MetalDetector v2.0: predicting the geometry of metal binding sites from protein sequence

MetalDetector identifies CYS and HIS involved in transition metal protein binding sites, starting from sequence alone. A major new feature of release 2.0 is the ability to predict which residues are jointly involved in the coordination of the same metal ion. The server is available at http://metaldetector.dsi.unifi.it/v2.0/

Molecular Modeling and 3D Analysis of Water Stress Responsive TaPase Phosphatase encoding Gene in Wheat (Triticum aestivum)

Acid phosphatases are key enzymes involved inorganic phosphorous acquisition under stress conditions thus implicated in crop productivity. The present study was designed to perform molecular modeling and 3D analysis of stress responsive acid phosphatase gene TaPase.  It has been observed that TaPase encodes protein which is soluble in nature. Based upon these results, a possible physiological role of TaPase in wheat was discussed. It was predicted that TaPase protein is disordered at N-terminal, posses signal peptide, rich in random coils, basic in nature and having polar amino acids. In addition, TaPase protein was modeled using QUARK server followed by validation using VADAR and QMEAN servers which revealed good nature of 3D structure. Functional analysis using PROFUNC server revealed potential role of TaPase under stress conditions along with hydrolytic activity. PDB sum motif analysis revealed Beta–Turns rich profiles in TaPase protein. Based on these findings the possible role of TaPase gene was discusse

Gene amplification and sequencing of hemicellulase using culture dependent technique

The present study aims to discover hemicellulase genes from thermophilic bacteria of Anoxybacillus gonensis G2T and Halothermobacillus malaysiensis RA using culture dependent technique. The hemicellulase genes with the amplicon size of 2118 bp, 1143 bp, 1554 bp and 2190 bp from A. gonensis G2T and H. malaysiensis RA were successfully obtained. Bioinformatics analyse of primary, secondary structure and tertiary structure of these proteins were performed by using bioinformatics tools. Beta-xylosidase from A. gonensis G2T and alpha-glucuronidase from H. malaysiensis RA are intracellular proteins. While, endo-1,4-beta-xylanase and alpha-N-arabinofuranosidase from H. malaysiensis RA have signal peptide and secreted as extracellular protein. I-TASSER and Swiss Model server were used to model the tertiary structure of hemicellulase. Good structures were generated after energy minimization through YASARA server. Using Neighbor-Joining method, the phylogenetic analysis reveals that beta-xylosidase from A. gonensis G2T (bxAg) is closely related with beta-xylosidase from Geobacillus sp. WSUCF1; endo-1,4-beta-xylanase from H. malaysiensis RA (ebxHm) is distantly related with endo-1,4-beta-xylanase from Xanthomonas translucens pv translucens, family GH10 Maribacter dokdonensis DSW-8 and Saccharicrinis fermentans DSM 9555JCM 21142; alpha-N-arabinofuranosidase from H. malaysiensis RA (anaHm) is closely related with alpha-N-arabinofuranosidase from Gemmatimonas sp. SG8 28; and alpha-glucuronidase from H. malaysiensis RA (agHm) is distantly related with alpha-glucuronidase from Sphingomonas sp.WG, Caulobacter vibrioides, Xanthomonas oryzae pv. oryzae and Stenotrophomonas maltophilia

The lifestyle switch protein Bd0108 of Bdellovibrio bacteriovorus is an intrinsically disordered protein

Bdellovibrio bacteriovorus is a δ-proteobacterium that preys upon Salmonella spp., E. coli, and other Gram-negative bacteria. Bdellovibrio can grow axenically (host-independent, HI, rare and mutation-driven) or subsist via a predatory lifecycle (host-dependent, HD, the usual case). Upon contact with prey, B. bacteriovorus enters the host periplasm from where it slowly drains the host cytosol of nutrients for its own replication. At the core of this mechanism is a retractile pilus, whose architecture is regulated by the protein Bd0108 and its interaction with the neighboring gene product Bd0109. Deletion of bd0108 results in negligible pilus formation, whereas an internal deletion (the one that instigates host-independence) causes mis-regulation of pilus length. These mutations, along with a suite of naturally occurring bd0108 mutant strains, act to control the entry to HI growth. To further study the molecular mechanism of predatory regulation, we focused on the apparent lifecycle switch protein Bd0108. Here we characterize the solution structure and dynamics of Bd0108 using nuclear magnetic resonance (NMR) spectroscopy complemented with additional biophysical methods. We then explore the interaction between Bd0108 and Bd0109 in detail utilizing isothermal titration calorimetry (ITC) and NMR spectroscopy. Together our results demonstrate that Bd0108 is an intrinsically disordered protein (IDP) and that the interaction with Bd0109 is of low affinity. Furthermore, we observe that Bd0108 retains an IDP nature while binding Bd0109. From our data we conclude that Bdellovibrio bacteriovorus utilizes an intrinsically disordered protein to regulate its pilus and control predation signaling

Dynamic Regulation of Phosphatidylinositol 3,5-bisphosphate and its Upstream Lipid Kinase Fab1/PIKfyve

Eukaryotes maintain homeostatic balance in part via signal transduction cascades, which generate cellular adaptations in response to extracellular cues. Some of these cascades are regulated in part via phosphorylated phosphoinositide lipids (PPIs), which are low-abundance signaling molecules. The PPI phosphatidylinositol (3,5)-bisphosphate (PI(3,5)P2) is generated from phosphatidylinositol 3-phosphate (PI3P) by the conserved lipid kinase Fab1/PIKfyve, which resides in a multiprotein complex known as the Fab1 complex. PI(3,5)P2 controls multiple pathways including calcium storage, lysosomal pH, and TORC1 signaling. PI(3,5)P2 is essential for multiple organ systems. Moreover, defects in the dynamic regulation of PI(3,5)P2 are linked to human diseases, especially those of the nervous system. However, few mechanisms that regulate PI(3,5)P2 have been identified. Here, we report a new mechanism that regulates Fab1 and dynamically controls cellular PI(3,5)P2 synthesis. Using multiple sequence alignment and secondary structure prediction we report a defined domain architecture of each member of the Fab1 complex. We identify new domains in Fab1/PIKfyve and Vac7, a Fab1 activator. Using a forward genetic screen optimized for the isolation of dominant-active Fab1 alleles, we identify point mutations within each domain of Fab1 that alter the dynamic regulation of PI(3,5)P2 levels. We characterize a subset of these dominant-active alleles and show that they disrupt a newly identified, inhibitory, intramolecular interaction between the yeast Fab1 kinase region and an upstream conserved cysteine-rich (CCR) domain. We report preliminary evidence that this mechanism may be conserved in a second lipid kinase—the PI4 5-kinase, Mss4. Point mutations in Mss4 were generated based on Fab1 dominant-active alleles. These Mss4 mutations increase Mss4 activity under basal conditions and in response to stimulus-induced Mss4 activation. These studies identify a mechanism of regulation that may be conserved among lipid kinases to dynamically control PPI levels in response to external stimuli.PHDCell and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/135757/1/langmj_1.pd

Learning to Predict Combinatorial Structures

The major challenge in designing a discriminative learning algorithm for predicting structured data is to address the computational issues arising from the exponential size of the output space. Existing algorithms make different assumptions to ensure efficient, polynomial time estimation of model parameters. For several combinatorial structures, including cycles, partially ordered sets, permutations and other graph classes, these assumptions do not hold. In this thesis, we address the problem of designing learning algorithms for predicting combinatorial structures by introducing two new assumptions: (i) The first assumption is that a particular counting problem can be solved efficiently. The consequence is a generalisation of the classical ridge regression for structured prediction. (ii) The second assumption is that a particular sampling problem can be solved efficiently. The consequence is a new technique for designing and analysing probabilistic structured prediction models. These results can be applied to solve several complex learning problems including but not limited to multi-label classification, multi-category hierarchical classification, and label ranking.Comment: PhD thesis, Department of Computer Science, University of Bonn (submitted, December 2009

Struktur und Funktion von Flokkulinen aus Saccharomyces cerevisiae sowie weiterer pilzlicher Zellwandproteine

Die Zellwand ist die primäre Interaktionsfläche von Hefezellen mit ihrer Umwelt. Sie besteht in ihrer inneren Schicht aus Chitin, β1,3- und β1,6-Glucanen, die für ihre Stabilität entscheidend sind. In der äußeren Schicht der Hefezellwand finden sich verschiedene Glyco-proteine, die für die Erkennung und Bindung von Zellen untereinander und die Interaktion mit ihrer weiteren Umgebung notwendig sind. Der Aufbau und Umbau der Zellwand selbst während des Zellzyklus der Hefe ist ein essentieller Vorgang, in den ebenfalls verschiedene Proteine, die in der Zellwand vorkommen und Glucane prozessieren, involviert sind. In dieser Arbeit wurden Adhäsine und das innere Zellwandprotein Sun4 aus der Bäckerhefe Saccharomyces cerevisiae und der humanpathogenen Hefe Candida glabrata strukturell und funktionell untersucht. Die untersuchten pilzlichen Adhäsine Flo5, Flo11 und Epa1 vermitteln vegetative Adhäsion der Hefen entweder an weitere Hefezellen, abiotische Substrate oder Wirtszellen. Sie bilden eine Familie mit einer gemeinsamen Superstruktur, die aus einer adhäsionsvermittelnden, N-terminalen A-Domäne, einer mittleren, hochglycosylierten B Domäne und einer C-terminalen C-Domäne mit Glycosylphosphatidylinositolanker besteht. In dieser Arbeit war es möglich, die molekularen Strukturen der adhäsionsvermittelnden Domänen Flo5A, Epa1A und Flo11A bei atomaren Auflösungen (0.89-1.5 Å) zu bestimmen. Dabei wurde deutlich, dass die mit der PA14-Domäne aus dem Anthrax-protektiven Antigen verwandten A-Domänen von Flo5 und Epa1 eine C-Typ-Lektin-artige Aktivität aufweisen und Glycanstrukturen auf anderen Hefezellen (Flo5A) oder Wirtszellen (Epa1A) binden. Native, für die biologische Funktion relevante Liganden konnten in den Strukturen beobachtet und Bindungskonstanten bestimmt werden. Die Flo11A-Domäne zeigt einen vollständig neuen Faltungstyp, offenbarte ihre Ligandspezifität jedoch nicht durch strukturelle Untersuchungen. Allerdings konnte ein lektinartiger Mechanismus ausgeschlossen und ein auf aromatischen Bändern beruhender, homotypischer Mechanismus vorgeschlagen werden. Die Struktur der in Pilzen hochkonservierten C-terminalen SUN-Domäne des inneren Zellwandproteins Sun4 aus S. cerevisiae konnte ebenfalls in atomarer Auflösung aufgeklärt und Hinweise auf eine β-Glucanase Aktivität dieser Domäne gefunden werden. Durch strukturbasiertes Design sollte die Entwicklung von fungiziden Medikamenten gegen diese in Pilzen ubiquitäre, aber exklusiv vorkommende Domäne möglich sein. Ein heterotypischer Gadolinium-Oxo-Supercluster, der während der Phasierung auf der Oberfläche von Flo5A beobachtet wurde, könnte als Templat für die Inkorporation von Gadolinium in verschiedene Proteine und so langfristig für die Entwicklung eines biokompatiblen und gewebespezifischen MRT-Kontrastmittels dienen