Comparison of abundance of translation factors between initiation and elongation conditions.

<p>Growth conditions for initiation conditions (glu +—+ 1 min) were obtained by adding glucose back to glucose depleted cells for 1 min, at which time cycloheximide was added and cells were harvested. Elongation conditions (glu +) were cells grown on glucose growth conditions. A-F. GFP fusions were as indicated.</p

AU-FDS and AU-A₂₃₀ analyses of extracts containing GFP fusions to translation components.

<p>For each set of data shown in Fig 1, the AUC analysis was from the same centrifuge run. Cells were grown on glucose-containing medium except as indicated. glu + - 10 min: growth was on glucose-containing medium followed by growth on medium depleted for glucose for 10 min; and glu +—+: growth was the same as glu + - 10 min except glucose was added back for 1 min. In panel A, it should be noted that the AU-A₂₃₀ abundance for the RPS4B-GFP sample was twice that of the STM1-GFP sample. A. RPL25A-Flag pull downs were conducted on strains carrying either RPS4B-GFP or STM1-GFP; B-D. Flag-PAB1 pull downs were conducted on strains carrying the GFP fusions as indicated. Data displayed in panels B and D were done on the same day on extracts split between the two centrifuge runs.</p

Analysis of Flag-SBP1 purified translation complexes.

<p>A. AU-A₂₆₀ analysis was conducted instead of AU-A₂₃₀ analysis for better detection of complexes with this particular Flag-tagged protein. A-F. Growth conditions were as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150616#pone.0150616.g001" target="_blank">Fig 1</a>. C. No flag refers to a strain lacking Flag-SBP1 plasmid. The differences between the no flag control and the Flag-SBP1 pull downs in panel C-F were not considered significant for eIF4E-GFP and eIF4G2-GFP.</p

AU-FDS analysis of two initiation conditions and polysomes.

<p>A and B. Comparison of two different initiation conditions. C-E. Expanded c(s) values to identify ribosomal-GFP protein migrations in polysomal material (greater than 90S).</p

Summary of dynamic changes in closed-loop factors in translating ribosomes dependent on polysomal, polyadenylation, initiation, and elongation states.

<p>Only the translating ribosomes at initiation containing closed-loop structures are summarized. For simplicity, the dynamic changes of closed-loop factors in translating ribosomes containing other combinations of closed-loop factors are not represented, and these changes in stoichiometry are explained more completely in the text. Also, based on our results only one PAB1 is represented as binding to each poly(A) tail.</p

Relative levels of proteins in the 77S monosomal translating complex during different stages of translation.

<p>Relative levels of proteins in the 77S monosomal translating complex during different stages of translation.</p

Analysis of eIF4E-Flag purified translation complexes.

<p>Growth conditions and analysis were conducted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150616#pone.0150616.g001" target="_blank">Fig 1</a>. A and B. Strains were either transformed with eIF4E-Flag plasmid (eIF4E-Flag) or with no eIF4E plasmid (eIF4E).</p

Relative levels of proteins in the polysomal translating complexes during different stages of translation.

<p>Relative levels of proteins in the polysomal translating complexes during different stages of translation.</p

Specific-Ion Effects on the Aggregation Mechanisms and Protein–Protein Interactions for Anti-streptavidin Immunoglobulin Gamma‑1

Non-native protein aggregation is common in the biopharmaceutical industry and potentially jeopardizes product shelf life, therapeutic efficacy, and patient safety. The present article focuses on the relationship(s) among protein–protein interactions, aggregate growth mechanisms, aggregate morphologies, and specific-ion effects for an anti-streptavidin (AS) immunoglobulin gamma 1 (IgG1). Aggregation mechanisms of AS-IgG1 were determined as a function of pH and NaCl concentration with sodium acetate buffer and compared to previous work with sodium citrate. Aggregate size and shape were determined using a combination of laser light scattering and small-angle neutron or X-ray scattering. Protein–protein interactions were quantified in terms of the protein–protein Kirkwood–Buff integral (<i>G</i>₂₂) determined from static light scattering and in terms of the protein effective charge (<i>Z</i>_eff) measured using electrophoretic light scattering. Changing from citrate to acetate resulted in significantly different protein–protein interactions as a function of pH for low NaCl concentrations when the protein displayed positive <i>Z</i>_eff. Overall, the results suggest that electrostatic repulsions between proteins were lessened because of preferential accumulation of citrate anions, compared to acetate anions, at the protein surface. The predominant aggregation mechanisms correlated well with <i>G</i>₂₂, indicating that ion-specific effects beyond traditional mean-field descriptions of electrostatic protein–protein interactions are important for predicting qualitative shifts in protein aggregation state diagrams. Interestingly, while solution conditions dictated which mechanisms predominated, aggregate average molecular weight and size displayed a common scaling behavior across both citrate- and acetate-based systems

Sedimentation Velocity Analysis with Fluorescence Detection of Mutant Huntingtin Exon 1 Aggregation in <i>Drosophila melanogaster</i> and <i>Caenorhabditis elegans</i>

At least nine neurodegenerative diseases that are caused by the aggregation induced by long tracts of glutamine sequences have been identified. One such polyglutamine-containing protein is huntingtin, which is the primary factor responsible for Huntington’s disease. Sedimentation velocity with fluorescence detection is applied to perform a comparative study of the aggregation of the huntingtin exon 1 protein fragment upon transgenic expression in <i>Drosophila melanogaster</i> and <i>Caenorhabditis elegans</i>. This approach allows the detection of aggregation in complex mixtures under physiologically relevant conditions. Complementary methods used to support this biophysical approach included fluorescence microscopy and semidenaturing detergent agarose gel electrophoresis, as a point of comparison with earlier studies. New analysis tools developed for the analytical ultracentrifuge have made it possible to readily identify a wide range of aggregating species, including the monomer, a set of intermediate aggregates, and insoluble inclusion bodies. Differences in aggregation in the two animal model systems are noted, possibly because of differences in levels of expression of glutamine-rich sequences. An increased level of aggregation is shown to correlate with increased toxicity for both animal models. Co-expression of the human Hsp70 in <i>D. melanogaster</i> showed some mitigation of aggregation and toxicity, correlating best with inclusion body formation. The comparative study emphasizes the value of the analytical ultracentrifuge equipped with fluorescence detection as a useful and rigorous tool for <i>in situ</i> aggregation analysis to assess commonalities in aggregation across animal model systems