Structure prediction of Bestrophin for the induced - fit docking of anthocyanins

Bestrophin, an integral membrane protein existing in basolateral region of the retina is a propitious target for drug discovery. Mutations in the Bestrophin protein cause Best Vitelliform Macular Dystrophy (BVMD) leading to retinal damages and loss of visual acuity. Owing to the lack of three dimensional structure and related structural homologs in the protein data bank, we modeled the bestrophin protein using Robetta ab initio method. Further, no treatment is available for the disease. In this situation, anthocyanins from natural sources are reported to combat retinal damages. Hence, we identified anthocyanins from Syzygium cumini fruit skin using Electrospray Ionization tandem mass spectrometry. These compounds were docked into the predicted bestrophin model to study the interactions within the active site. The results may provide a valuable insight into the structure of bestrophin and efficacy of anthocyanins in molecular docking studies.

Abbreviations

PTP - Putative transmembrane proteins, VMD - Vitelliform macular dystrophy, BVMD - Best''s vitelliform macular dystrophy, RPE - Retinal pigment epithelium, ESI-MS/MS - Electrospray Ionization Tandem Mass Spectrometry, UNIPROT - Universal Protein Resource, PSIPRED - Protein secondary structure prediction, TMH - Transmembrane Helices, SCFS - Syzygium cumini fruit skin DP - Declustering Potential IFD - Induced Fit Docking.     

Structure and interactions of the helical and U-box domains of CHIP, the C terminus of HSP70 interacting protein

The heat-shock proteins Hsp70 and Hsp90 play a crucial role in regulating protein quality control both by refolding and by preventing the aggregation of misfolded proteins. It has recently been shown that Hsp70 and Hsp90 act not only in protein refolding but also cooperate with the C terminus of Hsp70 interacting protein (CHIP), a multidomain ubiquitin ligase, to mediate the degradation of unfolded proteins. We present the crystal structure of the helical linker domain and U-box domain of zebrafish CHIP (DrCHIP-HU). The structure of DrCHIP-HU shows a symmetric homodimer. The conformation of the helical linker domains and the relative positions of the helical and U-box domains differ substantially in DrCHIP-HU from those in a recently published structure of an asymmetric dimer of mammalian (mouse) CHIP. We used an in vitro ubiquitination assay to identify residues, located on two long loops and a central alpha helix of the CHIP U-box domain, that are important for interacting with the ubiquitin-conjugating enzyme UbcH5b. In addition, we used NMR spectroscopy to define a complementary interaction surface located on the N-terminal alpha helix and the L4 and L7 loops of UbcH5b. Our results provide insights into conformational variability in the domain arrangement of CHIP and into U-box-mediated recruitment of UbcH5b for the ubiquitination of Hsp70 and Hsp90 substrates.     

Molecular characterization of beta-lactamase genes blaA and blaB of Yersinia enterocolitica biovar 1A

The beta-lactamase genes blaA and blaB were detected by PCR amplification in strains of Yersinia enterocolitica biovar 1A isolated from India, Germany, France and the USA. Both genes were detected in all strains. Polymerase chain reaction-restriction fragment length polymorphism revealed genetic heterogeneity in blaA but not in blaB. Cluster analysis of blaA restriction profiles grouped the strains into three groups. The blaA gene of Y. enterocolitica biovar 1A showed a high degree of sequence homology to that of Y. enterocolitica 8081 (biovar 1B) and Y. enterocolitica Y-56 (biovar 4), whereas homology was low with class A beta-lactamase genes of other members of the family Enterobacteriaceae. The pI 8.7 of enzyme Bla-A of Y. enterocolitica biovar 1A was similar to that of biovars 2, 3 and 4. The enzyme Bla-B focused at 6.8 and 7.1, indicating that biovar 1A strains produced a 'B-like' enzyme. This is the first study to have investigated the genetic heterogeneity of the beta-lactamase genes of Y. enterocolitica.     

Occurrence of Macrosiphum hellebori Theobald & Walton (Hemiptera: Aphididae) in Australia

The European aphid species, Macrosiphum hellebori , the hellebore aphid, is recorded from Australia, having been found in Melbourne, Victoria and in Canberra, Australian Capital Territory, colonising ornamental hellebores, Helleborus species (Ranunculaceae). Insect associates of M. hellebori are recorded, including the hymenopterous primary parasite Lysiphlebus testaceipes (Cresson) (Braconidae: Aphidiinae). Keys are provided for the identification of the three species of Macrosiphum Passerini known from Australia.     

Does post-fire abiotic habitat filtering create divergent plant communities in black spruce forests of eastern Canada?

We investigated the role of post-fire residual organic matter (ROM) thickness as a driver of community assembly in eastern Newfoundland. We hypothesized that if post-fire community assembly is predominantly controlled by ROM thickness (an abiotic habitat filter), then post-fire species composition and functional traits should correspond to the depth and distribution of ROM. However, if species interactions (biotic filter) are the primary constraints on community assembly, then post-fire species composition and their functional traits should be independent of the depth and distribution of ROM. We tested these predictions in three relatively mature plant communities, Kalmia angustifolia heath, black spruce (Picea mariana)-Kalmia shrub savannah and black spruce forest. Through pre-fire stand reconstruction, we found evidence that the three communities originated from black spruce forest. ROM thickness in heath was almost twice that of shrub savannah and six times more than forest, suggesting a gradient in fire severity. Distribution of ROM corresponded to patterns in vegetation dominance, where thick ROM (>2 cm) filtered out black spruce in favour of Kalmia. ROM thickness was a strong predictor of vegetation composition and function between heath and forest, but this was not found between the shrub savannah and forest. We attribute this to species interactions and allelopathy, which may have become important when ROM thickness was suitable for both seed (black spruce) and vegetative (Kalmia) regenerating species. Thus, priority effects or “who came first” may have lead to shrub savannah formation when ROM thickness was ~2 cm. We conclude that abiotic habitat filtering of thick ROM (>2 cm) on (primarily) species’ regeneration traits was the primary driver of community divergence from forest to heath and shrub savannah.     

Stabilization of anionic and neutral forms of a fluorophoric ligand at the active site of human carbonic anhydrase I

We synthesized a fluorogenic dansylamide derivative (JB2-48), which fills the entire (15 Å deep) active site pocket of human carbonic anhydrase I, and investigated the contributions of sulfonamide and hydrophobic regions of the ligand structure on the spectral, kinetic, and thermodynamic properties of the enzyme–ligand complex. The steady-state and fluorescence lifetime data revealed that the deprotonation of the sulfonamide moiety of the enzyme bound ligand increases the fluorescence emission intensity as well as the lifetime of the fluorophores. This is manifested via the electrostatic interaction between the active site resident Zn²⁺ cofactor and the negatively charged sulfonamide group of the ligand, and such interaction contributes to about 2.2 kcal/mol (ΔΔG°) and 0.89 kcal/mol (ΔΔG^‡) energy in stabilizing the ground and the putative transition states, respectively. We provide evidence that the anionic and neutral forms of JB2-48 are stabilized by the complementary microscopic/conformational states of the enzyme. The implication of the mechanistic studies presented herein in rationale design of carbonic anhydrase inhibitors is discussed.     

Pathway Palette: A rich internet application for peptide‐, protein‐ and network‐oriented analysis of MS data

Recent improvements in proteomic technologies have collectively yielded data sets that far exceed the capabilities of typical low‐throughput interpretation strategies. Unfortunately, tools designed to leverage the “peptide‐centric” content of MS‐based proteomics lag the current rate of data production. Here, we describe Pathway Palette ( http://blaispathways.dfci.harvard.edu ), a freely accessible internet application that enables researchers to easily transition from peptides to biological pathways, while simultaneously retaining the qualitative and quantitative aspects of the underlying MS data.     

Identification of aphid species (Hemiptera: Aphididae: Aphidinae) using a rapid polymerase chain reaction restriction fragment length polymorphism method based on the cytochrome oxidase subunit I gene

Abstract  The identification of immature aphids is often difficult or impossible. This can be a problem when there is a need for a rapid and accurate diagnosis of any aphid life stage, such as for quarantine inspections and horticultural surveys. A polymerase chain reaction restriction fragment length polymorphism technique is described on the mitochondrial cytochrome oxidase subunit I (COI) gene to develop a molecular identification key for immature aphids from Victoria, Australia. The restriction enzymes HpyCH4 IV, Dra I, Hinf I, Taq I and Ssp I characterised 26 haplotypes that corresponded to 25 aphid species commonly found in southern Australian aphid surveys, including the currant-lettuce aphid Nasonovia ribis-nigri (Mosley) that has recently invaded Australia, presumably from New Zealand. Overseas specimens of Aulacorthum solani (Kaltenbach) and N. ribis-nigri showed no significant sequence difference when compared with their Australian counterparts. The COI gene provides a useful marker for diagnostic aphid surveys.