An Experimental Investigation of Conformational Fluctuations in Proteins G and L

SummaryThe B1 domains of streptococcal proteins G and L are structurally similar, but they have different sequences and they fold differently. We have measured their NMR spectra at variable temperature using a range of concentrations of denaturant. Many residues have curved amide proton temperature dependence, indicating that they significantly populate alternative, locally unfolded conformations. The results, therefore, provide a view of the locations of low-lying, locally unfolded conformations. They indicate approximately 4–6 local minima for each protein, all within ca. 2.5 kcal/mol of the native state, implying a locally rough energy landscape. Comparison with folding data for these proteins shows that folding involves most molecules traversing a similar path, once a transition state containing a β hairpin has been formed, thereby defining a well-populated pathway down the folding funnel. The hairpin that directs the folding pathway differs for the two proteins and remains the most stable part of the folded protein

Structural Basis for the Recognition of Cellular mRNA Export Factor REF by Herpes Viral Proteins HSV-1 ICP27 and HVS ORF57

The herpesvirus proteins HSV-1 ICP27 and HVS ORF57 promote viral mRNA export by utilizing the cellular mRNA export machinery. This function is triggered by binding to proteins of the transcription-export (TREX) complex, in particular to REF/Aly which directs viral mRNA to the TAP/NFX1 pathway and, subsequently, to the nuclear pore for export to the cytoplasm. Here we have determined the structure of the REF-ICP27 interaction interface at atomic-resolution and provided a detailed comparison of the binding interfaces between ICP27, ORF57 and REF using solution-state NMR. Despite the absence of any obvious sequence similarity, both viral proteins bind on the same site of the folded RRM domain of REF, via short but specific recognition sites. The regions of ICP27 and ORF57 involved in binding by REF have been mapped as residues 104–112 and 103–120, respectively. We have identified the pattern of residues critical for REF/Aly recognition, common to both ICP27 and ORF57. The importance of the key amino acid residues within these binding sites was confirmed by site-directed mutagenesis. The functional significance of the ORF57-REF/Aly interaction was also probed using an ex vivo cytoplasmic viral mRNA accumulation assay and this revealed that mutants that reduce the protein-protein interaction dramatically decrease the ability of ORF57 to mediate the nuclear export of intronless viral mRNA. Together these data precisely map amino acid residues responsible for the direct interactions between viral adaptors and cellular REF/Aly and provide the first molecular details of how herpes viruses access the cellular mRNA export pathway

Detecting the influence of initial pioneers on succession at deep-sea vents

© The Author(s), 2012. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in PLoS One 7 (2012): e50015, doi:10.1371/journal.pone.0050015.Deep-sea hydrothermal vents are subject to major disturbances that alter the physical and chemical environment and eradicate the resident faunal communities. Vent fields are isolated by uninhabitable deep seafloor, so recolonization via dispersal of planktonic larvae is critical for persistence of populations. We monitored colonization near 9°50′N on the East Pacific Rise following a catastrophic eruption in order to address questions of the relative contributions of pioneer colonists and environmental change to variation in species composition, and the role of pioneers at the disturbed site in altering community structure elsewhere in the region. Pioneer colonists included two gastropod species: Ctenopelta porifera, which was new to the vent field, and Lepetodrilus tevnianus, which had been rare before the eruption but persisted in high abundance afterward, delaying and possibly out-competing the ubiquitous pre-eruption congener L. elevatus. A decrease in abundance of C. porifera over time, and the arrival of later species, corresponded to a decrease in vent fluid flow and in the sulfide to temperature ratio. For some species these successional changes were likely due to habitat requirements, but other species persisted (L. tevnianus) or arrived (L. elevatus) in patterns unrelated to their habitat preferences. After two years, disturbed communities had started to resemble pre-eruption ones, but were lower in diversity. When compared to a prior (1991) eruption, the succession of foundation species (tubeworms and mussels) appeared to be delayed, even though habitat chemistry became similar to the pre-eruption state more quickly. Surprisingly, a nearby community that had not been disturbed by the eruption was invaded by the pioneers, possibly after they became established in the disturbed vents. These results indicate that the post-eruption arrival of species from remote locales had a strong and persistent effect on communities at both disturbed and undisturbed vents.The authors received funding from National Science Foundation grant OCE-0424953, WHOI Deep Ocean Exploration Institute, WHOI Summer Student Fellow program, Woods Hole Partnership in Education Program, IFREMER and CNRS, Fondation TOTAL Chair Extreme Marine Environment, Biodiversity and Global change

Competitive and Cooperative Interactions Mediate RNA Transfer from Herpesvirus Saimiri ORF57 to the Mammalian Export Adaptor ALYREF

The essential herpesvirus adaptor protein HVS ORF57, which has homologs in all other herpesviruses, promotes viral mRNA export by utilizing the cellular mRNA export machinery. ORF57 protein specifically recognizes viral mRNA transcripts, and binds to proteins of the cellular transcription-export (TREX) complex, in particular ALYREF. This interaction introduces viral mRNA to the NXF1 pathway, subsequently directing it to the nuclear pore for export to the cytoplasm. Here we have used a range of techniques to reveal the sites for direct contact between RNA and ORF57 in the absence and presence of ALYREF. A binding site within ORF57 was characterized which recognizes specific viral mRNA motifs. When ALYREF is present, part of this ORF57 RNA binding site, composed of an a-helix, binds preferentially to ALYREF. This competitively displaces viral RNA from the a-helix, but contact with RNA is still maintained by a flanking region. At the same time, the flexible N-terminal domain of ALYREF comes into contact with the viral RNA, which becomes engaged in an extensive network of synergistic interactions with both ALYREF and ORF57. Transfer of RNA to ALYREF in the ternary complex, and involvement of individual ORF57 residues in RNA recognition, were confirmed by UV cross-linking and mutagenesis. The atomic-resolution structure of the ORF57-ALYREF interface was determined, which noticeably differed from the homologous ICP27-ALYREF structure. Together, the data provides the first site-specific description of how viral mRNA is locked by a herpes viral adaptor protein in complex with cellular ALYREF, giving herpesvirus access to the cellular mRNA export machinery. The NMR strategy used may be more generally applicable to the study of fuzzy protein-protein-RNA complexes which involve flexible polypeptide regions

Diversity of Meiofauna from the 9°50′N East Pacific Rise across a Gradient of Hydrothermal Fluid Emissions

Background: We studied the meiofauna community at deep-sea hydrothermal vents along a gradient of vent fluid emissions in the axial summit trought (AST) of the East Pacific Rise 9 degrees 50'N region. The gradient ranged from extreme high temperatures, high sulfide concentrations, and low pH at sulfide chimneys to ambient deep-sea water conditions on bare basalt. We explore meiofauna diversity and abundance, and discuss its possible underlying ecological and evolutionary processes. Methodology/Principal Findings: After sampling in five physico-chemically different habitats, the meiofauna was sorted, counted and classified. Abundances were low at all sites. A total of 52 species were identified at vent habitats. The vent community was dominated by hard substrate generalists that also lived on bare basalt at ambient deep-sea temperature in the axial summit trough (AST generalists). Some vent species were restricted to a specific vent habitat (vent specialists), but others occurred over a wide range of physico-chemical conditions (vent generalists). Additionally, 35 species were only found on cold bare basalt (basalt specialists). At vent sites, species richness and diversity clearly increased with decreasing influence of vent fluid emissions from extreme flow sulfide chimney (no fauna), high flow pompei worm (S: 4-7, H-loge': 0.11-0.45), vigorous flow tubeworm (S: 8-23; H-loge': 0.44-2.00) to low flow mussel habitats (S: 28-31; H-loge': 2.34-2.60). Conclusions/Significance: Our data suggest that with increasing temperature and toxic hydrogen sulfide concentrations and increasing amplitude of variation of these factors, fewer species are able to cope with these extreme conditions. This results in less diverse communities in more extreme habitats. The finding of many species being present at sites with and without vent fluid emissions points to a non endemic deep-sea hydrothermal vent meiofaunal community. This is in contrast to a mostly endemic macrofauna but similar to what is known for meiofauna from shallow-water vents

Ecology and Biogeography of Free-Living Nematodes Associated with Chemosynthetic Environments in the Deep Sea: A Review

Background: Here, insight is provided into the present knowledge on free-living nematodes associated with chemosynthetic environments in the deep sea. It was investigated if the same trends of high standing stock, low diversity, and the dominance of a specialized fauna, as observed for macro-invertebrates, are also present in the nematodes in both vents and seeps. Methodology: This review is based on existing literature, in combination with integrated analysis of datasets, obtained through the Census of Marine Life program on Biogeography of Deep-Water Chemosynthetic Ecosystems (ChEss). Findings: Nematodes are often thriving in the sulphidic sediments of deep cold seeps, with standing stock values ocassionaly exceeding largely the numbers at background sites. Vents seem not characterized by elevated densities. Both chemosynthetic driven ecosystems are showing low nematode diversity, and high dominance of single species. Genera richness seems inversely correlated to vent and seep fluid emissions, associated with distinct habitat types. Deep-sea cold seeps and hydrothermal vents are, however, highly dissimilar in terms of community composition and dominant taxa. There is no unique affinity of particular nematode taxa with seeps or vents. Conclusions: It seems that shallow water relatives, rather than typical deep-sea taxa, have successfully colonized the reduced sediments of seeps at large water depth. For vents, the taxonomic similarity with adjacent regular sediments is much higher, supporting rather the importance of local adaptation, than that of long distance distribution. Likely the ephemeral nature of vents, its long distance offshore and the absence of pelagic transport mechanisms, have prevented so far the establishment of a successful and typical vent nematode fauna. Some future perspectives in meiofauna research are provided in order to get a more integrated picture of vent and seep biological processes, including all components of the marine ecosystem

Multiorgan MRI findings after hospitalisation with COVID-19 in the UK (C-MORE): a prospective, multicentre, observational cohort study

Introduction: The multiorgan impact of moderate to severe coronavirus infections in the post-acute phase is still poorly understood. We aimed to evaluate the excess burden of multiorgan abnormalities after hospitalisation with COVID-19, evaluate their determinants, and explore associations with patient-related outcome measures. Methods: In a prospective, UK-wide, multicentre MRI follow-up study (C-MORE), adults (aged ≥18 years) discharged from hospital following COVID-19 who were included in Tier 2 of the Post-hospitalisation COVID-19 study (PHOSP-COVID) and contemporary controls with no evidence of previous COVID-19 (SARS-CoV-2 nucleocapsid antibody negative) underwent multiorgan MRI (lungs, heart, brain, liver, and kidneys) with quantitative and qualitative assessment of images and clinical adjudication when relevant. Individuals with end-stage renal failure or contraindications to MRI were excluded. Participants also underwent detailed recording of symptoms, and physiological and biochemical tests. The primary outcome was the excess burden of multiorgan abnormalities (two or more organs) relative to controls, with further adjustments for potential confounders. The C-MORE study is ongoing and is registered with ClinicalTrials.gov, NCT04510025. Findings: Of 2710 participants in Tier 2 of PHOSP-COVID, 531 were recruited across 13 UK-wide C-MORE sites. After exclusions, 259 C-MORE patients (mean age 57 years [SD 12]; 158 [61%] male and 101 [39%] female) who were discharged from hospital with PCR-confirmed or clinically diagnosed COVID-19 between March 1, 2020, and Nov 1, 2021, and 52 non-COVID-19 controls from the community (mean age 49 years [SD 14]; 30 [58%] male and 22 [42%] female) were included in the analysis. Patients were assessed at a median of 5·0 months (IQR 4·2–6·3) after hospital discharge. Compared with non-COVID-19 controls, patients were older, living with more obesity, and had more comorbidities. Multiorgan abnormalities on MRI were more frequent in patients than in controls (157 [61%] of 259 vs 14 [27%] of 52; p<0·0001) and independently associated with COVID-19 status (odds ratio [OR] 2·9 [95% CI 1·5–5·8]; padjusted=0·0023) after adjusting for relevant confounders. Compared with controls, patients were more likely to have MRI evidence of lung abnormalities (p=0·0001; parenchymal abnormalities), brain abnormalities (p<0·0001; more white matter hyperintensities and regional brain volume reduction), and kidney abnormalities (p=0·014; lower medullary T1 and loss of corticomedullary differentiation), whereas cardiac and liver MRI abnormalities were similar between patients and controls. Patients with multiorgan abnormalities were older (difference in mean age 7 years [95% CI 4–10]; mean age of 59·8 years [SD 11·7] with multiorgan abnormalities vs mean age of 52·8 years [11·9] without multiorgan abnormalities; p<0·0001), more likely to have three or more comorbidities (OR 2·47 [1·32–4·82]; padjusted=0·0059), and more likely to have a more severe acute infection (acute CRP >5mg/L, OR 3·55 [1·23–11·88]; padjusted=0·025) than those without multiorgan abnormalities. Presence of lung MRI abnormalities was associated with a two-fold higher risk of chest tightness, and multiorgan MRI abnormalities were associated with severe and very severe persistent physical and mental health impairment (PHOSP-COVID symptom clusters) after hospitalisation. Interpretation: After hospitalisation for COVID-19, people are at risk of multiorgan abnormalities in the medium term. Our findings emphasise the need for proactive multidisciplinary care pathways, with the potential for imaging to guide surveillance frequency and therapeutic stratification

Structural studies of the polysaccharide degrading apparatus of bacteria and fungi

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