Evelyne Lebrun

1H-13C nuclear magnetic resonance assignment and structural characterization of HIV-1 Tat protein.

Tat is a viral protein essential for activation of the HIV genes and plays an important role in the HIV-induced immunodeficiency. We chemically synthesized a Tat protein (86 residues) with its six glycines C alpha labelled with 13C. This synthetic protein has the full Tat activity. Heteronuclear nuclear magnetic resonance (NMR) spectra and NOE back-calculation made possible the sequential assignment of the 86 spin systems. Consequently, 915 NMR restraints were identified and 272 of them turned out to be long range ([i-j] > 4), providing structural information on the whole Tat protein. The poor spectral dispersion of Tat NMR spectra does not allow an accurate structure to be determined as for other proteins studied by 2D NMR. Nevertheless, we were able to determine the folding for Tat protein at a 1-mM protein concentration in a 100 mM, pH 4.5 phosphate buffer. The two main Tat functional regions, the basic region and the cysteine-rich region, are well exposed to solvent while a part of the N-terminal region and the C-terminal region constitute the core of Tat Bru. The basic region adopts an extended structure while the cysteine-rich region is made up of two loops. Resolution of this structure was determinant to develop a drug design approach against Tat. The chemical synthesis of the drugs allowed the specific binding and the inhibition of Tat to be verified.

Phylogeny of Rieske/cytb Complexes with a Special Focus on the Haloarchaeal Enzymes

Rieske/cytochrome b (Rieske/cytb) complexes are proton pumping quinol oxidases that are present in most bacteria and Archaea. The phylogeny of their subunits follows closely the 16S-rRNA phylogeny, indicating that chemiosmotic coupling was already present in the last universal common ancestor of Archaea and bacteria. Haloarchaea are the only organisms found so far that acquired Rieske/cytb complexes via interdomain lateral gene transfer. They encode two Rieske/cytb complexes in their genomes; one of them is found in genetic context with nitrate reductase genes and has its closest relatives among Actinobacteria and the Thermus/Deinococcus group. It is likely to function in nitrate respiration. The second Rieske/cytb complex of Haloarchaea features a split cytochrome b sequence as do Cyanobacteria, chloroplasts, Heliobacteria, and Bacilli. It seems that Haloarchaea acquired this complex from an ancestor of the above-mentioned phyla. Its involvement in the bioenergetic reaction chains of Haloarchaea is unknown. We present arguments in favor of the hypothesis that the ancestor of Haloarchaea, which relied on a highly specialized bioenergetic metabolism, that is, methanogenesis, and was devoid of quinones and most enzymes of anaerobic or aerobic bioenergetic reaction chains, integrated laterally transferred genes into its genome to respond to a change in environmental conditions that made methanogenesis unfavorable.

Inhibition of bovine dihydrofolate reductase and enhancement of methotrexate sensitivity by N4-(2-acetoxyethoxymethyl)-2acetylpyridine thiosemicarbazone

N4-(2-Acetoxyethoxymethyl)-2-acetylpyridine thiosemicarbazone (AATSC) belongs to a series of molecules known to have broad antimicrobial inhibitory activity. These molecules contain the 2-acetoxyethoxy moiety which could conceivably take up a conformation analogous to that of the ribosyl group. Moreover, the thiosemicarbazone moiety, when in the presence of a suitable enzymatic site, could mimic the triazine group, which is found in a number of antifolate drugs. AATSC, which has both bacterial inhibitory activity and water solubility, was accordingly evaluated for its antifolate activity against the bovine liver dihydrofolate reductase. AATSC is shown to be a fully uncompetitive inhibitor of that enzyme. Furthermore, AATSC enhances the activity of methotrexate. Such a potentiation could be useful for therapeutic purposes.

Iron-Sulfur Centers Involved in Photosynthetic Light Reactions

Publisher Summary Since the beginning of 199Os, the trend appears to have reverted to strong interactions between the fields of photosynthetic studies and bioinorganic chemistry largely because of the impact of molecular biology, particularly, site-specific mutagenesis and heterologous expression. This chapter summarizes these recent advances with emphasis on the part played by iron–sulfur centers in photosynthesis. If the inorganic chemistry of iron–sulfur centers has evolved tremendously since 1962, the modern notion of photosynthesis equally has little to do with its early versions. It is known that plant-type photosynthesis occurred first in eubacteria or more specifically in the ancestors of extant cyanobacteria, and was subsequently imported into eukaryotes via the events of endosymbiosis. As no photosynthetic mechanisms based on chlorophylls have so far been found in archaebacteria, the evolution of photosynthesis is considered to have exclusively occurred within the domain of eubacteria. In this domain, two further (anoxygenic) photosynthetic principles have been observed, each involving a cytochrome bc complex and only a single reaction center (RC) that resembles either photosystem I or photosystem II of oxygenic photosynthesis. The iron–sulfur centers participating in the light reactions of the three different photosynthetic principles are discussed in the chapter.

Crystallization and preliminary X-ray diffraction studies of the mitogenic lentil (Lens culinaris) lectin

The lentil lectin extracted from Lens culinaris seeds crystallizes in the orthorhombic space group P 2 1 2 1 2 1 with unit cell dimensions a =50·1 A, b =67·8 A and c =130·2 A. The asymmetric unit contains one, protein molecule. The crystals are suitable for high resolution work.

1H NMR Study of the Fe4S4 Center in Ferredoxin I fromDesulfovibrio desulfuricansNorway:Sequence-Specific Assignment of theCluster-Ligated Cysteines

The oxidized and reduced forms of the [Fe4–S4] ferredoxin I from Desulfovibrio desulfuricans Norway were investigated by 1H NMR spectroscopy with the aim of obtaining the complete assignment of the cysteines ligating the cluster. A combination of TOCSY and NOESY measurements together with information from the x‐ray crystallographic structure of related ferredoxins provided the sequence‐specific assignment of the four cysteines coordinated to the cluster. Through EXSY experiments, the hyperfine shifted resonance signals in the reduced ferredoxin were also assigned. The temperature dependence of the contact‐shifted cysteinyl residues of the reduced ferredoxin reveals that two cysteines exhibit anti‐Curie behavior whereas the other two cysteines display Curie behavior; that identifies Cys 9 (I) and Cys 15 (III) as ligated to the mixed‐valence iron ions.

ANALYSIS OF NMR DATA AND GLOBAL FOLD OF THE FE4-S4 FERREDOXIN I FROM DESULFOVIBRIO DESULFURICANS NORWAY

The oxidized form of the [Fe4–S4] ferredoxin I from Desulfovibrio desulfuricans Norway (DdN Fd I) was investigated by 1H and 13C NMR spectroscopy. The sequence‐specific 1H assignments of 93% of the amino acid residues of the whole protein, a complete determination of its secondary structure and an identification of a disulfide bridge are reported. The secondary structure of DdN Fd I was determined from both sequential and spatial NOEs. These NOEs reveal two anti‐parallel β‐sheets including Thr1–Ile4 and Glu59–Ile56, Val22–Ile26 and Thr35–Lys31, one helical segment (ranging from Ala41 to Asp49) and three tight turns. Three‐dimensional features of DdN Fd I were evidenced from long‐range NOE cross peaks between the secondary structural elements of the protein. Among them, a possible disulfide bridge, located between the pair of cysteines which are not coordinated to the cluster, was indicated by a 13C–1H HSQC experiment at natural abundance. The comparison of secondary structural elements and tertiary contacts of DdN Fd I protein with those of ferredoxins from mesophilic bacteria Desulfovibrio gigas and Desulfovibrio africanus and that from the hyperthermostable archaeon Thermococcus litoralis confirms that DdN Fd I exhibits the same global protein folding topology as the others. The chemical shifts of DdN Fd I were compared with those of other monocluster‐type ferredoxins. They show a peculiar conservation of the hydrophobic core of these ferredoxins.