Prediction and analysis of protein structure

This thesis, which contains an introduction and four manuscripts, summarises my efforts during my the past four years to understand proteins, their structure and dynamics. The first manuscript presents a protocol that refines models as part of a protein structure prediction pipeline. To achieve this, we used spatial information from determined structures and sequence information from multiple alignments. The protocol was used to improve the quality of rough models containing only one point per residue. In the second manuscript we investigated protein fold space. We compared models with known fold to determined structures and found that out models contained many folds that were not seen in the present pool of structures in the PDB. Comparison of structural features revealed no reason why the model folds could not exist. We investigated how well geometric comparison methods distinguished fold in the third manuscript. We presented a novel measure of topological similarity and showed that geometric methods have trouble distinguishing fold differences between both models and PDB structures. In the last manuscript we showed that the architecture is the most important factor for dynamics as measured by normal modes. Protein fold has some effect and cannot be discarded completely, but larger differences in fold does not necessarily correspond to larger differences in flexibility if the architecture is the same

CFU (mean ± s. e.) of <i>E</i>. <i>coli</i> O157:H7 EDL933 and isogenic mutants inoculated at matched MOI when co-cultured with <i>T</i>. <i>pyriformis</i> (solid lines) and <i>P</i>. <i>caudatum</i> (dashed lines) at ambient laboratory temperature.

<p>(A) <i>E</i>. <i>coli</i> O157:H7 EDL933 (squares) and EDL933ΔStx 2a subunit A (triangles). (B) <i>E</i>. <i>coli</i> O157:H7 EDL933 (squares) and EDL933ΔStx 1 phage (diamonds). (C) <i>E</i>. <i>coli</i> O157:H7 EDL933 (squares) and EDL933ΔStx 2a phage (circles). (D) <i>E</i>. <i>coli</i> O157:H7 EDL933 (squares) and EDL933ΔStx 1 & Stx 2a phages (asterisks).</p

Sequence of primers for PCR amplification used throughout these studies.

<p>Sequence of primers for PCR amplification used throughout these studies.</p

Density (mean ± s. e.) of <i>P</i>. <i>caudatum</i> when co-cultured with matched MOI of <i>E</i>. <i>coli</i> O157:H7 EDL933 and cattle commensal <i>E</i>. <i>coli</i> (186) for three days at ambient laboratory temperature.

<p>Squares, <i>P</i>. <i>caudatum</i> with added <i>E</i>. <i>coli</i>; no marker, <i>P</i>. <i>caudatum</i> in trough water without added bacteria.</p

<i>P</i>. <i>caudatum</i> grazing of <i>E</i>. <i>coli</i> O157:H7 EDL933 and cattle commensal <i>E</i>. <i>coli</i> (186) over three days at ambient laboratory temperature.

<p>CFU (mean ± s. e.) for three grazing trials each containing a matched MOI of <i>E</i>. <i>coli</i> O157:H7 EDL933 (squares) and cattle commensal <i>E</i>. <i>coli</i> (triangles).</p

<i>E</i>. <i>coli</i> strains used in these studies.

<p><i>E</i>. <i>coli</i> strains used in these studies.</p

Microbiological status and pathologic lesions of animals in experiments 2 and 3.

<p>Microbiological status and pathologic lesions of animals in experiments 2 and 3.</p

Clinical scores exhibited by bighorn sheep commingled with <i>M</i>. <i>ovipneumoniae</i> carrier domestic goats in Experiment 1.

<p>Three-day rolling average summed clinical scores based on observed anorexia, nasal discharge, cough, dyspnea, head shaking, ear paresis and weakness/incoordination. <i>M</i>. <i>ovipneumoniae</i> colonized DG were comingled with naïve BHS on day 0.</p

Exposure of bighorn sheep to domestic goats colonized with <i>Mycoplasma ovipneumoniae</i> induces sub-lethal pneumonia

<div><p>Background</p><p>Bronchopneumonia is a population limiting disease of bighorn sheep (<i>Ovis canadensis</i>) that has been associated with contact with domestic Caprinae. The disease is polymicrobial but is initiated by <i>Mycoplasma ovipneumoniae</i>, which is commonly carried by both domestic sheep (<i>O</i>. <i>aries</i>) and goats (<i>Capra aegagrus hircus</i>). However, while previous bighorn sheep comingling studies with domestic sheep have resulted in nearly 100% pneumonia mortality, only sporadic occurrence of fatal pneumonia was reported from previous comingling studies with domestic goats. Here, we evaluated the ability of domestic goats of defined <i>M</i>. <i>ovipneumoniae</i> carriage status to induce pneumonia in comingled bighorn sheep.</p><p>Methodology/Principal findings</p><p>In experiment 1, three bighorn sheep naïve to <i>M</i>. <i>ovipneumoniae</i> developed non-fatal respiratory disease (coughing, nasal discharge) following comingling with three naturally <i>M</i>. <i>ovipneumoniae</i>-colonized domestic goats. Gross and histological lesions of pneumonia, limited to small areas on the ventral and lateral edges of the anterior and middle lung lobes, were observed at necropsies conducted at the end of the experiment. A control group of three bighorn sheep from the same source housed in isolation during experiment 1 remained free of observed respiratory disease. In experiment 2, three bighorn sheep remained free of observed respiratory disease while comingled with three <i>M</i>. <i>ovipneumoniae-</i>free domestic goats. In experiment 3, introduction of a domestic goat-origin strain of <i>M</i>. <i>ovipneumoniae</i> to the same comingled goats and bighorn sheep used in experiment 2 resulted in clinical signs of respiratory disease (coughing, nasal discharge) in both host species. At the end of experiment 3, gross and histological evidence of pneumonia similar to that observed in experiment 1 bighorn sheep was observed in both affected bighorn sheep and domestic goats.</p><p>Conclusions/Significance</p><p><i>M</i>. <i>ovipneumoniae</i> strains carried by domestic goats were transmitted to comingled bighorn sheep, triggering development of pneumonia. However, the severity of the disease was markedly milder than that seen in similar experiments with domestic sheep strains of the bacterium.</p></div

Outcomes and microbiological findings of lambs born during experiment 1.

<p>Outcomes and microbiological findings of lambs born during experiment 1.</p