Structural studies on the enzyme complex isopropylmalate isomerase (LeuCD) from Mycobacterium tuberculosis

The absence of the leucine biosynthesis pathway in humans makes the enzymes of this pathway in pathogenic bacteria such as Mycobacterium tuberculosis potential candidates for developing novel antibacterial drugs. One of these enzymes is isopropylmalate isomerase (IPMI). IPMI exists as a complex of two subunits: the large (LeuC) and the small (LeuD) subunit. The functional LeuCD complex catalyzes the stereospecific conversion reaction of α‐isopropylmalate to β‐isopropylmalate. Three C‐terminally truncated variants of LeuD have been analyzed by X‐ray crystallography to resolutions of 2.0 Å (LeuD_1–156), 1.2 Å (LeuD_1–168), and 2.5 Å (LeuD_1–186), respectively. The two most flexible parts of the structure are the regions of residues 30–37, the substrate discriminating loop, and of residues 70–74, the substrate binding loop. The three determined structures were also compared with the structures of other bacterial LeuDs. This comparison suggests the presence of two LeuD subfamilies. A model for the structure of the inactive enzyme complex has been obtained from solution X‐ray scattering experiments. The crystal structure of LeuD was shown to be compatible with the solution X‐ray scattering data from the small subunit. In contrast, the solution scattering results suggest that the large subunit LeuC and the LeuCD complex have overall shapes, which are radically different from the ones observed in the crystals of the functional homolog mitochondrial aconitase. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Genome sequence of Chthoniobacter flavus Ellin428, an aerobic heterotrophic soil bacterium

Chthoniobacter flavus Ellin428 is the first isolate from the class Spartobacteria of the bacterial phylum Verrucomicrobia. C. flavus Ellin428 can metabolize many of the saccharide components of plant biomass but is incapable of growth on amino acids or organic acids other than pyruvate.     

Inhibition of RNase L and RNA-dependent protein kinase (PKR) by sunitinib impairs antiviral innate immunity

RNase L and RNA-dependent protein kinase (PKR) are effectors of the interferon antiviral response that share homology in their pseudokinase and protein kinase domains, respectively. Sunitinib is an orally available, ATP-competitive inhibitor of VEGF and PDGF receptors used clinically to suppress angiogenesis and tumor growth. Sunitinib also impacts IRE1, an endoplasmic reticulum protein involved in the unfolded protein response that is closely related to RNase L. Here, we report that sunitinib is a potent inhibitor of both RNase L and PKR with IC(50) values of 1.4 and 0.3 μM, respectively. In addition, flavonol activators of IRE1 inhibited RNase L. Sunitinib treatment of wild type (WT) mouse embryonic fibroblasts resulted in about a 12-fold increase in encephalomyocarditis virus titers. However, sunitinib had no effect on encephalomyocarditis virus growth in cells lacking both PKR and RNase L. Furthermore, oral delivery of sunitinib in WT mice resulted in 10-fold higher viral titers in heart tissues while suppressing by about 2-fold the IFN-β levels. In contrast, sunitinib had no effect on viral titers in mice deficient in both RNase L and PKR. Also, sunitinib reduced mean survival times from 12 to 6 days in virus-infected WT mice while having no effect on survival of mice lacking both RNase L and PKR. Results indicate that sunitinib treatments prevent antiviral innate immune responses mediated by RNase L and PKR.     

Design Principles of FRET‐Based Dye‐Sensitized Solar Cells with Buried Quantum Dot Donors

In this article, the physics of FRET is demonstrated for an architecture of dye‐sensitized solar cells, in which the quantum dot “antennas” that serve as donors are incorporated into the solid titania electrode, providing isolation from electrolyte quenching, and potentially increased photostability. The energy transferred to the dye acceptor from the quantum dot donor, in addition to the direct light absorption by the dye, finally induce dye excitation and electron injection to the metal oxide semiconductor electrode. We use time‐resolved photoluminescence measurements to directly show achievement of FRET efficiencies of up to 70%, corresponding to over 80% internal quantum efficiency when considering radiative energy transfer as well. The various parameters governing the FRET efficiency and the requirements for high efficiency FRET‐based cells are discussed. Since both buried donors inside the electrode and donors solubilized in the electrolyte have both been shown to achieve high energy transfer efficiencies, and as the two methods take advantage of different available volumes of the electrode to introduce donors providing the excess absorption, synergy of the two methods is highly promising for achieving panchromatic absorption within a thin electrode.     

Genomic profiling identifies novel mutations and SNPs in ABCD1 gene: a molecular, biochemical and clinical analysis of X-ALD cases in India

X-linked adrenoleukodystrophy (X-ALD) affects the nervous system white matter and adrenal cortex secondary to mutations in the ABCD1 gene that encode the peroxisomal membrane protein. We conducted a genomic and protein expression study of susceptibility gene with its clinical and biochemical analysis. To the best of our knowledge this is the first preliminary comprehensive study in Indian population that identified novel mutations and SNPs in a relatively large group. We screened 17 Indian indigenous X-linked adrenoleukodystrophy cases and 70 controls for mutations and SNPs in the exonic regions (including flanking regions) of ABCD1 gene by direct sequencing with ABI automated sequencer along with Western blot analysis of its endogenous protein, ALDP, levels in peripheral blood mononuclear cells. Single germ line mutation was identified in each index case in ABCD1 gene. We detected 4 novel mutations (2 missense and 2 deletion/insertion) and 3 novel single nucleotide polymorphisms. We observed a variable protein expression in different patients. These findings were further extended to biochemical and clinical observations as it occurs with great clinical expression variability. This is the first major study in this population that presents a different molecular genetic spectrum as compared to Caucasian population due to geographical distributions of ethnicity of patients. It enhances our knowledge of the causative mutations of X-ALD that grants holistic base to develop effective medicine against X-ALD.     

Green-fluorescent protein from the bioluminescent jellyfish Clytia gregaria is an obligate dimer and does not form a stable complex with the Ca(2+)-discharged photoprotein clytin

Green-fluorescent protein (GFP) is the origin of the green bioluminescence color exhibited by several marine hydrozoans and anthozoans. The mechanism is believed to be Fo?rster resonance energy transfer (FRET) within a luciferase-GFP or photoprotein-GFP complex. As the effect is found in vitro at micromolar concentrations, for FRET to occur this complex must have an affinity in the micromolar range. We present here a fluorescence dynamics investigation of the recombinant bioluminescence proteins from the jellyfish Clytia gregaria, the photoprotein clytin in its Ca(2+)-discharged form that is highly fluorescent (λ(max) = 506 nm) and its GFP (cgreGFP; λ(max) = 500 nm). Ca(2+)-discharged clytin shows a predominant fluorescence lifetime of 5.7 ns, which is assigned to the final emitting state of the bioluminescence reaction product, coelenteramide anion, and a fluorescence anisotropy decay or rotational correlation time of 12 ns (20 °C), consistent with tight binding and rotation with the whole protein. A 34 ns correlation time combined with a translational diffusion constant and molecular brightness from fluorescence fluctuation spectroscopy all confirm that cgreGFP is an obligate dimer down to nanomolar concentrations. Within the dimer, the two chromophores have a coupled excited-state transition yielding fluorescence depolarization via FRET with a transfer correlation time of 0.5 ns. The 34 ns time of cgreGFP showed no change upon addition of a 1000-fold excess of Ca(2+)-discharged clytin, indicating no stable complexation below 0.2 mM. It is proposed that any bioluminescence FRET complex with micromolar affinity must be one formed transiently by the cgreGFP dimer with a short-lived (millisecond) intermediate in the clytin reaction pathway.     

A general approach for detecting folding intermediates from steady-state and time-resolved fluorescence of single-tryptophan-containing proteins

During denaturant-induced equilibrium (un)folding of wild-type apoflavodoxin from Azotobacter vinelandii, a molten globule-like folding intermediate is formed. This wild-type protein contains three tryptophans. In this study, we use a general approach to analyze time-resolved fluorescence and steady-state fluorescence data that are obtained upon denaturant-induced unfolding of a single-tryptophan-containing variant of apoflavodoxin [i.e., W74/F128/F167 (WFF) apoflavodoxin]. The experimental data are assembled in matrices, and subsequent singular-value decomposition of these matrices (i.e., based on either steady-state or time-resolved fluorescence data) shows the presence of three significant, and independent, components. Consequently, to further analyze the denaturation trajectories, we use a three-state protein folding model in which a folding intermediate and native and unfolded protein molecules take part. Using a global analysis procedure, we determine the relative concentrations of the species involved and show that the stability of WFF apoflavodoxin against global unfolding is ～4.1 kcal/mol. Analysis of time-resolved anisotropy data of WFF apoflavodoxin unfolding reveals the remarkable observation that W74 is equally well fixed within both the native protein and the molten globule-like folding intermediate. Slight differences between the direct environments of W74 in the folding intermediate and native protein cause different rotameric populations of the indole in both folding species as fluorescence lifetime analysis reveals. Importantly, thermodynamic analyses of the spectral denaturation trajectories of the double-tryptophan-containing protein variants WWF apoflavodoxin and WFW apoflavodoxin show that these variants are significantly more stable (5.9 kcal/mol and 6.8 kcal/mol, respectively) than WFF apoflavodoxin (4.1 kcal/mol) Hence, tryptophan residues contribute considerably to the 10.5 kcal/mol thermodynamic stability of native wild-type apoflavodoxin.     

Meningococcal Factor H Binding Protein fHbpd184 Polymorphism Influences Clinical Course of Meningococcal Meningitis

Factor H Binding protein (fHbp) is an important meningococcal virulence factor, enabling the meningococcus to evade the complement system, and a main target for vaccination. Recently, the structure of fHBP complexed with factor H (fH) was published. Two fHbp glutamic acids, E₂₈₃ and E₃₀₄, form salt bridges with fH, influencing interaction between fHbp and fH. Fifteen amino acids were identified forming hydrogen bonds with fH. We sequenced fHbp of 254 meningococcal isolates from adults with meningococcal meningitis included in a prospective clinical cohort to study the effect of fHbp variants on meningococcal disease severity and outcome. All fHbp of subfamily A had E304 substituted with T304. Of the 15 amino acids in fHbp making hydrogen bonds to fH, 3 were conserved, 11 show a similar distribution between the two fHbp subfamilies as the polymorphism at position 304. The proportion of patients infected with meningococci with fHbp of subfamily A with unfavorable outcome was 2.5-fold lower than that of patients infected with meningococci with fHbp of subfamily B (2 of 40 (5%) vs. 27 of 213 (13%) (P = 0.28). The charge of 2 of 15 amino acids (at position 184 and 306) forming hydrogen bonds was either basic or acidic. The affinity of fHbp_K184 and of fHbp_D184 for recombinant purified human fH was assessed by Surface Plasmon Resonance and showed average K_D of 2.60×10⁻⁸ and 1.74×10⁻⁸, respectively (ns). Patients infected with meningococci with fHbp_D184 were more likely to develop septic shock during admission (11 of 42 [26%] vs. 19 of 211 [9%]; P = 0.002) resulting in more frequent unfavorable outcome (9 of 42 [21%] vs. 20 of 211 [10%]; P = 0.026). In conclusion, we dentified fHBP_D184 to be associated with septic shock in patients with meningococcal meningitis.     

Phylogeography of Sardinian cave salamanders (genus Hydromantes) is mainly determined by geomorphology

Detecting the factors that determine the interruption of gene flow between populations is key to understanding how speciation occurs. In this context, caves are an excellent system for studying processes of colonization, differentiation and speciation, since they represent discrete geographical units often with known geological histories. Here, we asked whether discontinuous calcareous areas and cave systems represent major barriers to gene flow within and among the five species of Sardinian cave salamanders (genus Hydromantes) and whether intraspecific genetic structure parallels geographic distance within and among caves. We generated mitochondrial cytochrome b gene sequences from 184 individuals representing 48 populations, and used a Bayesian phylogeographic approach to infer possible areas of cladogenesis for these species and reconstruct historical and current dispersal routes among distinct populations. Our results show deep genetic divergence within and among all Sardinian cave salamander species, which can mostly be attributed to the effects of mountains and discontinuities in major calcareous areas and cave systems acting as barriers to gene flow. While these salamander species can also occur outside caves, our results indicate that there is a very poor dispersal of these species between separate cave systems.