The Non-catalytic ``Cap Domain'' of a Mycobacterial Metallophosphoesterase Regulates Its Expression and Localization in the Cell

Despite highly conserved core catalytic domains, members of the metallophosphoesterase (MPE) superfamily perform diverse and crucial functions ranging from nucleotide and nucleic acid metabolism to phospholipid hydrolysis. Unique structural elements outside of the catalytic core called ``cap domains'' are thought to provide specialization to these enzymes; however, no directed study has been performed to substantiate this. The cap domain of Rv0805, an MPE from Mycobacterium tuberculosis, is located C-terminal to its catalytic domain and is dispensable for the catalytic activity of this enzyme in vitro. We show here that this C-terminal extension (CTE) mediates in vivo localization of the protein to the cell membrane and cell wall as well as modulates expression levels of Rv0805 in mycobacteria. We also demonstrate that Rv0805 interacts with the cell wall of mycobacteria, possibly with the mycolyl-arabinogalactan-peptidoglycan complex, by virtue of its C terminus, a hitherto unknown property of this MPE. Using a panel of mutant proteins, we identify interactions between active site residues of Rv0805 and the CTE that determine its association with the cell wall. Finally, we show that Rv0805 and a truncated mutant devoid of the CTE produce different phenotypic effects when expressed in mycobacteria. Our study thus provides a detailed dissection of the functions of the cap domain of an MPE and suggests that the repertoire of cellular functions of MPEs cannot be understood without exploring the modulatory effects of these subdomains.

Sequence and conformational preferences at termini of alpha-helices in membrane proteins: Role of the helix environment

-helices are amongst the most common secondary structural elements seen in membrane proteins and are packed in the form of helix bundles. These -helices encounter varying external environments (hydrophobic, hydrophilic) that may influence the sequence preferences at their N and C-termini. The role of the external environment in stabilization of the helix termini in membrane proteins is still unknown. Here we analyze -helices in a high-resolution dataset of integral -helical membrane proteins and establish that their sequence and conformational preferences differ from those in globular proteins. We specifically examine these preferences at the N and C-termini in helices initiating/terminating inside the membrane core as well as in linkers connecting these transmembrane helices. We find that the sequence preferences and structural motifs at capping (Ncap and Ccap) and near-helical (N' and C') positions are influenced by a combination of features including the membrane environment and the innate helix initiation and termination property of residues forming structural motifs. We also find that a large number of helix termini which do not form any particular capping motif are stabilized by formation of hydrogen bonds and hydrophobic interactions contributed from the neighboring helices in the membrane protein. We further validate the sequence preferences obtained from our analysis with data from an ultradeep sequencing study that identifies evolutionarily conserved amino acids in the rat neurotensin receptor. The results from our analysis provide insights for the secondary structure prediction, modeling and design of membrane proteins. Proteins 2014; 82:3420-3436. (c) 2014 Wiley Periodicals, Inc.

Design of symmetric TIM barrel proteins from first principles

Background: Computational protein design is a rapidly maturing field within structural biology, with the goal of designing proteins with custom structures and functions. Such proteins could find widespread medical and industrial applications. Here, we have adapted algorithms from the Rosetta software suite to design much larger proteins, based on ideal geometric and topological criteria. Furthermore, we have developed techniques to incorporate symmetry into designed structures. For our first design attempt, we targeted the (alpha/beta)(8) TIM barrel scaffold. We gained novel insights into TIM barrel folding mechanisms from studying natural TIM barrel structures, and from analyzing previous TIM barrel design attempts. Methods: Computational protein design and analysis was performed using the Rosetta software suite and custom scripts. Genes encoding all designed proteins were synthesized and cloned on the pET20-b vector. Standard circular dichroism and gel chromatographic experiments were performed to determine protein biophysical characteristics. 1D NMR and 2D HSQC experiments were performed to determine protein structural characteristics. Results: Extensive protein design simulations coupled with ab initio modeling yielded several all-atom models of ideal, 4-fold symmetric TIM barrels. Four such models were experimentally characterized. The best designed structure (Symmetrin-1) contained a polar, histidine-rich pore, forming an extensive hydrogen bonding network. Symmetrin-1 was easily expressed and readily soluble. It showed circular dichroism spectra characteristic of well-folded alpha/beta proteins. Temperature melting experiments revealed cooperative and reversible unfolding, with a T-m of 44 degrees C and a Gibbs free energy of unfolding (Delta G degrees) of 8.0 kJ/mol. Urea denaturing experiments confirmed these observations, revealing a C-m of 1.6 M and a Delta G degrees of 8.3 kJ/mol. Symmetrin-1 adopted a monomeric conformation, with an apparent molecular weight of 32.12 kDa, and displayed well resolved 1D-NMR spectra. However, the HSQC spectrum revealed somewhat molten characteristics. Conclusions: Despite the detection of molten characteristics, the creation of a soluble, cooperatively folding protein represents an advancement over previous attempts at TIM barrel design. Strategies to further improve Symmetrin-1 are elaborated. Our techniques may be used to create other large, internally symmetric proteins.

Dissecting Ubiquitin Folding Using the Self-Organized Polymer Model

Folding of Ubiquitin (Ub), a functionally important protein found in eukaryotic organisms, is investigated at low and neutral pH at different temperatures using simulations of the coarse-grained self-organized-polymer model with side chains (SOP-SC). The melting temperatures (T-m's), identified with the peaks in the heat capacity curves, decrease as pH decreases, in qualitative agreement with experiments. The calculated radius of gyration, showing dramatic variations with pH, is in excellent agreement with scattering experiments. At T-m Ub folds in a two-state manner at low and neutral pH. Clustering analysis of the conformations sampled in equilibrium folding trajectories at T-m with multiple transitions between the folded and unfolded states, shows a network of metastable states connecting the native and unfolded states. At low and neutral pH, Ub folds with high probability through a preferred set of conformations resulting in a pH-dependent dominant folding pathway. Folding kinetics reveal that Ub assembly at low pH occurs by multiple pathways involving a combination of nucleation-collapse and diffusion collision mechanism. The mechanism by which Ub folds is dictated by the stability of the key secondary structural elements responsible for establishing long-range contacts and collapse of Ub. Nucleation collapse mechanism holds if the stability of these elements are marginal, as would be the case at elevated temperatures. If the lifetimes associated with these structured microdomains are on the order of hundreds of microseconds, then Ub folding follows the diffusion collision mechanism with intermediates, many of which coincide with those found in equilibrium. Folding at neutral pH is a sequential process with a populated intermediate resembling that sampled at equilibrium. The transition state structures, obtained using a P-fold analysis, are homogeneous and globular with most of the secondary and tertiary structures being native-like. Many of our findings for both the thermodynamics and kinetics of folding are not only in agreement with experiments but also provide missing details not resolvable in standard experiments. The key prediction that folding mechanism varies dramatically with pH is amenable to experimental tests.

The putative role of some conserved water molecules in the structure and function of human transthyretin

Human transthyretin (hTTR) is a multifunctional protein that is involved in several neurodegenerative diseases. Besides the transportation of thyroxin and vitamin A, it is also involved in the proteolysis of apolipoprotein A1 and A beta peptide. Extensive analyses of 32 high-resolution X-ray and neutron diffraction structures of hTTR followed by molecular-dynamics simulation studies using a set of 15 selected structures affirmed the presence of 44 conserved water molecules in its dimeric structure. They are found to play several important roles in the structure and function of the protein. Eight water molecules stabilize the dimeric structure through an extensive hydrogen-bonding network. The absence of some of these water molecules in highly acidic conditions (pH <= 4.0) severely affects the interfacial hydrogen-bond network, which may destabilize the native tetrameric structure, leading to its dissociation. Three pairs of conserved water molecules contribute to maintaining the geometry of the ligand-binding cavities. Some other water molecules control the orientation and dynamics of different structural elements of hTTR. This systematic study of the location, absence, networking and interactions of the conserved water molecules may shed some light on various structural and functional aspects of the protein. The present study may also provide some rational clues about the conserved water-mediated architecture and stability of hTTR.

Anomalous reduction in domain wall displacement at the morphotropic phase boundary of the piezoelectric alloy system PbTiO3-BiScO3

A comparative study of field-induced domain switching and lattice strain was carried out by in situ electric-field-dependent high-energy synchrotron x-ray diffraction on a morphotropic phase boundary (MPB) and a near-MPB rhombohedral/pseudomonoclinic composition of a high-performance piezoelectric alloy (1-x) PbTiO3-(x)BiScO3. It is demonstrated that the MPB composition showing large d(33) similar to 425 pC/N exhibits significantly reduced propensity of field-induced domain switching as compared to the non-MPB rhombohedral composition (d(33) similar to 260 pC/N). These experimental observations contradict the basic premise of the martensitic-theory-based explanation which emphasizes on enhanced domain wall motion as the primary factor for the anomalous piezoelectric response in MPB piezoelectrics. Our results favor field-induced structural transformation to be the primary mechanism contributing to the large piezoresponse of the critical MPB composition of this system.

La teoría de la justicia de Amartya Sen y los orígenes del concepto católico de justicia social en Antonio Rosmini

Resumen: El autor propone en este artículo reflexionar sobre algunos aspectos del libro The Idea of Justice de Amartya Sen a la luz d e la obra del filósofo italiano Antonio Rosmini, difusor de la expresión “justicia social” en los orígenes del pensamiento social católico. En primer lugar, el autor muestra las coincidencias de Sen y Rosmini sobre el lugar de la razón en el análisis de la idea de justicia social frente a los planteos puramente emocionales y pragmáticos. En segundo lugar, propone una comparación entre las críticas que desarrollan Sen y Rosmini a las denominadas por el primero “teorías trascendentales” de la justicia y a los llamado s “cambios estructurales” así como la necesidad de cambios graduales para llegar a una sociedad justa. En tercer lugar, intenta mostrar cómo la opción por las personas y sus capacidades elegida por Sen puede ser iluminada y profundizada desde la teoría rosminiana de las capacidades. En cuarto lugar, describe el giro cognitivo que propone Sen para las instituciones y las políticas públicas. Finalmente, el autor enuncia algunas objeciones que pudieran hacerse al planteo propuesto en el artículo junto con unos comentarios finales sobre las mismas.

Umbrales del relato y autorreferencia en los libros de viajes españoles contemporáneos

Número monográfico: El viaje y sus discursos

Asistencia, recuperación y restauración de las víctimas

[ES] Junto con formas simples de restitución y compensación, se han detectado variantes de las primeras prácticas de justicia restaurativa en las antiguas civilizaciones de Israel, Sumeria, Babilonia y Roma y entre los pueblos aborígenes de Norteamérica y Oceanía. Por su parte, en el siglo XX, principalmente en respuesta a la ola criminal de los años 60 y 70, y buscando alternativas a los métodos tradicionales de tratamiento de los delitos de esa época, comenzaron a emerger programas experimentales que usaban los principios de justicia restaurativa a comienzos de los 70 en Canadá, Estados Unidos, Inglaterra, Australia y Nueva Zelanda.

Accelerogram processing using reliability bounds and optimal correction methods

This study addresses the problem of obtaining reliable velocities and displacements from accelerograms, a concern which often arises in earthquake engineering. A closed-form acceleration expression with random parameters is developed to test any strong-motion accelerogram processing method. Integration of this analytical time history yields the exact velocities, displacements and Fourier spectra. Noise and truncation can also be added. A two-step testing procedure is proposed and the original Volume II routine is used as an illustration. The main sources of error are identified and discussed. Although these errors may be reduced, it is impossible to extract the true time histories from an analog or digital accelerogram because of the uncertain noise level and missing data. Based on these uncertainties, a probabilistic approach is proposed as a new accelerogram processing method. A most probable record is presented as well as a reliability interval which reflects the level of error-uncertainty introduced by the recording and digitization process. The data is processed in the frequency domain, under assumptions governing either the initial value or the temporal mean of the time histories. This new processing approach is tested on synthetic records. It induces little error and the digitization noise is adequately bounded. Filtering is intended to be kept to a minimum and two optimal error-reduction methods are proposed. The "noise filters" reduce the noise level at each harmonic of the spectrum as a function of the signal-to-noise ratio. However, the correction at low frequencies is not sufficient to significantly reduce the drifts in the integrated time histories. The "spectral substitution method" uses optimization techniques to fit spectral models of near-field, far-field or structural motions to the amplitude spectrum of the measured data. The extremes of the spectrum of the recorded data where noise and error prevail are then partly altered, but not removed, and statistical criteria provide the choice of the appropriate cutoff frequencies. This correction method has been applied to existing strong-motion far-field, near-field and structural data with promising results. Since this correction method maintains the whole frequency range of the record, it should prove to be very useful in studying the long-period dynamics of local geology and structures.

Sterochemically modified polyamides for recognition in the minor groove of DNA

The design of synthetic molecules that recognize specific sequences of DNA is an ongoing challenge in molecular medicine. Cell-permeable small molecules targeting predetermined DNA sequences offer a potential approach for offsetting the abnormal effects of misregulated gene-expression. Over the past twenty years, Professor Peter B. Dervan has developed a set of pairing rules for the rational design of minor groove binding polyamides containing pyrrole (Py), imidazole (Im), and hydroxypyrrole (Hp). Polyamides have illustrated the capability to permeate cells and inhibit transcription of specific genes in vivo. This provides impetus to identify structural elements that expand the repetoire of polyamide motifs with recognition properties comparable to naturally occurring DNA binding proteins. Through the introduction of chiral amino acids, we have developed chiral polyamides with stereochemically regulated binding characteristics. In addition, chiral substituents have facilitated the development of new polyamide motifs that broaden binding site sizes targetable by this class of ligands.

Autonomia da vontade, consensualismo e arbitragem: A extensão da cláusula compromissória a partes não-signatárias fundamentada na teoria dos grupos de sociedades. A prática da Corte Internacional de Arbitragem da Câmara de Comércio Internacional (CCI) e sua compatibilidade com o ordenamento jurídico brasileiro

A prática da arbitragem comercial internacional tem se deparado, há pelos menos quatro décadas, com a problemática da extensão da cláusula compromissória a uma parte não-signatária, integrante do mesmo grupo de sociedades a que pertence uma das partes integrantes da convenção, em razão do comportamento adotado pela parte não-signatária nas fases de negociação do contrato, execução ou extinção. Nesse sentido, a prática da Corte Internacional de Arbitragem da Câmara de Comércio Internacional dos últimos trinta anos e reiteradas decisões judiciais em países de diferentes tradições jurídicas como a França, Suíça e Estados Unidos têm se manifestado favoravelmente a essa extensão subjetiva da convenção de arbitragem. O estudo da doutrina nacional e da jurisprudência do Superior Tribunal de Justiça sobre os grupos de sociedades e seus efeitos, e a análise detida de diversos precedentes do Superior Tribunal de Justiça e do Supremo Tribunal Federal sobre a homologação de sentenças arbitrais estrangeiras, revelam a compatibilidade da referida prática arbitral internacional com o ordenamento jurídico brasileiro.

Verificação à fadiga de pontes rodoviárias mistas (aço-concreto)

As pontes rodoviárias mistas (aço-concreto) estão sujeitadas às ações dinâmicas variáveis, por exemplo, devido ao tráfego de veículos sobre a superfície irregular do pavimento. Estas ações dinâmicas podem gerar a nucleação de fraturas ou mesmo a sua propagação sobre a estrutura. A correta consideração desses aspectos objetivou o desenvolvimento de uma metodologia de análise, com a finalidade de avaliar os níveis dos esforços e tensões oriundos do tráfego dos veículos sobre a superfície irregular do pavimento e, bem como, proceder uma verificação à fadiga de obras de arte rodoviárias em aço e mistas (aço-concreto). Para tal, as técnicas para a contagem de ciclos de tensão e a aplicação das regras de dano acumulado foram analisadas através de curvas do tipo S-N, associadas a diversas normas de projeto. A ponte rodoviária mista (aço-concreto) investigada neste estudo é constituída por quatro vigas de aço longitudinais e por um tabuleiro de concreto armado. O modelo numérico-computacional, desenvolvido para a análise dinâmica da ponte, foi concebido com base em técnicas usuais de discretização através do método dos elementos finitos. Simulam-se as almas das vigas de aço e as lajes de concreto do tabuleiro através de elementos finitos de casca. As mesas dessas vigas, transversinas e os enrijecedores são modelados por elementos de viga tridimensionais. Os veículos são representados a partir de sistemas "massa-mola-amortecedor". O tráfego dessas viaturas é considerado mediante a simulação de comboios semi-infinitos, deslocando-se com velocidade constante sobre a ponte. As conclusões da presente investigação versam acerca da vida útil de serviço dos elementos estruturais de pontes mistas (aço-concreto).

Modelagem numérica de elementos tracionados em aço inoxidável com parafusos defasados

Atualmente, a utilização do aço inoxidável em elementos estruturais é considerada uma solução cara para os problemas da engenharia estrutural. Todavia, mudanças de atitudes dentro da construção civil, uma transição global para um desenvolvimento sustentável e redução em impactos ambientais têm seguramente provocado um aumento na utilização do aço inoxidável. As normas de projeto de aço inoxidável atuais são, em grande parte, baseadas em analogias assumidas com o comportamento de estruturas desenvolvidas com aço carbono. Todavia, o aço inoxidável apresenta quatro curvas não-lineares tensão versus deformação (tensão e compressão, paralela e perpendicular a laminação do material), sem patamar de escoamento e região de encruamento claramente definidos, modificando assim, o comportamento global das estruturas que o utilizam. Em elementos estruturais submetidos a forças axiais de tração, a ruptura da seção líquida representa um dos estados limites últimos a serem verificados. Com o objetivo de se avaliar a resistência a tração de elementos estruturais aparafusados em aço inoxidável S304, este trabalho apresenta um modelo numérico baseado no método dos elementos finitos através do programa Ansys (versão 11). A não-linearidade do material foi considerada através do critério de plastificação de Von Mises e curvas tensão versus deformação verdadeira. A não-linearidade geométrica foi introduzida no modelo através da Formulação de Lagrange atualizado. O modelo numérico foi calibrado com resultados experimentais obtidos em ensaios de laboratório, a partir de ligações aparafusadas alternadas rígidas, onde não se ocorre nenhuma rotação entre os membros, transferindo nenhum momento fletor, apenas esforço normal e cisalhante.

O cantar da Germânia: política e cultura na Alemanha na passagem dos séculos XII e XIII

Esta tese desenvolvida na linha de pesquisa Política e Cultura destaca o interesse pelos fenômenos da transmissão do conhecimento intelectual. A tese reconstrói a expressão da cultura política medieval germânica pela ótica de dois poetas, Walther von der Vogelweide e Wolfram von Eschenbach, integrantes da aristocracia guerreira. Os suportes teóricos dos Sprüche, sentenças de natureza política de Vogelweide e do Parzifal de Eschenbach, foram fundamentais para compreender o posicionamento político e cultural alemão frente às transformações que afetaram a Cristandade no período de fins do século XII ao início do século XIII. Esse período foi marcado pelo confronto de Papas e Imperadores em torno do direito de exercer a autoridade no âmbito do Sacro Império Romano-Germânico. O movimento cruzadista, o renascimento urbano e a ascensão de novos atores sociais burgueses integram o conjunto dos elementos a serem levados em conta na elaboração da tese. A ideia norteadora do trabalho tornou necessário recorrer a obras literárias com objetivo de elucidar questões de natureza histórica, tendo claro que a Literatura não é um contraponto da História e o texto resultante contribua para ressignificar a produção política da cultura medieval e um melhor entendimento dos mecanismos do poder na Alemanha medieval. No plano teórico-metodológico recorremos à chamada História Cultural fornecendo uma visão integradora dos planos político, social e econômico.