Structural and dynamical studies on human epidermal-type fatty acid binding protein using high-resolution NMR spectroscopy

Human epidermal-type fatty acid binding protein (E-FABP) belongs to a family of intracellular non-enzymatic 14-15 kDa lipid binding proteins (LBP) that specifically bind and facilitate the transport of fatty acids, bile acids or retinoids. Their functions have also been associated with fatty acid signalling, cell growth, regulation and differentiation. As a contribution to better understand the structure-function relationship of this protein, the features of its solution structure determined by NMR spectroscopy are reported here. Both unlabeled and 15N-enriched samples of recombinant human E-FABP were used for multidimensional high-resolution NMR. The sequential backbone as well as side-chain resonance assignments have been completed. They are reported here and are also available at the BioMagResBank under the accession number BMRB-5083. The presence of six cysteines in the amino acid sequence of human E-FABP is highly unusual for LBPs. Four of the six cysteines are unique to the E-FABPs: C43, C47, C67 and C87. In the three-dimensional structure of E-FABP, two cysteine pairs (C67/C87 and C120/C127) were identified by X-ray analysis to be close enough to allow disulfide bridge formation, but a S-S bond was actually found only between C120 and C127 [Hohoff et al., 1999]. Since the exclusion of a disulfide bridge between C67 and C87 improved the Rfree factor of the crystallographic model, the existence of a covalent bond between these two side- chains was considered unlikely. This agrees with the NMR data, where SCH resonances have been observed for the cysteine residues C43, C67 (tentative assignment) and C87, thus excluding the possibility of a second disulfide bridge in solution. Based on the NOE and hydrogen exchange data, an ensemble of 20 energy-minimized conformers representing the solution structure of human E-FABP complexed with stearic acid has been obtained. The analysis of homonuclear 2D NOESY and 15N-edited 3D NOESY spectra led to a total of 2926 NOE-derived distance constraints. Furthermore, 37 slow- exchanging backbone amide protons were identified to be part of the hydrogen-bonding network in the >-sheet and subsequently converted into 74 additional distance constraints. Finally, the disulfide bridge between C120 and C127 was defined by 3 upper and 3 lower distance bounds. The structure calculation program DYANA regarded 998 of these constraints as irrelevant, i.e., they did not restrict the distance between two protons. Out of the remaining 2008 non-trivial distance constraints, 371 were intraresidual (i = j), 508 sequential (|i - j| = 1), 233 medium-range (1 4) NOEs. The protein mainly consists of 10 antiparallel -strands forming a >-barrel structure with a large internal cavity. The three-dimensional solution structure of human E-FABP has been determined with a root-mean-square deviation of 0.92 ± 0.11 Å and 1.46 ± 0.10 Å for the backbone and heavy atoms, respectively, excluding the terminal residues. Without the portal region (i.e., for residues 4-26, 40-56, 63-75 and 83-134; the portal region apparently represents the only opening in the protein surface through which the fatty acid ligand can enter and exit the internal binding cavity), an average backbone RMSD of 0.85 ± 0.10 Å was obtained, thus reflecting the higher conformational dispersion in the portal region. Superposition with the X-ray structure of human E-FABP (excluding the terminal residues) yielded average backbone RMSD values of 1.00 ± 0.07 Å for the entire residue range and 0.98 ± 0.06 Å without the portal region. This indicates a close similarity of the crystallographic and the solution structures. The structure coordinates have been deposited at the RCSB data bank under PDB ID code 1JJJ. The measurement of 15N relaxation experiments (T1, T2 and heteronuclear NOE) at three different fields (500, 600 and 800 MHz) provided information on the internal dynamics of the protein backbone. Nearly all non-terminal backbone amide groups showed order parameters S2 > 0.8, with an average value of 0.88 ± 0.04, suggesting a uniformly low backbone mobility in the nanosecond-to-picosecond time range throughout the entire protein sequence. Moreover, hydrogen/deuterium exchange experiments indicated a direct correlation between the stability of the hydrogen-bonding network in the >-sheet structure and the conformational exchange (Rex) in the millisecond-to-microsecond time range. The features of E-FABP backbone dynamics elaborated here differ from those of the phylogenetically closely related heart-type FABP and the more distantly related ileal lipid binding protein. The results on protein dynamics obtained in this work allow to conclude that the different LBP family members E-FABP, H-FABP and ILBP are characterized by varying stabilities in the protein backbone structures. Hydrogen/deuterium exchange experiments displayed significant differences in the chemical exchange with the solvent for the backbone amide protons belonging to the hydrogen-bonding network in the >-sheets. The >-barrel structure of H- FABP appears to be the most rigid, with exchange processes presumably slower than the millisecond-to-microsecond time range. ILBP, on the other hand, shows the fastest hydrogen exchange as well as a significant number of exchange parameters (Rex), implying a decreased stability in the >-sheet structure. E-FABP, finally, appears to rank between these two proteins based on the hydrogen/deuterium exchange, with Rex terms in the >-strands indicating millisecond-to-microsecond exchange processes like in ILBP

Dulce patria, un poemario sobre la dictadura chilena

Las dictaduras del Cono Sur originaron numerosos textos literarios que narran la violencia ocurrida, queriendo también servir de testimonios. En el presente texto presentamos el poemario Dulce patria, de nuestra autoría, publicado en 2012, y que, recreando voces de diversos grupos sociales, relata la historia de la dictadura chilena. La violencia atravesó el mundo político, el económico, las relaciones de trabajo y la cultura, habiéndose manifestado también en otros campos, como los vínculos familiares y el exilio. Interesa en el presente texto comentar principalmente las estrategias narrativas utilizadas en la confección del poemario.As ditaduras do Cone Sul originaram numerosos textos literários que narram a violência ocorrida, querendo servir também de testemunhos. No presente texto apresentamos o poemário Dulce patria, de nossa autoria, publicado em 2012, e que, recriando vozes de diversos grupos sociais, relata a história da ditadura chilena. A violência atravessou o mundo político, o econômico, as relações de trabalho e a cultura, mas manifestou-se também em outros campos, como nos vínculos familiares e o exílio. Interessa no presente texto comentar principalmente as estratégias narrativas utilizadas na confecção do poemário.The dictatorships of the Southern Cone originated numerous literary texts that narrate the violence that occurred, and also purport to serve as a testimony thereof. In the present text we present the poem collection Dulce patria (Sweet Fatherland) of our authorship, and published in 2012, which recreates voices from various social groups, and thereby tells a history of the Chilean dictatorship. Violence crossed over the political and economic world, as well as labor relations and culture, and also manifested itself in other fields, such as family ties and exile. The present text aims to comment mainly on the narrative strategies used in creating the poems

Mestizo exaltation: the invention of Chilean roto

This article examines the process of transformation of the roto, a Chilean personage of urban origin, into a national symbol of chilenidade. As a descendent of Spaniards and Natives, the mestizo was rejected in the Nineteenth century in the light of racial theories that were current at that time. However, at the beginning of the Twentieth century it seemed that intellectuals discovered the mestizo qualities, and tried to raise its status as one of a national symbol

O Tráfico de Escravos de Moçambique para as ilhas do índico, 1720-1902

O Tráfico de Escravos de Moçambique para as ilhas do índico, 1720-1902. Maputo; Núcleo Editorial da Universidade Eduardo Mondlane, 1987

O tráfico de crianças escravas para o Brasil durante o século XVIII

Estudando listas de escravos transportados do porto de Luanda entre 1734 e 1769, o autor estima em cerca de 10% a presença de crianças entre os escravos. Analisa os decretos de impostos de 1758, que discerniam as crias de pé e as crias de peito. Chega a uma média anual de 534 crianças, das quais 73% seriam crias de pé, declinando o número de crianças na segunda metade do século XVIII. O artigo também analisa as circunstâncias de capturas das crianças e o modo como eram alojadas nos navios, bem como sua taxa de mortalidade na travessia. Procura definir a importância de Luanda com relação a outros portos de Angola e comparar as porcentagens relativas de crianças nos portos do Rio de Janeiro, Bahia e Pernambuco. Finalmente, compara o tráfico brasileiro com o tráfico inglês, dinamarquês e espanhol nas Antilhas, que apresentavam um número bastante superior de crianças.The author analyses lists of slaves imported from Luanda between 1734 and 1769, estimating the presence of 10% of children in the slave trafic to Brazil. Perusing tax decrees of 1758, notices that different amounts of taxes were payed by "crias de pé" and children still in their mothers lap. The author establishes an annual rate of 534 children, that declined after the middle of the 18th century. The article also describes the manner they were captured in the African coast, their lodgement on board the slave ships, their rate of mortality; compares Luanda to other Angolan and Brazilian ports. Finally, the author compares the brazilian trade in slave children to the higher numbers registered for the British, Dutch and ish trade in the Caribbean region