the physical, psychological and psychosocial comprehensive personal child development besides on their sports development "(Pradas, 2009, p. 151), is therefore an ideal moment to analyze which sports are practiced in schools or why are practiced some more than others. "The table tennis as well as being a sport for everyone, is presented as an attractive game, where its practice is funny at any age, both children and adults, mainly because it has simple game rules, not enclosing danger for the physical integrity of its practitioners during their game" (Pradas, 2009, p. 83). It is a sport that is "open to all, regardless of age or sex, as high-level sport, as family or social practice" (Gatien, 1993, p. 16). However, "there are few books on table tennis. Few books, both reflexion or popularization about table tennis, adorn the shelves of bookstores and libraries." (Erb, 1999, p.14) and add "So, the school environment suffers from lack of explanatory and educational work related to this issue." (Erb, 1999, p.14) In particular, it is intended to achieve the following objectives within the Community of Madrid: • To determine the percentage of schools that have spaces and materials suitable for practicing table tennis and identify, from the different Directorates of Land Area (DAT), which has the best equipped schools in both facilities and materials to develop programs to promote table tennis. • Find out the possible causes that explained why table tennis is not practiced as much as other sports, analyzing impediments that limit the implementation of table tennis as a regular sport in primary schools. Analyze the opinion of teachers in terms of materials and facilities needed for table tennis. • Analyze the level of knowledge about table tennis among professionals who teach the subject of Physical Education and their needs to include teaching units about table tennis in their teaching programs. • Knowing the profile of the ideal teacher who recommends the use of table tennis and figure out the interest of teachers to receive specific training of table tennis. • Analyze the professional opinion on the suitability of table tennis in Primary Education taking into account the objectives pursued, to develop the skills, content, evaluation criteria and learning standards that can work and injuries involved. Methodology The investigation was characterized by using an inductive methodology, arising from the professional experience of the author, was also transverse to analyze reality in a particular time and quantitative type. The population under study were all the state schools in Madrid region, being the physical education teachers responsible for providing the requested data. These data were obtained using as data collection instrument a self-administered questionnaire with multiple choice questions, because it facilitates the analysis thereof. In terms of obtained data, on a population of 798 schools, a sample of 276 was achieved, this represented a response rate of 34.59%, assuming the worst case scenario (p = q) and a level 95% confidence for the total of the 276 completed questionnaires, the maximum error was ± 4.78%. Results In terms of the results, they were set according to three dimensions: center level, professorate level and Sport level and trying to find out whether the five objectives were achieved. After analyzing the results, it was found that schools possessed sufficient facilities for table tennis, however, lacked specific materials and training by teachers, as well as teaching resources and a program to promote table tennis. A clear interest was noticed by teachers in order to receive specific training in table tennis since most recommended the use of table tennis in the subject of physical education in primary education. Finally, the results proved the number of motor, affective, cognitive and social objectives developed by table tennis and its contribution to the acquisition of basic skills and the objective "k" of primary education, in addition to the low rate of injury it causes. Discussion and conclusions Table tennis is an ideal sport to be practiced and taught in the subject of Physical Education in Primary Education due to the large amount of content that are likely to be worked through this sport and due to the large number of individual and social values that can foster the practice of table tennis. The causes of that so far, table tennis is not a sport practiced regularly in schools despite working many specific contents of the subject of Physical Education may be due to factors outside the sport of table tennis and subject to solved with adequate investment in specific materials, teacher training and educational resources. By endowing the centers with the necessary teaching materials and resources and providing training to teachers, they would introduce teaching units of table tennis within their annual programs. Madrid and the Spanish Federation of table tennis should develop a promotional program by endowing materials and resources to the centers, as did other federations such as badminton and basketball, among others.

Nagele: un nuevo asentamiento en un paisaje artificial

Nagele es un asentamiento urbano situado en el Noordoostpolder, territorio neerlandés ganado al mar. Fue diseñado por arquitectos de los grupos De 8 en Opbouw entre los que destacaron Rietveld, Van Eesteren, Van Eyck, Bakema, Stam y Ruys. El proyecto se desarrolló entre 1947 y 1956, un periodo de tiempo con formas de proyectar muy ricas en interpretaciones. Los arquitectos pusieron en crisis los planteamientos historicistas de las nuevas poblaciones de los pólderes. Propusieron un nuevo prototipo, una morfología compacta y concéntrica que transmitiría igualdad a una comunidad agrícola, entendida como una sociedad urbana del siglo XX. La administración apoyó la propuesta que convertiría el proyecto en un arriesgado reto por su falta de antecedentes. La vigencia de las formulaciones permanece hoy en día en la ciudad construida, aunque con alteraciones. En los dibujos del proceso se encuentran los principales enunciados teóricos que este trabajo pretende descubrir. El trabajo aborda aspectos no suficientemente explorados, como su relación con el pólder, la evolución de las estrategias proyectivas, la ordenación paisajista y los elementos urbanos. El Noordoostpolder es la culminación de una serie de experiencias multidisciplinares en el reclamo de tierras a gran escala. Se estudia su estructura urbana policéntrica, la parcelación agrícola que origina el proyecto urbano y la vinculación de la vegetación con la infraestructura, proporcionando orientación, protección climática y escala humana, conceptos que impregnan las estrategias del proyecto urbano. La primera fase de la ordenación configuró áreas monofuncionales que respondían a cada una de las cuatro necesidades básicas del método científico de la ciudad higienista. El acontecimiento que marcó el final de la primera fase fue su presentación en el séptimo CIAM de 1949, cuyo título fue Aplicación de la Carta de Atenas. El programa residencial se dividió en clusters organizados en torno a una pradera vecinal central, vinculándose el orden vecinal, urbano y territorial. La segunda fase fue un catalizador de nuevos planteamientos. El proyecto se transformó en un In-between Realm, un escenario teórico donde coexisten fenómenos tradicionalmente antagónicos que Van Eyck denominó Twin Phenomena, convirtiéndose la ciudad en una réplica formal de la ambivalencia de la mente humana. La indefinición espacial no programada en la propuesta anterior se transformó en un conjunto de espacios urbanos, con límites y dimensiones adaptados a la escala humana. El proyecto es anterior a la obra escrita de Van Eyck por lo que estimuló sus enunciados teóricos. Unas ideas también reconocidas en los tres CIAM posteriores en los que también se expuso el proyecto. El diseño paisajista se integra en el proyecto urbano desde sus orígenes. El límite se compone de una barrera boscosa que protege climáticamente, proporciona escala humana y control visual frente a las llanuras infinitas del pólder. Van Eyck sintetizó el proyecto como una habitación verde sin techo, afirmación que dilucida su equivalencia con el de un interior doméstico. Exteriormente la ciudad se convierte en una unidad autónoma del territorio. Interiormente, un sistema jerarquizado de vegetación vinculado a la arquitectura y la infraestructura constituye espacios urbanos de diferentes escalas. La propuesta fue transformada por Boer y Ruys en un nuevo espacio urbano único, no asimilando los conceptos anteriores. El proyecto y construcción de los primeros elementos urbanos consistió en un reparto de tareas a De 8 en Opbouw, hecho que estimula estudiar su relación con el proyecto urbano. La estructura policéntrica organiza las aulas de las escuelas de Van Eyck, las diferentes áreas confesionales del cementerio de Ruys y las unidades residenciales, diseñadas por Stam, Rietveld y Stam-Besse. Los Twin Phenomena alcanzan un acuerdo en el corredor comercial, diseñado por Bakema y Van der Broek. La generación de espacios dentro de otros aparece también en el cementerio, a través de una nueva barrera boscosa y en el sistema de pliegues del muro que configura la iglesia de Bakema y Van der Broek. El proyecto se vincula a un planteamiento holístico, mediante el que el diseño de cada uno de sus elementos tiene en cuenta las estrategias proyectivas del todo del cual forma parte, convirtiéndose, al igual que las obras de De Stijl, en parte de una composición infinita que acerca arte y diseño en la vida cotidiana de la sociedad. La diversidad generacional e ideológica de estos arquitectos convirtió el proyecto en un tablero de juego sobre el que se aplicaron diferentes formas de proyectar la ciudad, ubicando a Nagele en un punto de inflexión del Movimiento Moderno. ABSTRACT The research focuses on the Nagele project, a Dutch urban settlement located in the Noordoostpolder, a territory which was entirely reclaimed from the IJsselmeer lake. It was designed by a group of architects from the De 8 and Opbouw teams, the leading protagonists being Rietveld, Van Eesteren, Van Eyck, Bakema, Stam y Ruys. It was designed from 1947 to 1956, a fruitful period in urban planning. These architects questioned the traditionalist urban design applied to the new populations in the IJsselmeer polders. Facing their principles, the work group proposed a new prototype; a compact and concentric urban pattern to foster equality in a new community of farm labourers, which was recognized by the architects as a twentieth century urban society. The government supported their new proposals. The lack of implementation of the innovatory conceptual statements subjected the project into a high-risk challenge. However, in spite of these difficulties, the basic concepts remain though partially transformed, in the actual city. The project drawings reflect the principle concepts that this work aims to discover. Some approaches that have not been sufficiently studied are tackled in this thesis. Firstly, the project´s relationship with the polder. Secondly, the evolution of projective strategies during the period of urban planning, the landscape design and the design of urban elements. The Noordoostpolder is the culmination of a series of multi-disciplinary experiences in large scale land reclamation, whose polycentric urban structure and agricultural subdivision provide the framework of Nagele. Linking the vegetation to infrastructure fostered orientation, climate protection and human scale; strategies which were repeated, though on a smaller scale, in the actual city. The first phase of the project was composed of mono-functional urban areas which responded to each of the four basic human needs indicated by the scientific method of the functional city. The presentation of the project at the seventh CIAM in 1949 was the event which marked the end of the first phase of the planning. This congress was entitled Implementation of the Athens Charter. The residential program was divided into housing clusters surrounding a central prairie, a pattern which was related to its urban and territorial whole. The second phase of the plan was subjected to a new theoretical approach. The urban planning became an In-between Realm, a theoretical scenario where traditionally antagonistic concepts coexist. Van Eyck named these concepts Twin Phenomena. The city thus conceived of as a counterform of the ambivalence of the human mind where spatial indefinition in the previous proposals was transformed into a Bunch of Places with defined boundaries and dimensions, all of which reflecting human scale. The landscape design was integrated into the urban project from its inception. The limits consist of a green wind-barrier which not only provides climate protection but also provides human scale and visual control towards the unlimited plains of the polder. Van Eyck summarised the project as a green room without a roof. This statement elucidates its equivalence to a domestic interior. Outwardly, the city becomes an autonomous unit on the territory. Inwardly a hierarchical vegetation system is linked to architecture and infrastructure. Together, they configure different scales of urban spaces. The proposal was transformed by Boer and Ruys into a unique urban space without assimilating Van Eyck´s concepts. The study of the Nagele landscape project of Nagele and the writings of Van Eyck verify the fact that many of his theoretical foundations (In-between Realm, Twin Phenomena, Bunch of Places, Right Scale) can be applied not only to architecture and city but also to landscape design. The application of these principles led the Nagele project to become a counterform of Van Eyck´s thinking. The design and construction of the first urban elements involved a distribution of tasks to De 8 en Opbouw, which stimulated their relationship with the urban project. The polycentric structure organised the school classrooms outlined by Van Eyck, the different areas of the cemetery planned by Ruys and the housing clusters designed by Stam, Rietveld and Stam-Besse. The Twin Phenomena concept can be applied in Van der Broek´s shopping corridor. The concept space within another space is also implemented in the cemetery surrounded by a new green barrier, and in the church built by Van der Broek and Bakema, whose spaces are configured by a folding wall. The project takes a holistic approach, which considers the design of each element within the strategies of the whole, where they become parts of an infinite composition, as in the art works of De Stijl fostering art and design to ordinary people´s daily lives. The generational and ideological diversity of these architects turned the project into a game board on which different ways of planning the city were played, obtaining Nagele the distinction of being a turning point of Modernism.

Identification of Novel Components of the Unfolded Protein Response in Arabidopsis

Unfavorable environmental and developmental conditions may cause disturbances in protein folding in the endoplasmic reticulum (ER) that are recognized and counteracted by components of the Unfolded Protein Response (UPR) signaling pathways. The early cellular responses include transcriptional changes to increase the folding and processing capacity of the ER. In this study, we systematically screened a collection of inducible transgenic Arabidopsis plants expressing a library of transcription factors for resistance toward UPR-inducing chemicals. We identified 23 candidate genes that may function as novel regulators of the UPR and of which only three genes (bZIP10, TBF1, and NF-YB3) were previously associated with the UPR. The putative role of identified candidate genes in the UPR signaling is supported by favorable expression patterns in both developmental and stress transcriptional analyses. We demonstrated that WRKY75 is a genuine regulator of the ER-stress cellular responses as its expression was found to be directly responding to ER stress-inducing chemicals. In addition, transgenic Arabidopsis plants expressing WRKY75 showed resistance toward salt stress, connecting abiotic and ER-stress responses.

Single-pair fluorescence resonance energy transfer on freely diffusing molecules: Observation of Förster distance dependence and subpopulations

Photon bursts from single diffusing donor-acceptor labeled macromolecules were used to measure intramolecular distances and identify subpopulations of freely diffusing macromolecules in a heterogeneous ensemble. By using DNA as a rigid spacer, a series of constructs with varying intramolecular donor-acceptor spacings were used to measure the mean and distribution width of fluorescence resonance energy transfer (FRET) efficiencies as a function of distance. The mean single-pair FRET efficiencies qualitatively follow the distance dependence predicted by Förster theory. Possible contributions to the widths of the FRET efficiency distributions are discussed, and potential applications in the study of biopolymer conformational dynamics are suggested. The ability to measure intramolecular (and intermolecular) distances for single molecules implies the ability to distinguish and monitor subpopulations of molecules in a mixture with different distances or conformational states. This is demonstrated by monitoring substrate and product subpopulations before and after a restriction endonuclease cleavage reaction. Distance measurements at single-molecule resolution also should facilitate the study of complex reactions such as biopolymer folding. To this end, the denaturation of a DNA hairpin was examined by using single-pair FRET.

Mechanical and chemical unfolding of a single protein: A comparison

Is the mechanical unraveling of protein domains by atomic force microscopy (AFM) just a technological feat or a true measurement of their unfolding? By engineering a protein made of tandem repeats of identical Ig modules, we were able to get explicit AFM data on the unfolding rate of a single protein domain that can be accurately extrapolated to zero force. We compare this with chemical unfolding rates for untethered modules extrapolated to 0 M denaturant. The unfolding rates obtained by the two methods are the same. Furthermore, the transition state for unfolding appears at the same position on the folding pathway when assessed by either method. These results indicate that mechanical unfolding of a single protein by AFM does indeed reflect the same event that is observed in traditional unfolding experiments. The way is now open for the extensive use of AFM to measure folding reactions at the single-molecule level. Single-molecule AFM recordings have the added advantage that they define the reaction coordinate and expose rare unfolding events that cannot be observed in the absence of chemical denaturants.

Structure and function of the Bacillus hybrid enzyme GluXyn-1: Native-like jellyroll fold preserved after insertion of autonomous globular domain

The 1,3–1,4-β-glucanase from Bacillus macerans (wtGLU) and the 1,4-β-xylanase from Bacillus subtilis (wtXYN) are both single-domain jellyroll proteins catalyzing similar enzymatic reactions. In the fusion protein GluXyn-1, the two proteins are joined by insertion of the entire XYN domain into a surface loop of cpMAC-57, a circularly permuted variant of wtGLU. GluXyn-1 was generated by protein engineering methods, produced in Escherichia coli and shown to fold spontaneously and have both enzymatic activities at wild-type level. The crystal structure of GluXyn-1 was determined at 2.1 Å resolution and refined to R = 17.7% and R(free) = 22.4%. It shows nearly ideal, native-like folding of both protein domains and a small, but significant hinge bending between the domains. The active sites are independent and accessible explaining the observed enzymatic activity. Because in GluXyn-1 the complete XYN domain is inserted into the compact folding unit of GLU, the wild-type-like activity and tertiary structure of the latter proves that the folding process of GLU does not depend on intramolecular interactions that are short-ranged in the sequence. Insertion fusions of the GluXyn-1 type may prove to be an easy route toward more stable bifunctional proteins in which the two parts are more closely associated than in linear end-to-end protein fusions.

Mass spectrometry of ribosomes and ribosomal subunits

Nanoflow electrospray ionization has been used to introduce intact Escherichia coli ribosomes into the ion source of a mass spectrometer. Mass spectra of remarkable quality result from a partial, but selective, dissociation of the particles within the mass spectrometer. Peaks in the spectra have been assigned to individual ribosomal proteins and to noncovalent complexes of up to five component proteins. The pattern of dissociation correlates strongly with predicted features of ribosomal protein–protein and protein–RNA interactions. The spectra allow the dynamics and state of folding of specific proteins to be investigated in the context of the intact ribosome. This study demonstrates a potentially general strategy to probe interactions within complex biological assemblies.

Oligomerization domain-directed reassembly of active dihydrofolate reductase from rationally designed fragments

Reassembly of enzymes from peptide fragments has been used as a strategy for understanding the evolution, folding, and role of individual subdomains in catalysis and regulation of activity. We demonstrate an oligomerization-assisted enzyme reassembly strategy whereby fragments are covalently linked to independently folding and interacting domains whose interactions serve to promote efficient refolding and complementation of fragments, forming active enzyme. We show that active murine dihydrofolate reductase (E.C. 1.5.1.3) can be reassembled from complementary N- and C-terminal fragments when fused to homodimerizing GCN4 leucine zipper-forming sequences as well as heterodimerizing protein partners. Reassembly is detected by an in vivo selection assay in Escherichia coli and in vitro. The effects of mutations that disrupt fragment affinity or enzyme activity were assessed. The steady–state kinetic parameters for the reassembled mutant (Phe-31 → Ser) were determined; they are not significantly different from the full-length mutant. The strategy described here provides a general approach for protein dissection and domain swapping studies, with the capacity both for rapid in vivo screening as well as in vitro characterization. Further, the strategy suggests a simple in vivo enzyme-based detection system for protein–protein interactions, which we illustrate with two examples: ras–GTPase and raf–ras-binding domain and FK506-binding protein-rapamycin complexed with the target of rapamycin TOR2.

Enhanced transcription factor access to arrays of histone H3/H4 tetramer⋅DNA complexes in vitro: Implications for replication and transcription

Defined model systems consisting of physiologically spaced arrays of H3/H4 tetramer⋅5S rDNA complexes have been assembled in vitro from pure components. Analytical hydrodynamic and electrophoretic studies have revealed that the structural features of H3/H4 tetramer arrays closely resemble those of naked DNA. The reptation in agarose gels of H3/H4 tetramer arrays is essentially indistinguishable from naked DNA, the gel-free mobility of H3/H4 tetramer arrays relative to naked DNA is reduced by only 6% compared with 20% for nucleosomal arrays, and H3/H4 tetramer arrays are incapable of folding under ionic conditions where nucleosomal arrays are extensively folded. We further show that the cognate binding sites for transcription factor TFIIIA are significantly more accessible when the rDNA is complexed with H3/H4 tetramers than with histone octamers. These results suggest that the processes of DNA replication and transcription have evolved to exploit the unique structural properties of H3/H4 tetramer arrays.

Exploring the origins of topological frustration: Design of a minimally frustrated model of fragment B of protein A

Topological frustration in an energetically unfrustrated off-lattice model of the helical protein fragment B of protein A from Staphylococcus aureus was investigated. This Gō-type model exhibited thermodynamic and kinetic signatures of a well-designed two-state folder with concurrent collapse and folding transitions and single exponential kinetics at the transition temperature. Topological frustration is determined in the absence of energetic frustration by the distribution of Fersht φ values. Topologically unfrustrated systems present a unimodal distribution sharply peaked at intermediate φ, whereas highly frustrated systems display a bimodal distribution peaked at low and high φ values. The distribution of φ values in protein A was determined both thermodynamically and kinetically. Both methods yielded a unimodal distribution centered at φ = 0.3 with tails extending to low and high φ values, indicating the presence of a small amount of topological frustration. The contacts with high φ values were located in the turn regions between helices I and II and II and III, intimating that these hairpins are in large part required in the transition state. Our results are in good agreement with all-atom simulations of protein A, as well as lattice simulations of a three- letter code 27-mer (which can be compared with a 60-residue helical protein). The relatively broad unimodal distribution of φ values obtained from the all-atom simulations and that from the minimalist model for the same native fold suggest that the structure of the transition state ensemble is determined mostly by the protein topology and not energetic frustration.

Significance of chaperonin 10-mediated inhibition of ATP hydrolysis by chaperonin 60

Chaperonins are essential for the folding of proteins in bacteria, mitochondria, and chloroplasts. We have functionally characterized the yeast mitochondrial chaperonins hsp60 and hsp10. In the presence of ADP, one molecule of hsp10 binds to hsp60 with an apparent Kd of 0.9 nM and a second molecule of hsp10 binds with a Kd of 24 nM. In the presence of ATP, the purified yeast chaperonins mediate the refolding of mitochondrial malate dehydrogenase. Hsp10 inhibits the ATPase activity of hsp60 by about 40%. Hsp10(P36H) is a point mutant of hsp10 that confers temperature-sensitive growth to yeast. Consistent with the in vivo phenotype, refolding of mitochondrial malate dehydrogenase in the presence of purified hsp10(P36H) and hsp60 is reduced at 25°C and abolished at 30°C. The affinity of hsp10(P36H) to hsp60 as well as to Escherichia coli GroEL is reduced. However, this decrease in affinity does not correlate with the functional defect, because hsp10(P36H) fully assists the GroEL-mediated refolding of malate dehydrogenase at 30°C. Refolding activity, rather, correlates with the ability of hsp10(P36H) to inhibit the ATPase of GroEL but not that of hsp60. Based on our findings, we propose that the inhibition of ATP hydrolysis is mechanistically coupled to chaperonin-mediated protein folding.

A natural polymorphism in β-lactamase is a global suppressor

A M182T substitution was discovered as a second-site suppressor of a missense mutation in TEM-1 β-lactamase. The combination of the M182T substitution with other substitutions in the enzyme indicates the M182T substitution is a global suppressor of missense mutations in β-lactamase. The M182T substitution also is found in natural variants of TEM-1 β-lactamase with altered substrate specificity that have evolved in response to antibiotic therapy. The M182T substitution may have been selected in natural isolates as a suppressor of folding or stability defects resulting from mutations associated with drug resistance. This pathway of protein evolution may occur in other targets of antimicrobial drugs such as the HIV protease.

Proteins unfold in steps

I present results from an experiment on the dynamics of folding of a globular protein (bovine serum albumin). Employing a micro-mechanical technique, I perform the measurements on very few molecules (1–100). I observed a sequence of steps in time for both unfolding and refolding. The overall characteristic time of the process is thus built up of waiting times between successive steps. The pattern of steps is reproducible, demonstrating the existence of deterministic pathways for folding and unfolding. Certain symmetries in the patterns of steps may reflect the architecture of the protein’s structure.