Cómic, arquitectura narrativa. Describiendo cuatro dimensiones con dos

Cómic, arquitectura narrativa. Describiendo cuatro dimensiones con dos. La mayor parte de los estudios con un enfoque académico en torno a cómic y arquitectura, se han centrado más en las distintas realidades representadas dentro del medio, en sus decorados, que en la estructura del cómic en sí. Existe un aspecto gráfico que se ha venido obviando o al menos no ha adquirido el protagonismo que merece: los espacios representados en el cómic se encuentran en un medio que se concibe y estructura de una manera similar a como el arquitecto dibuja y concibe la arquitectura. En lo básico, históricamente, las herramientas y soportes para idear y representar la arquitectura son muy parecidas a aquellas que usa el autor de cómic. Lápiz, tinta y color sobre un papel, combinadas trabajando en un tablero, ese es lugar de nacimiento de ambos mundos. A lo largo de esta tesis se muestra cómo el dibujo une a creadores de narraciones gráficas y edificios. La relación va más allá de que el cómic pueda utilizar las disposiciones vitruvianas, ichnographia, ortographia o scenographia, (planta, alzado, perspectiva), la propia articulación que hace del espacio de la página, puede sintonizar con la manera en la que se concibe una estructura arquitectónica. La tesis inicia con un capítulo de recorrido en torno a las teorías del cómic existentes, buscando intersecciones posibles con la representación arquitectónica o el proyecto espacial. En este primer paso de estudio del estado de la cuestión han pesado más los trabajos de los últimos veinticinco años. Se trata de un periodo en el que, a la par que se ha producido una explosión creativa en el mundo del cómic, determinados teóricos han incidido en un análisis más formal y visual del medio. Tras recoger teorías en el primer capítulo, se plantea un recorrido histórico a la búsqueda de posibles resonancias de “arquitecturas mentales” en el pasado. Esta búsqueda de referentes más allá del siglo XIX, momento en el que nace el medio en su forma actual, es ya un aspecto comúb en distintos estudios de cómic que han servido de inspiración. Se ha preferido ubicar esta colección de antecedentes como segundo capítulo, evitando dar al trabajo un carácter de historia e incidiendo en su enfoque de tipo visual. El tercer capítulo analiza una serie de obras y autores de cómic en su forma actual, buscando distintas estrategias para la descripción de espacios y las narraciones que ocurren en ellos. El crecimiento de casos en estas últimas décadas ha demostrado hasta qué punto el desarrollo de un campo depende tanto de que exista una masa crítica en un contexto conectado y productivo, como de la aparición de individuos que abren puertas a los demás, con la cristalización personal que hacen de una situación. Se analiza en un cuarto capítulo la relación entre la arquitectura y el cómic modernos. Una relación que se presenta partiendo desde sus respectivos padres, para después tratar un elenco creciente de experiencias en el que la narración gráfica y el hecho arquitectónico estan íntimamente ligados. Inspirado en la estrategia que Gubern y Gasca utilizan en El discurso del cómic (Cátedra, 1988) se cierra la tesis con un discurso visual por las arquitecturas mentales y narrativas del cómic: la representación del espacio a través de juego con las dimensiones, el uso de recursos gráficos propios de la descripción arquitectónica, la sección, la planta, la utilización de elementos constructivos,etc, completando el abanico de estrategias gráficas presentadas. ABSTRACT Comic narrative architecture. Describing four dimensions with two Most of the studies with an academic focus around comics and architecture have centered their view more on the different realities represented in the media, in its sets, than in the structure of comics itself. There is a graphic aspect that has been ignored, or at least has not gained the prominence it deserves: the spaces rendered in the comics are set in a medium that is conceived and structured in a similar way as the architect draws and conceives architecture. In basics, tools for designing and representing architecture are very similar to those used by the cartoonist. Pencil, ink and color on paper, combined working on a board, that is birthplace of both worlds. Throughout this thesis its shown how drawing connects creators of graphic stories and buildings. The relationship goes beyond the use that comics can do of the Vitruvian provisions, ichnographia, ortographia or scenographia (plan, elevation, perspective), to the arrange itself that it makes with space on the page, in tune with the way in which architectural structures are conceived. The thesis starts with a chapter that collects existing comics theories and their possible intersection with architectural rendering. In this first step of study on the state of affairs, the focus has been over studies from the last twenty years. It is a period in which, at the same time that there has been a creative explosion in the world of comics, some theorists have worked with a more formal and visual approach. After collecting theories in the first chapter, a historical tour is made to search for possible resonances of “mental architectures” in the past. This search for references beyond the nineteenth century, when the medium was born in its current form, is already a regular process in various recent studies that have served as inspiration. We preferred to locate this as a second chapter, to avoid giving the work a character of History, and reenforcing its visual approach. The third chapter analyzes a series of comics and authors, looking for different strategies for describing spaces and stories that occur in them. The growth of cases in recent decades has shown how the development of a field depends both on the existence of a critical mass in a connected and productive enviroment, and the appearance of individuals who open doors to others with the personal crystallization that they make on that context. The relationship between architecture and the modern comic is analyzed in a fourth chapter. A relationship that is described starting from their parents, and then going through a growing cast of experiences in which graphic narrative and architecture are closely linked. Inspired by the strategy used Gubern and Gasca in El discurso del cómic (Cátedra, 1988), The Comics’ Speech, the thesis concludes with a visual discourse through this narrative comic architectures: the representation of space by playing with dimensions, the use of resources for architectural rendering, section, plant, etc., to complete the range of graphical strategies presented.

Un deseo moderno todavía pendiente : la casa patio, una posible técnica de agrupación de vivienda colectiva vertical

La idea de aportar a la vivienda en altura las cualidades de la casa patio, se ha estudiado durante años. Podemos decir que el origen de este análisis se inició en 1922 cuando Le Corbusier empezó a investigar a cerca de los Inmuebles Villa; sin duda sabemos que ha llegado hasta la actualidad. Ayudado por la labor de búsqueda de diferentes arquitectos a lo largo de la historia, podemos afirmar que todos los casos conforman una idea única de proyecto. La idea de agrupar casas patio en altura manteniendo las características propias de este tipo de viviendas, privacidad, espacio libre, apertura cenital, comunicación interior‐exterior… Los Inmuebles‐Villa serán repetidamente aludidos, citados o retomados como un tipo paradigmático entre las propuestas de agrupaciones de casas patio en altura. Si pensamos en el concepto tradicional de patio, denominarlo como un espacio de viviendas apiladas en altura no parece una elección adecuada, no obstante la noción de patio permite una gran cantidad de asociaciones. Su valor radica en el traslado de una idea conocida a un nuevo contexto, produciendo así una reformulación del problema. Elegimos para este proceso, la vivienda colectiva, entendiendo la colectividad como agrupación, pues es esta agrupación de vivienda la encargada de crear ciudad; así no sólo vale la pena dar importancia a una célula con su independencia, sino en la relación que estas establecen con sus semejantes. De ahí la idea de que esa colectividad de casas patio, pueda darse también en vertical, buscando las condiciones antes mencionadas. Un deseo que apareció en el Movimiento Moderno y que desde ese momento ha sido algo anhelado por la arquitectura hasta nuestros días. El patio se presenta en la mayoría de las ocasiones como un espacio con múltiples afecciones, desde el patio que aporta a la vivienda una zona de esparcimiento hasta un espacio destinado a la ventilación, aunque en definitiva, podemos decir que consiste en un espacio capaz de contener un lugar. Quizás por eso aparece de diversas formas en nuestra arquitectura, sin importar su ubicación o forma. Simplemente un espacio que aporta al interior unas características que esas viviendas nunca alcanzarían sin él. Por eso lo que conocemos como casa patio es un tipo de arquitectura que se extiende hasta la antigüedad. Ese concepto de espacio exterior incorporado a la vivienda es una de las piezas fundamentales en nuestra arquitectura. Una idea ampliamente estudiada que deja a un lado los diferentes tipos de “patio” que fueron apareciendo, así como las múltiples funciones que dichos espacios fueron capaces de llevar a cabo, primando en definitiva la forma en la que cambiaron la idea de habitar. A lo largo de este análisis se han ido hallando evidencias de los múltiples mecanismos empleados en los casos estudiados. Como se ha visto, en ellos aparecen características que únicamente pueden aparecer en un espacio exterior cuando se trata de un patio. Los patios estudiados se consideran patios tradicionales, independientemente de la cota a la que se encuentren, aunque no cumplan alguna de las características a priori necesarias para la definición de patio. Para captar la esencia de esos espacios, no es necesario traspasar sus límites, ya que ésta se irradia al interior e invade la atmósfera de los espacios contiguos. Se plantea como objetivo principal rastrear las huellas de una investigación proyectual que Le Corbusier inaugura y deja abierta; se trata de descubrir qué ecos, qué rebotes, qué repercusiones tiene el concepto de patio en altura a lo largo de su historia. En esta búsqueda de identificación de las características de estos espacios, hemos seleccionado tres casos, partiendo de las experiencias tanto en patios tradicionales como en patios en altura de tres maestros como son Le Corbusier, José Luis Sert y Charles Correa. Los casos estudiados podrían haber sido muchos más. Este número supone solo un ejemplo de un universo mayor, que ha sido operativo para el desarrollo de este trabajo. Ellos nos guiarán por su arquitectura para intentar resolver la idea de los patios en altura. Hemos analizado las influencias proyectuales existentes entre algunos de los casos que forman parte de esta reflexión, entre los que existen unos vínculos claros, que pueden ser esas claves que estamos buscando para resolver la agrupación de casa patio en altura. A través de este trabajo se ha puesto de manifiesto que la aplicación del concepto de casas patio en altura depende tanto de su ubicación, del clima y la cultura local, como el tipo de patio del que se parte. De estos conceptos surgen soluciones diferentes entre ellas y por tanto no pueden extrapolarse una sola idea a todos los casos. Algunas ideas están presentes durante toda la investigación: fachadas como resultado de la repetición de células, sucesión de espacios, plantas de casas patio tradicional, dobles alturas…Otros casos destacan por su singularidad, por haber buscado la solución del problema mediante la originalidad. En definitiva buscamos casas patio en altura. Y si esta tesis contribuye, en alguna medida, a obtener las claves para que esta tipología edificatoria funcione, de sus características, sus necesidades, y su resultado, es decir, de su búsqueda de ese deseo moderno que presentábamos al principio, será entonces que esa tipología ilusoria, sea una arquitectura real, y por lo tanto también podrá llevarse a cabo y dejar de ser un deseo para convertirse en realidad. Sera que el deseo moderno se ha cumplido. ABSTRACT The idea of giving housing the height of the patio housing qualities, it has been studied for ages. It can be said that the origin of this analysis was started in 1922, when Le Cobustier began investigating about The Villa Building: obviously it is known nowadays due to different architect’s labor of searching through the history. It’s easy to say that all the cases shape a single project idea , the idea of gathering patio houses with the same height keeping the inner characteristics of traditional patio houses such privacy, free space, zenithal opening, inside –outside communication…etc. If we thought about the traditional concept of patio, to define it, in the case of gathering because of the height, it is not suitable to consider it like a pile of houses with the same height. Nevertheless the concept of patio allows a wide variety of settlements. Its worth is based on the transference of a renowned idea into a new context, producing thus a reformulation of the problem. The patio is frequently disposed like a zone with multiple utilities, from the leisure zone patio to an air circulating space, definitively it can be said that it consists on a space which is able to content a place. Maybe that is why it appears in various forms in our architecture, regardless of its placement or shape. It is simply a space which provides many features which are impossible to reach without it. That is what we know as patio house is a type of architecture which is spread till antiquity. This concept of outdoors space joined housing is one of the main pieces of our architecture. It is an idea wide analyses which lefts aside the different types of patios which were developed, focusing mainly the way they changed the idea of inhabiting. We have chosen for this analysis process, the collective housing, we understood collective like group, thus it is the group of housing responsible to built up a city, not just it is worth to give importance to an independent cell, in addition to the relation which those ones can stablish with their similar ones. Due to the idea of collectivity of patio houses, it can be also developed in vertical, looking for the previously mention condition. A wish which appeared with the modern movement and since then it has been something desired by architecture just nowadays. It is understood as the main target to follow the trail about a project investigation which was begun and left open by Le Cobustier: the task is discovering the resonances, the bounces and repercussions which belong to the patio concept in height through its history. In this quest to distinguish the characteristics of these spaces, we have chosen three cases, starting from the experiences as traditional patios as in the height of patios of the masters such as Le Cobustier, Jose Luis Sert and Charles Correa. The studied cases could be even more. This number is just an example of a huger universe, which has been operational for the developing of this work. We will be managed through their architecture trying to solve the idea of patios according to their height. Over this analysis evidences there are found multiple mechanics used in the studied cases. As we could see later, in these ones there are characteristics which are only found in an outdoors space when it is a patio. The studied patios are considered traditional patios, independently of the level they have, although they do not fit at first some of the necessary characteristics included in a patio definition. To catch the essence of those spaces, it is not necessary to go through its limits, since it is transmitted to the interior and pervades the adjoining spaces. It has been analyzed the project influences exiting among several cases which build up part of this consideration , among them there are some clear links, which may be these keys we are looking for to solve the joint of patio houses in height. With this work it has been highlighted that the application of the patios houses in height concept depends not only on its placement, the weather and the local culture, but also on the type of patio we are using. From these considerations different solutions were given by themselves as consequence they can be extrapolated into a single idea for all the cases. Some ideas are present along the complete investigation: facades resulting from cells repetition, series of spaces, traditional patio house floor, double height… Some other cases stand out due to it singularity, because they have searched for a solution to the problem by means of originality. To sum up, we look for patio houses in height. And if this thesis contributes, in some way, to get the clues to make work this type of building, about its characteristics, its necessities and its result, in other words, its searching of this modern desire that we showed at the beginning, then this imaginary typo will be a real architecture therefore it can also come truth and can abandon the form of wish to became into a reality. So the modern desire has been fulfilled.

O Judaísmo Encalacrado: mística e religião em A Hora da Estrela, de Clarice Lispector

O objetivo deste trabalho foi o de demonstrar a importância e pertinência do diálogo entre literatura e religião, concentrando o âmbito de sua pesquisa a partir das relações entre a escritura de Clarice Lispector e as ressonâncias de uma tradição (judaísmo), que embora não tenha sido assumida pela autora, persiste, encalacrada em toda a sua obra. Tendo, como objeto de pesquisa o romance A Hora da Estrela, esta dissertação procurou entender como o drama da narrativa, em seu jogo de tensões e identidades, em sua pluralidade de títulos e referências, sinaliza também um profundo problema com a religião. Focando, de forma especial a personagem Macabéa, este estudo procurou compreender como as referencias da personagem, sua dificuldade com as palavras, seus desencontros do outro (tu) e sua rejeição social, revelam um grave problema de ordem teológica, demonstrando os equívocos de uma sociedade, que fundada sobre o prisma da religião, massacra os mais fracos. O desfecho do livro sinaliza uma possível epifania, mas, desvela-se num fracasso da religião e de seu poder salvífico. O trabalho procurou a partir dos principais temas do romance, perceber suas correspondências com o tema da religião. Pode com isso, a partir das contribuições de Scholem, ver como dilema existencial e subjetivo da personagem possui proximidade com o drama místico cabalístico, além disso, perceber as correlações entre a obra clariceana e textos da tradição judaíca, quer na sua referência explícita (nome da personagem), como também, em seu aspecto de busca, em sua forma de midrash e nas inúmeras exegeses e comentários que propicia. Finaliza seu esforço, levantando uma discussão ética sobre os valores teológicos da sociedade e seus profundos equívocos, ante a vida e morte da pobre Macabéa, uma mulher que viveu a religião nos limites da própria existência.

O Judaísmo Encalacrado: mística e religião em A Hora da Estrela, de Clarice Lispector

O objetivo deste trabalho foi o de demonstrar a importância e pertinência do diálogo entre literatura e religião, concentrando o âmbito de sua pesquisa a partir das relações entre a escritura de Clarice Lispector e as ressonâncias de uma tradição (judaísmo), que embora não tenha sido assumida pela autora, persiste, encalacrada em toda a sua obra. Tendo, como objeto de pesquisa o romance A Hora da Estrela, esta dissertação procurou entender como o drama da narrativa, em seu jogo de tensões e identidades, em sua pluralidade de títulos e referências, sinaliza também um profundo problema com a religião. Focando, de forma especial a personagem Macabéa, este estudo procurou compreender como as referencias da personagem, sua dificuldade com as palavras, seus desencontros do outro (tu) e sua rejeição social, revelam um grave problema de ordem teológica, demonstrando os equívocos de uma sociedade, que fundada sobre o prisma da religião, massacra os mais fracos. O desfecho do livro sinaliza uma possível epifania, mas, desvela-se num fracasso da religião e de seu poder salvífico. O trabalho procurou a partir dos principais temas do romance, perceber suas correspondências com o tema da religião. Pode com isso, a partir das contribuições de Scholem, ver como dilema existencial e subjetivo da personagem possui proximidade com o drama místico cabalístico, além disso, perceber as correlações entre a obra clariceana e textos da tradição judaíca, quer na sua referência explícita (nome da personagem), como também, em seu aspecto de busca, em sua forma de midrash e nas inúmeras exegeses e comentários que propicia. Finaliza seu esforço, levantando uma discussão ética sobre os valores teológicos da sociedade e seus profundos equívocos, ante a vida e morte da pobre Macabéa, uma mulher que viveu a religião nos limites da própria existência.

Complete resolution of the solid-state NMR spectrum of a uniformly 15N-labeled membrane protein in phospholipid bilayers

Complete resolution of the amide resonances in a three-dimensional solid-state NMR correlation spectrum of a uniformly 15N-labeled membrane protein in oriented phospholipid bilayers is demonstrated. The three orientationally dependent frequencies, 1H chemical shift, 1H–15N dipolar coupling, and 15N chemical shift, associated with each amide resonance are responsible for resolution among resonances and provide sufficient angular restrictions for protein structure determination. Because the protein is completely immobilized by the phospholipids on the relevant NMR time scales (10 kHz), the linewidths will not degrade in the spectra of larger proteins. Therefore, these results demonstrate that solid-state NMR experiments can overcome the correlation time problem and extend the range of proteins that can have their structures determined by NMR spectroscopy to include uniformly 15N-labeled membrane proteins in phospholipid bilayers.

Correlation of the structural and functional domains in the membrane protein Vpu from HIV-1

Vpu is an 81-residue membrane protein encoded by the HIV-1 genome. NMR experiments show that the protein folds into two distinct domains, a transmembrane hydrophobic helix and a cytoplasmic domain with two in-plane amphipathic α-helices separated by a linker region. Resonances in one-dimensional solid-state NMR spectra of uniformly 15N labeled Vpu are clearly segregated into two bands at chemical shift frequencies associated with NH bonds in a transmembrane α-helix, perpendicular to the membrane surface, and with NH bonds in the cytoplasmic helices parallel to the membrane surface. Solid-state NMR spectra of truncated Vpu2–51 (residues 2–51), which contains the transmembrane α-helix and the first amphipathic helix of the cytoplasmic domain, and of a construct Vpu28–81 (residues 28–81), which contains only the cytoplasmic domain, support this structural model of Vpu in the membrane. Full-length Vpu (residues 2–81) forms discrete ion-conducting channels of heterogeneous conductance in lipid bilayers. The most frequent conductances were 22 ± 3 pS and 12 ± 3 pS in 0.5 M KCl and 29 ± 3 pS and 12 ± 3 pS in 0.5 M NaCl. In agreement with the structural model, truncated Vpu2–51, which has the transmembrane helix, forms discrete channels in lipid bilayers, whereas the cytoplasmic domain Vpu28–81, which lacks the transmembrane helix, does not. This finding shows that the channel activity is associated with the transmembrane helical domain. The pattern of channel activity is characteristic of the self-assembly of conductive oligomers in the membrane and is compatible with the structural and functional findings.

Solid and liquid phase 59Co NMR studies of cobalamins and their derivatives

We describe the application of 59Co NMR to the study of naturally occurring cobalamins. Targets of these investigations included vitamin B12, the B12 coenzyme, methylcobalamin, and dicyanocobyrinic acid heptamethylester. These measurements were carried out on solutions and powders of different origins, and repeated at a variety of magnetic field strengths. Particularly informative were the solid-state central transition NMR spectra, which when combined with numerical line shape analyses provided a clear description of the cobalt coupling parameters. These parameters showed a high sensitivity to the type of ligands attached to the metal and to the crystallization history of the sample. 59Co NMR determinations also were carried out on synthetic cobaloximes possessing alkyl, cyanide, aquo, and nitrogenated axial groups, substituents that paralleled the coordination of the natural compounds. These analogs displayed coupling anisotropies comparable to those of the cobalamins, as well as systematic up-field shifts that can be rationalized in terms of their stronger binding affinity to the cobalt atom. Cobaloximes also displayed a higher regularity in the relative orientations of their quadrupole and shielding coupling tensors, reflecting a higher symmetry in their in-plane coordination. For the cobalamines, poor correlations were observed between the values measured for the quadrupole couplings in the solid and the line widths observed in the corresponding solution 59Co NMR resonances.

Noninvasive measurement of gene expression in skeletal muscle

We have developed a noninvasive detection method for expression of viral-mediated gene transfer. A recombinant adenovirus was constructed by using the gene for arginine kinase (AK), which is the invertebrate correlate to the vertebrate ATP-buffering enzyme, creatine kinase. Gene expression was noninvasively monitored using 31P-magnetic resonance spectroscopy (31P-MRS). The product of the AK enzyme, phosphoarginine (PArg), served as an MRS-visible reporter of AK expression. The recombinant adenovirus coding for arginine kinase (rAdCMVAK) was injected into the right hindlimbs of neonatal mice. Two weeks after injection of rAdCMVAK, a unique 31P-MRS resonance was observed. It was observable in all rAdCMVAK injected hindlimbs and was not present in the contralateral control or the vehicle injected limb. PArg and phosphocreatine (PCr) concentrations were calculated to be 11.6 ± 0.90 and 13.6 ± 1.1 mM respectively in rAdCMVAK injected limbs. AK activity was demonstrated in vivo by monitoring the decreases in PArg and ATP resonances during prolonged ischemia. After 1 h of ischemia intracellular pH was 6.73 ± 0.06, PCr/ATP was decreased by 77 ± 8%, whereas PArg/ATP was decreased by 50 ± 15% of basal levels. PArg and PCr returned to basal levels within 5 min of the restoration of blood flow. AK activity persisted for at least 8 mo after injection, indicating that adenoviral-mediated gene transfer can produce stable expression for long periods of time. Therefore, the cDNA encoding AK provides a useful reporter gene that allows noninvasive and repeated monitoring of gene expression after viral mediated gene transfer to muscle.

NMR and mutagenesis evidence for an I domain allosteric site that regulates lymphocyte function-associated antigen 1 ligand binding

The leukocyte integrin, lymphocyte function-associated antigen 1 (LFA-1) (CD11a/CD18), mediates cell adhesion and signaling in inflammatory and immune responses. To support these functions, LFA-1 must convert from a resting to an activated state that avidly binds its ligands such as intercellular adhesion molecule 1 (ICAM-1). Biochemical and x-ray studies of the Mac-1 (CD11b/CD18) I domain suggest that integrin activation could involve a conformational change of the C-terminal α-helix. We report the use of NMR spectroscopy to identify CD11a I domain residues whose resonances are affected by binding to ICAM-1. We observed two distinct sites in the CD11a I domain that were affected. As expected from previous mutagenesis studies, a cluster of residues localized around the metal ion-dependent adhesion site (MIDAS) was severely perturbed on ICAM-1 binding. A second cluster of residues distal to the MIDAS that included the C-terminal α-helix of the CD11a I domain was also affected. Substitution of residues in the core of this second I domain site resulted in constitutively active LFA-1 binding to ICAM-1. Binding data indicates that none of the 20 substitution mutants we tested at this second site form an essential ICAM-1 binding interface. We also demonstrate that residues in the I domain linker sequences can regulate LFA-1 binding. These results indicate that LFA-1 binding to ICAM-1 is regulated by an I domain allosteric site (IDAS) and that this site is structurally linked to the MIDAS.

NMR characterization and solution structure determination of the oxidized cytochrome c7 from Desulfuromonas acetoxidans

The solution structure of the three-heme electron transfer protein cytochrome c7 from Desulfuromonas acetoxidans is reported. The determination of the structure is obtained through NMR spectroscopy on the fully oxidized, paramagnetic form. The richness of structural motifs and the presence of three prosthetic groups in a protein of 68 residues is discussed in comparison with the four-heme cytochromes c3 already characterized through x-ray crystallography. In particular, the orientation of the three hemes present in cytochrome c7 is similar to that of three out of four hemes of cytochromes c3. The reduction potentials of the individual hemes, which have been obtained through the sequence-specific assignment of the heme resonances, are discussed with respect to the properties of the protein matrix. This information is relevant for any attempt to understand the electron transfer pathway.

Unusual 1H NMR chemical shifts support (His) Cɛ1—H⋅⋅⋅O⩵C H-bond: Proposal for reaction-driven ring flip mechanism in serine protease catalysis

13C-selective NMR, combined with inhibitor perturbation experiments, shows that the Cɛ1—H proton of the catalytic histidine in resting α-lytic protease and subtilisin BPN′ resonates, when protonated, at 9.22 ppm and 9.18 ppm, respectively, which is outside the normal range for such protons and ≈0.6 to 0.8 ppm further downfield than previously reported. They also show that the previous α-lytic protease assignments [Markley, J. L., Neves, D. E., Westler, W. M., Ibanez, I. B., Porubcan, M. A. & Baillargeon, M. W. (1980) Front. Protein Chem. 10, 31–61] were to signals from inactive or denatured protein. Simulations of linewidth vs. pH demonstrate that the true signal is more difficult to detect than corresponding signals from inactive derivatives, owing to higher imidazole pKa values and larger chemical shift differences between protonated and neutral forms. A compilation and analysis of available NMR data indicates that the true Cɛ1—H signals from other serine proteases are similarly displaced downfield, with past assignments to more upfield signals probably in error. The downfield displacement of these proton resonances is shown to be consistent with an H-bond involving the histidine Cɛ1—H as donor, confirming the original hypothesis of Derewenda et al. [Derewenda, Z. S., Derewenda, U. & Kobos, P. M. (1994) J. Mol. Biol. 241, 83–93], which was based on an analysis of literature x-ray crystal structures of serine hydrolases. The invariability of this H-bond among enzymes containing Asp-His-Ser triads indicates functional importance. Here, we propose that it enables a reaction-driven imidazole ring flip mechanism, overcoming a major dilemma inherent in all previous mechanisms, namely how these enzymes catalyze both the formation and productive breakdown of tetrahedral intermediates.

A nicked duplex decamer DNA with a PEG6 tether

A dumbbell double-stranded DNA decamer tethered with a hexaethylene glycol linker moiety (DDSDPEG), with a nick in the centre of one strand, has been synthesised. The standard NMR methods, E.COSY, TOCSY, NOESY and HMQC, were used to measure 1H, 31P and T1 spectral parameters. Molecular modelling using rMD-simulated annealing was used to compute the structure. Scalar couplings and dipolar contacts show that the molecule adopts a right-handed B-DNA helix in 38 mM phosphate buffer at pH 7. Its high melting temperature confirms the good base stacking and stability of the duplex. This is partly attributed to the presence of the PEG6 linker at both ends of the duplex that restricts the dynamics of the stem pentamers and thus stabilises the oligonucleotide. The inspection of the global parameters shows that the linker does not distort the B-DNA geometry. The computed structure suggests that the presence of the nick is not disturbing the overall tertiary structure, base pair geometry or duplex base pairing to a substantial extent. The nick has, however, a noticeable impact on the local geometry at the nick site, indicated clearly by NMR analysis and reflected in the conformational parameters of the computed structure. The 1H spectra also show much sharper resonances in the presence of K+ indicating that conformational heterogeneity of DDSDPEG is reduced in the presence of potassium as compared to sodium or caesium ions. At the same time the 1H resonances have longer T1 times. This parameter is suggested as a sensitive gauge of stabilisation.

Dissection of the ion-induced folding of the hammerhead ribozyme using 19F NMR

We have used 19F NMR to analyze the metal ion-induced folding of the hammerhead ribozyme by selective incorporation of 5fluorouridine. We have studied the chemical shift and linewidths of 19F resonances of 5-fluorouridine at the 4 and 7 positions in the ribozyme core as a function of added Mg2+. The data fit well to a simple two-state model whereby the formation of domain 1 is induced by the noncooperative binding of Mg2+ with an association constant in the range of 100 to 500 M−1, depending on the concentration of monovalent ions present. The results are in excellent agreement with data reporting on changes in the global shape of the ribozyme. However, the NMR experiments exploit reporters located in the center of the RNA sections undergoing the folding transitions, thereby allowing the assignment of specific nucleotides to the separate stages. The results define the folding pathway at high resolution and provide a time scale for the first transition in the millisecond range.

Observation via one-dimensional 13Calpha NMR of local conformational substates in thermal unfolding equilibria of a synthetic analog of the GCN4 leucine zipper.

Synthesis of a 33-residue, capped leucine zipper analogous to that in GCN4 is reported. Histidine and arginine residues are mutated to lysine to reduce the unfolding temperature. CD and ultracentrifugation studies indicate that the molecule is a two-stranded coiled coil under benign conditions. Versions of the same peptide are made with 99% 13Calpha at selected sites. One-dimensional 13C NMR spectra are assigned by inspection and used to study thermal unfolding equilibria over the entire transition from 8 to 73 degrees C. Spectra at the temperature extremes establish the approximate chemical shifts for folded and unfolded forms at each labeled site. Resonances for each amino acid appear at both locations at intermediate T, indicating that folded and unfolded forms interconvert slowly (> >2 ms) on the NMR time scale. Moreover, near room temperature, the structured form's resonance is double at several, but not all, sites, indicating at least two slowly interconverting, structured, local conformational substates. Analysis of the dynamics is possible. For example, near room temperature at the Val-9, Ala-24, and Gly-31 positions, the equilibrium constant for interconversion of the two structured forms is near unity and the time scale is > or= 10-20 ms.

Nuclear magnetic dipole interactions in field-oriented proteins: information for structure determination in solution.

The measurement of dipolar contributions to the splitting of 15N resonances of 1H-15N amide pairs in multidimensional high-field NMR spectra of field-oriented cyanometmyoglobin is reported. The splittings appear as small field-dependent perturbations of normal scalar couplings. Assignment of more than 90 resonances to specific sequential sites in the protein allows correlation of the dipolar contributions with predictions based on the known susceptibility and known structure of the protein. Implications as an additional source of information for protein structure determination in solution are discussed.