Structural features of halophilicity derived from the crystal structure of dihydrofolate reductase from the Dead Sea halophilic archaeon, Haloferax volcanii

AbstractBackground: The proteins of halophilic archaea require high salt concentrations both for stability and for activity, whereas they denature at low ionic strength. The structural basis for this phenomenon is not yet well understood. The crystal structure of dihydrofolate reductase (DHFR) from Haloferax volcanii (hv-DHFR) reported here provides the third example of a structure of a protein from a halophilic organism. The enzyme is considered moderately halophilic, as it retains activity and secondary structure at monovalent salt concentrations as low as 0.5 M.Results: The crystal structure of hv-DHFR has been determined at 2.6 å resolution and reveals the same overall fold as that of other DHFRs. The structure is in the apo state, with an open conformation of the active-site gully different from the open conformation seen in other DHFR structures. The unique feature of hv-DHFR is a shift of the α helix encompassing residues 46–51 and an accompanied altered conformation of the ensuing loop relative to other DHFRs. Analysis of the charge distribution, amino acid composition, packing and hydrogen-bonding pattern in hv-DHFR and its non-halophilic homologs has been performed.Conclusions: The moderately halophilic behavior of hv-DHFR is consistent with the lack of striking structural features expected to occur in extremely halophilic proteins. The most notable feature of halophilicity is the presence of clusters of non-interacting negatively charged residues. Such clusters are associated with unfavorable electrostatic energy at low salt concentrations, and may account for the instability of hv-DHFR at salt concentrations lower than 0.5 M. With respect to catalysis, the open conformation seen here is indicative of a conformational transition not reported previously. The impact of this conformation on function and/or halophilicity is unknown

Establishing live-cell single-molecule localization microscopy imaging and single-particle tracking in the archaeon Haloferax volcanii

In recent years, fluorescence microscopy techniques for the localization and tracking of single molecules in living cells have become well-established and indispensable tools for the investigation of cellular biology and in vivo biochemistry of many bacterial and eukaryotic organisms. Nevertheless, these techniques are still not established for imaging archaea. Their establishment as a standard tool for the study of archaea will be a decisive milestone for the exploration of this branch of life and its unique biology. Here we have developed a reliable protocol for the study of the archaeon Haloferax volcanii. We have generated an autofluorescence-free H. volcanii strain, evaluated several fluorescent proteins for their suitability to serve as single-molecule fluorescence markers and codon-optimized them to work under optimal H. volcanii cultivation conditions. We found that two of them, Dendra2Hfx and PAmCherry1Hfx, provide state-of-the-art single-molecule imaging. Our strategy is quantitative and allows dual-color imaging of two targets in the same field of view as well as DNA co-staining. We present the first single-molecule localization microscopy (SMLM) images of the subcellular organization and dynamics of two crucial intracellular proteins in living H. volcanii cells, FtsZ1, which shows complex structures in the cell division ring, and RNA polymerase, which localizes around the periphery of the cellular DNA. This work should provide incentive to develop SMLM strategies for other archaeal organisms in the near future.</jats:p

Identification of essential and non-essential single-stranded DNA-binding proteins in a model archaeal organism

Single-stranded DNA-binding proteins (SSBs) play vital roles in all aspects of DNA metabolism in all three domains of life and are characterized by the presence of one or more OB fold ssDNA-binding domains. Here, using the genetically tractable euryarchaeon Haloferax volcanii as a model, we present the first genetic analysis of SSB function in the archaea. We show that genes encoding the OB fold and zinc finger-containing RpaA1 and RpaB1 proteins are individually non-essential for cell viability but share an essential function, whereas the gene encoding the triple OB fold RpaC protein is essential. Loss of RpaC function can however be rescued by elevated expression of RpaB, indicative of functional overlap between the two classes of haloarchaeal SSB. Deletion analysis is used to demonstrate important roles for individual OB folds in RpaC and to show that conserved N- and C-terminal domains are required for efficient repair of DNA damage. Consistent with a role for RpaC in DNA repair, elevated expression of this protein leads to enhanced resistance to DNA damage. Taken together, our results offer important insights into archaeal SSB function and establish the haloarchaea as a valuable model for further studies

RadB acts in homologous recombination in the archaeon Haloferax volcanii, consistent with a role as recombination mediator

Homologous recombination plays a central role in the repair of double-strand DNA breaks, the restart of stalled replication forks and the generation of genetic diversity. Regulation of recombination is essential since defects can lead to genome instability and chromosomal rearrangements. Strand exchange is a key step of recombination – it is catalysed by RecA in bacteria, Rad51/Dmc1 in eukaryotes and RadA in archaea. RadB, a paralogue of RadA, is present in many archaeal species. RadB has previously been proposed to function as a recombination mediator, assisting in RadA-mediated strand exchange. In this study, we use the archaeon Haloferax volcanii to provide evidence to support this hypothesis. We show that RadB is required for efficient recombination and survival following treatment with DNA-damaging agents, and we identify two point mutations in radA that suppress the ΔradB phenotype. Analysis of these point mutations leads us to propose that the role of RadB is to act as a recombination mediator, which it does by inducing a conformational change in RadA and thereby promoting its polymerisation on DNA

GOLIAH: A Gaming Platform for Home-Based Intervention in Autism – Principles and Design

Meeting the required intensive intervention hour for treating children with autism is challenging in terms of trained manpower needed and costs. Advances in Information Communication Technology and computer gaming may help in this respect by creating a nomadically deployable closed loop intervention system involving the child and active participation of parents and therapists.An automated serious gaming platform enabling intensive intervention in nomadic settings has been developed by mapping two pivotal skills in autism spectrum disorder: Imitation and Joint Attention (JA). Eleven games – seven Imitation and four JA – were derived from the Early Start Denver Model. The games involved application of visual and audio stimuli with multiple difficulty levels and a wide variety of tasks and actions pertaining to the Imitation and JA. The platform runs on mobile devices and allows the therapist to (1) characterize the child’s initial difficulties/strengths, ensuring tailored and adapted intervention by choosing appropriate games and (2) investigate and track the temporal evolution of the child’s progress through a set of automatically extracted quantitative performance metrics. The platform allows the therapist to change the game or its difficulty levels during the intervention depending on the child’s progress. Performance of the platform was assessed in a 3-month open trial with 10 children with autism. The children and the parents participated in 80% of the sessions both at home (77.5%) and at hospital (90%). All children went through all games but, given the diversity of the games and the heterogeneity of children profiles and abilities, for a given game the number of sessions dedicated to the game varied and could be tailored through automatic scoring. Parents (N = 10) highlighted enhancement in the child’s concentration, flexibility and self-esteem in 78%, 89% and 44% of the cases respectively and 56% observed an enhanced parents-child relationship. This pilot study shows the feasibility of using the developed gaming platform for home-based intensive intervention. However, the overall capability of the platform in delivering intervention needs to be assessed in a bigger open trial

Investigation of eighth-grade students' understanding of the slope of the linear function

This study aimed to investigate eighth-grade students' difficulties and misconceptions and their performance of translation between the different representation modes related to the slope of linear functions. The participants were 115 Turkish eighth-grade students in a city in the eastern part of the Black Sea region of Turkey. Data was collected with an instrument consisting of seven written questions and a semi-structured interview protocol conducted with six students. Students' responses to questions were categorized and scored. Quantitative data was analyzed using the SPSS 17.0 statistical packet program with cross tables and one-way ANOVA. Qualitative data obtained from interviews was analyzed using descriptive analytical techniques. It was found that students' performance in articulating the slope of the linear function using its algebraic representation form was higher than their performance in using transformation between graphical and algebraic representation forms. It was also determined that some of them had difficulties and misunderstood linear function equations, graphs, and slopes and could not comprehend the connection between slope and the x- and y-intercepts

Structural Basis for the Aminoacid Composition of Proteins from Halophilic Archea

In order to survive in highly saline environments, proteins from halophilic archea have evolved with biased amino acid compositions that have the capacity to reduce contacts with the solvent