Advanced database mining integrating sequence and structure bioinformatics with microfluidics challenges enzyme engineering

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Engineering the protein dynamics of an ancestral luciferase.

Protein dynamics are often invoked in explanations of enzyme catalysis, but their design has proven elusive. Here we track the role of dynamics in evolution, starting from the evolvable and thermostable ancestral protein AncHLD-RLuc which catalyses both dehalogenase and luciferase reactions. Insertion-deletion (InDel) backbone mutagenesis of AncHLD-RLuc challenged the scaffold dynamics. Screening for both activities reveals InDel mutations localized in three distinct regions that lead to altered protein dynamics (based on crystallographic B-factors, hydrogen exchange, and molecular dynamics simulations). An anisotropic network model highlights the importance of the conformational flexibility of a loop-helix fragment of Renilla luciferases for ligand binding. Transplantation of this dynamic fragment leads to lower product inhibition and highly stable glow-type bioluminescence. The success of our approach suggests that a strategy comprising (i) constructing a stable and evolvable template, (ii) mapping functional regions by backbone mutagenesis, and (iii) transplantation of dynamic features, can lead to functionally innovative proteins

KP-LAB Knowledge Practices Laboratory -- Specification of end-user applications

deliverablesThe present deliverable provides a high-level view on the new specifications of end user applications defined in the WPII during the M37-M46 period of the KP-Lab project. This is the last in the series of four deliverables that cover all the tools developed in the project, the previous ones being D6.1, D6.4 and D6.6. This deliverable presents specifications for the new functionalities for supporting the dedicated research studies defined in the latest revision of the KP-Lab research strategy. The tools addressed are: the analytic tools (Data export, Time-line-based analyser, Visual analyser), Clipboard, Search, Versioning of uploadable content items, Visual Model Editor (VME) and Visual Modeling Language Editor (VMLE). The main part of the deliverable provides the summary of tool specifications and the description of the Knowledge Practices Environment architecture, as well as an overview of the revised technical design process, of the tools’ relationship with the research studies, and of the driving objectives and the high-level requirements relevant for the present specifications. The full specifications of tools are provided in the annexes 1-9

KP-LAB Knowledge Practices Laboratory -- External release of end-user applications

deliverablesThis deliverable describes the M24 release of the End user applications for knowledge practices software v2.0.0. The deliverable includes the technical development performed until M24 (January 2008) within WP6 according to Description of Work 2.1 and D6.4 M21 specification of end-user applications. The current release is comprised of two set of tools: 1. Shared Space Tool The shared space and the accompanying support material can be found on the Internet at: http://2d.mobile.evtek.fi:8080/shared-space 2. Map-It. The installer program for Map-It v2.0.0 is available at: http://www.kp-lab.org/intranet/testable-tools/kp-lab-tools/map-it/map-it-2-0.0 Please consult the "Getting Started" Note before installing and using Map-It: http://www.kp-lab.org/intranet/testable-tools/kp-lab-tools/map-it/getting-started-with-map-it 3. Change Laboratory tools The release targeted for the end users participating in the trials planned to be conducted in the CL Working Knot can be accessed via the following link: http://2d.mobile.evtek.fi:8080/shared-space/cl.html Anyone who wishes to try the software out but is not participating in the Change Laboratory trials should use the development deployment on: http://mielikki.mobile.evtek.fi/shared-space/cl.html The M24 release of Semantic Multimedia Annotation tools is still delayed. The release of CASS Memo Client has been postponed to be included in the M28 release in DoW3

Engineering the protein dynamics of an ancestral luciferase

Abstract: Protein dynamics are often invoked in explanations of enzyme catalysis, but their design has proven elusive. Here we track the role of dynamics in evolution, starting from the evolvable and thermostable ancestral protein AncHLD-RLuc which catalyses both dehalogenase and luciferase reactions. Insertion-deletion (InDel) backbone mutagenesis of AncHLD-RLuc challenged the scaffold dynamics. Screening for both activities reveals InDel mutations localized in three distinct regions that lead to altered protein dynamics (based on crystallographic B-factors, hydrogen exchange, and molecular dynamics simulations). An anisotropic network model highlights the importance of the conformational flexibility of a loop-helix fragment of Renilla luciferases for ligand binding. Transplantation of this dynamic fragment leads to lower product inhibition and highly stable glow-type bioluminescence. The success of our approach suggests that a strategy comprising (i) constructing a stable and evolvable template, (ii) mapping functional regions by backbone mutagenesis, and (iii) transplantation of dynamic features, can lead to functionally innovative proteins

In vitro growth-inhibitory effect of plant-derived extracts and compounds against Paenibacillus larvae and their acute oral toxicity to adult honey bees

In total, 26 natural compounds of various chemical classes (flavonoids, alkaloids, terpenoids) and 19 crude extracts from selected plants were tested in vitro for antibacterial activity against three strains of P. larvae, the causal agent of American Foulbrood Disease of honey bees (AFB) by the broth microdilution method. Among the individual substances, sanguinarine (MIC 4 μg/ml), followed by thymoquinone, capsaicin, trans-2-hexenal and nordihydroguaiaretic acid (MIC 4–32 μg/ml) possessed the strongest antibacterial effect. In case of extracts, common hop (Humulus lupulus L.) and myrtle (Myrtus communis L.) methanolic-dichloromethane extracts exhibited the highest growth-inhibitory effect with MICs ranging from 2 to 8 μg/ml. Acute oral toxicity of the most active natural products was determined on adult honey bees, showing them as non-toxic at concentrations as high as 100 μg peer bee. Our study leads to identification of highly potent natural products effective against AFB in vitro with very low MICs compared to those reported in literature, low toxicity to adult honey bees and commercial availability suggesting them as perspective, low cost and consumer-acceptable agents for control of AFB

Nanoscale Dynamic Readout of a Chemical Redox Process Using Radicals Coupled with Nitrogen-Vacancy Centers in Nanodiamonds

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