Position Effect Takes Precedence Over Target Sequence in Determination of Adenine Methylation Patterns in the Nuclear Genome of a Eukaryote, \u3cem\u3eTetrahymena thermophila\u3c/em\u3e

Approximately 0.8% of the adenine residues in the macronuclear DNA of the ciliated protozoan Tetrahymena thermophila are modified to N6-methyladenine. DNA methylation is site specific and the pattern of methylation is constant between clonal cell lines. In vivo, modification of adenine residues appears to occur exclusively in the sequence 5′-NAT-3′, but no consensus sequence for modified sites has been found. In this study, DNA fragments containing a site that is uniformly methylated on the 50 copies of the macronuclear chromosome were cloned into the extrachromosomal rDNA. In the novel location on the rDNA minichromosome, the site was unmethylated. The result was the same whether the sequences were introduced in a methylated or unmethylated state and regardless of the orientation of the sequence with respect to the origin of DNA replication. The data show that sequence is insufficient to account for site-specific methylation in Tetrahymena and argue that other factors determine the pattern of DNA methylation

Distinct ubiquitin binding modes exhibited by SH3 domains: Molecular determinants and functional implications

SH3 domains constitute a new type of ubiquitin-binding domains. We previously showed that the third SH3 domain (SH3-C) of CD2AP binds ubiquitin in an alternative orientation. We have determined the structure of the complex between first CD2AP SH3 domain and ubiquitin and performed a structural and mutational analysis to decipher the determinants of the SH3-C binding mode to ubiquitin. We found that the Phe-to-Tyr mutation in CD2AP and in the homologous CIN85 SH3-C domain does not abrogate ubiquitin binding, in contrast to previous hypothesis and our findings for the first two CD2AP SH3 domains. The similar alternative binding mode of the SH3-C domains of these related adaptor proteins is characterised by a higher affinity to C-terminal extended ubiquitin molecules. We conclude that CD2AP/CIN85 SH3-C domain interaction with ubiquitin constitutes a new ubiquitin-binding mode involved in a different cellular function and thus changes the previously established mechanism of EGF-dependent CD2AP/CIN85 mono-ubiquitination. © 2013 Ortega Roldan et al

Biocatalytic, one-pot diterminal oxidation and esterification of n-alkanes for production of α,ω-diol and α,ω-dicarboxylic acid esters

Direct and selective terminal oxidation of medium-chain n-alkanes is a major challenge in chemistry. Efforts to achieve this have so far resulted in low specificity and overoxidized products. Biocatalytic oxidation of medium-chain n-alkanes – with for example the alkane monooxygenase AlkB from P. putida GPo1- on the other hand is highly selective. However, it also results in overoxidation. Moreover, diterminal oxidation of medium-chain n-alkanes is inefficient. Hence, α,ω-bifunctional monomers are mostly produced from olefins using energy intensive, multi-step processes. By combining biocatalytic oxidation with esterification we drastically increased diterminal oxidation upto 92 mol% and reduced overoxidation to 3% for n-hexane. This methodology allowed us to convert medium-chain n-alkanes into α,ω-diacetoxyalkanes and esterified α,ω-dicarboxylic acids. We achieved this in a one-pot reaction with resting-cell suspensions of genetically engineered Escherichia coli. The combination of terminal oxidation and esterification constitutes a versatile toolbox to produce α,ω-bifunctional monomers from n-alkanes

Development of enzymatic activity during protein folding. Detection of a spectroscopically silent native-like intermediate of muscle acylphosphatase.

The recovery of enzymatic activity during the folding of muscle acylphosphatase and two single residue mutants (proline 54 to alanine and proline 71 to alanine) from 7 M urea has been monitored and compared with the development of intrinsic fluorescence emission. Fluorescence measurements reveal the presence in the wild-type protein of a major rapid refolding phase followed by a second low amplitude slow phase. The slow phase is absent in the fluorescence trace acquired with the proline 54 to alanine mutant, suggesting the involvement of this proline residue in the fluorescence-detected slow phase of the wild-type protein. The major kinetic phase is associated with a considerable recovery of enzymatic activity, indicating that a large fraction of molecules refolds with effective two-state behavior. The use of time-resolved enzymatic activity as a probe to follow the folding process reveals, however, the presence of another exponential slow phase arising from proline 71. This slow phase is not observable by utilizing optical probes, indicating that, unlike proline 54, the cis to trans isomerization of proline 71 can take place in an intermediate possessing a native-like fold. We suggest that, although spectroscopically silent and structurally insignificant, the cis-trans interconversion of proline residues in native-like intermediates may be crucial for the generation of enzymatic activity of functional enzymes

Accurate characterization of weak macromolecular interactions by titration of NMR residual dipolar couplings: application to the CD2AP SH3-C:ubiquitin complex

The description of the interactome represents one of key challenges remaining for structural biology. Physiologically important weak interactions, with dissociation constants above 100 μM, are remarkably common, but remain beyond the reach of most of structural biology. NMR spectroscopy, and in particular, residual dipolar couplings (RDCs) provide crucial conformational constraints on intermolecular orientation in molecular complexes, but the combination of free and bound contributions to the measured RDC seriously complicates their exploitation for weakly interacting partners. We develop a robust approach for the determination of weak complexes based on: (i) differential isotopic labeling of the partner proteins facilitating RDC measurement in both partners; (ii) measurement of RDC changes upon titration into different equilibrium mixtures of partially aligned free and complex forms of the proteins; (iii) novel analytical approaches to determine the effective alignment in all equilibrium mixtures; and (iv) extraction of precise RDCs for bound forms of both partner proteins. The approach is demonstrated for the determination of the three-dimensional structure of the weakly interacting CD2AP SH3-C:Ubiquitin complex (Kd = 132 ± 13 μM) and is shown, using cross-validation, to be highly precise. We expect this methodology to extend the remarkable and unique ability of NMR to study weak protein–protein complexes

Structural and Biophysical Characterization of Staphylococcus Aureus SaMazF Shows Conservation of Functional Dynamics

The Staphylococcus aureus genome contains three toxin-antitoxin modules, including one mazEF module, SamazEF. Using an on-column separation protocol we are able to obtain large amounts of wild-type SaMazF toxin. The protein is well-folded and highly resistant against thermal unfolding but aggregates at elevated temperatures. Crystallographic and nuclear magnetic resonance (NMR) solution studies show a well-defined dimer. Differences in structure and dynamics between the X-ray and NMR structural ensembles are found in three loop regions, two of which undergo motions that are of functional relevance. The same segments also show functionally relevant dynamics in the distantly related CcdB family despite divergence of function. NMR chemical shift mapping and analysis of residue conservation in the MazF family suggests a conserved mode for the inhibition of MazF by MazE

Fire Promotes Pollinator Visitation: Implications for Ameliorating Declines of Pollination Services

Pollinators serve critical roles for the functioning of terrestrial ecosystems, and have an estimated annual value of over $150 billion for global agriculture. Mounting evidence from agricultural systems reveals that pollinators are declining in many regions of the world, and with a lack of information on whether pollinator communities in natural systems are following similar trends, identifying factors which support pollinator visitation and services are important for ameliorating the effects of the current global pollinator crisis. We investigated how fire affects resource structure and how that variation influences floral pollinator communities by comparing burn versus control treatments in a southeastern USA old-field system. We hypothesized and found a positive relationship between fire and plant density of a native forb, Verbesina alternifolia, as well as a significant difference in floral visitation of V. alternifolia between burn and control treatments. V. alternifolia density was 44% greater and floral visitation was 54% greater in burned treatments relative to control sites. When the density of V. alternifolia was experimentally reduced in the burn sites to equivalent densities observed in control sites, floral visitation in burned sites declined to rates found in control sites. Our results indicate that plant density is a proximal mechanism by which an imposed fire regime can indirectly impact floral visitation, suggesting its usefulness as a tool for management of pollination services. Although concerns surround the negative impacts of management, indirect positive effects may provide an important direction to explore for managing future ecological and conservation issues. Studies examining the interaction among resource concentration, plant apparency, and how fire affects the evolutionary consequences of altered patterns of floral visitation are overdue. DOI: 10.1371/journal.pone.007985

Identifying context factors explaining physician's low performance in communication assessment: an explorative study in general practice

Contains fulltext : 97982.pdf (postprint version ) (Open Access)ABSTRACT: BACKGROUND: Communication is a key competence for health care professionals. Analysis of registrar and GP communication performance in daily practice, however, suggests a suboptimal application of communication skills. The influence of context factors could reveal why communication performance levels, on average, do not appear adequate. The context of daily practice may require different skills or specific ways of handling these skills, whereas communication skills are mostly treated as generic. So far no empirical analysis of the context has been made. Our aim was to identify context factors that could be related to GP communication. METHODS: A purposive sample of real-life videotaped GP consultations was analyzed (N = 17). As a frame of reference we chose the MAAS-Global, a widely used assessment instrument for medical communication. By inductive reasoning, we analyzed the GP behaviour in the consultation leading to poor item scores on the MAAS-Global. In these cases we looked for the presence of an intervening context factor, and how this might explain the actual GP communication behaviour. RESULTS: We reached saturation after having viewed 17 consultations. We identified 19 context factors that could potentially explain the deviation from generic recommendations on communication skills. These context factors can be categorized into doctor-related, patient-related, and consultation-related factors. CONCLUSIONS: Several context factors seem to influence doctor-patient communication, requiring the GP to apply communication skills differently from recommendations on communication. From this study we conclude that there is a need to explicitly account for context factors in the assessment of GP (and GP registrar) communication performance. The next step is to validate our findings