A Multilaboratory Comparison of Calibration Accuracy and the Performance of External References in Analytical Ultracentrifugation

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies

A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation.

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies

Structural Characterization of Phosducin and Its Complex with the 14-3-3 Protein

Phosducin (Pdc), a highly conserved phosphoprotein involved in the regulation of retinal phototransduction cascade, transcriptional control, and modulation of blood pressure, is controlled in a phosphorylation-dependent manner, including the binding to the 14-3-3 protein. However, the molecular mechanism of this regulation is largely unknown. Here, the solution structure of Pdc and its interaction with the 14-3-3 protein were investigated using small angle x-ray scattering, time-resolved fluorescence spectroscopy, and hydrogen-deuterium exchange coupled to mass spectrometry. The 14-3-3 protein dimer interacts with Pdc using surfaces both inside and outside its central channel. The N-terminal domain of Pdc, where both phosphorylation sites and the 14-3-3-binding motifs are located, is an intrinsically disordered protein that reduces its flexibility in several regions without undergoing dramatic disorder-to-order transition upon binding to 14-3-3. Our data also indicate that the C-terminal domain of Pdc interacts with the outside surface of the 14-3-3 dimer through the region involved in G$_t$βγ binding. In conclusion, we show that the 14-3-3 protein interacts with and sterically occludes both the N- and C-terminal Gtβγ binding interfaces of phosphorylated Pdc, thus providing a mechanistic explanation for the 14-3-3-dependent inhibition of Pdc function

Role of the EF-hand-like motif in the 14-3-3 protein-mediated activation of yeast neutral trehalase Nth1

Trehalases hydrolyze the non-reducing disaccharide trehalose amassed by cells as a universal protectant and storage carbohydrate. Recently, it has been shown that the activity of neutral trehalase Nth1 from Saccharomyces cerevisiae is mediated by the 14-3-3 protein binding that modulates the structure of both the catalytic domain and the region containing the EF-hand-like motif, whose role in the activation of Nth1 is unclear. In this work, the structure of the Nth1·14-3-3 complex and the importance of the EF-hand-like motif were investigated using site-directed mutagenesis, hydrogen/deuterium exchange coupled to mass spectrometry, chemical cross-linking, and small angle x-ray scattering. The low resolution structural views of Nth1 alone and the Nth1·14-3-3 complex show that the 14-3-3 protein binding induces a significant structural rearrangement of the whole Nth1 molecule. The EF-hand-like motif-containing region forms a separate domain that interacts with both the 14-3-3 protein and the catalytic trehalase domain. The structural integrity of the EF-hand like motif is essential for the 14-3-3 protein-mediated activation of Nth1, and calcium binding, although not required for the activation, facilitates this process by affecting its structure. Our data suggest that the EF-hand like motif-containing domain functions as the intermediary through which the 14-3-3 protein modulates the function of the catalytic domain of Nth1

Structural Insight into the 14-3-3 Protein-dependent Inhibition of Protein Kinase ASK1 (Apoptosis Signal-regulating kinase 1)

Apoptosis signal-regulating kinase 1 (ASK1, also known as MAP3K5), a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, regulates diverse physiological processes. The activity of ASK1 is triggered by various stress stimuli and is involved in the pathogenesis of cancer, neurodegeneration, inflammation, and diabetes. ASK1 forms a high molecular mass complex whose activity is, under non-stress conditions, suppressed through interaction with thioredoxin and the scaffolding protein 14-3-3. The 14-3-3 protein binds to the phosphorylated Ser-966 motif downstream of the ASK1 kinase domain. The role of 14-3-3 in the inhibition of ASK1 has yet to be elucidated. In this study we performed structural analysis of the complex between the ASK1 kinase domain phosphorylated at Ser-966 (pASK1-CD) and the 14-3-3ζ protein. Small angle x-ray scattering (SAXS) measurements and chemical cross-linking revealed that the pASK1-CD·14-3-3ζ complex is dynamic and conformationally heterogeneous. In addition, structural analysis coupled with the results of phosphorus NMR and time-resolved tryptophan fluorescence measurements suggest that 14-3-3ζ interacts with the kinase domain of ASK1 in close proximity to its active site, thus indicating this interaction might block its accessibility and/or affect its conformation

Examples for the determination of radial magnification errors.

<p>(A) Radial intensity profile measured in scans of the precision mask. Blue lines are experimental scans, and shaded areas indicate the regions expected to be illuminated on the basis of the known mask geometry. In this example, the increasing difference between the edges corresponds to a calculated radial magnification error of -3.1%. (B—D) Examples for differences between the experimentally measured positions of the light/dark transitions (blue circles, arbitrarily aligned for absolute mask position) and the known edge distances of the mask. The solid lines indicate the linear or polynomial fit. (B) Approximately linear magnification error with a slope corresponding to an error of -0.04%. Also indicated as thin lines are the confidence intervals of the linear regression. (C) A bimodal shift pattern of left and right edges, likely resulting from out-of-focus location of the mask, with radial magnification error of -1.7%. (D) A non-linear distortion leading to a radial magnification error of -0.53% in the <i>s</i>-values from the analysis of back-transformed data. The thin grey lines in C and D indicate the best linear fit through all data points.</p

Examples of transient changes in the console temperature reading during the SV experiment, as saved in the scan file data.

<p>For comparison, the maximum adiabatic cooling of -0.3°C would be expected after approximately 300 sec, recovering to the equilibrium temperature after approximately 1,200 s (see Fig 3 in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126420#pone.0126420.ref033" target="_blank">33</a>]).</p

Histogram and box-and-whisker plot of <i>s</i>-values of the BSA monomer after different corrections: Raw experimental <i>s</i>-values (black, with grey histogram), scan time corrected <i>s</i>_<i>t</i>-values (blue), rotor temperature corrected <i>s</i>_<i>20T</i>-values (green), or radial magnification corrected <i>s</i>_<i>r</i>-values (cyan), and fully corrected <i>s</i>_{<i>20T</i>,<i>t</i>,<i>r</i>,<i>v</i>}-values (red with red histogram).

<p>The box-and-whisker plots indicate the central 50% of the data as solid line and draw the smaller and larger 25% percentiles as individual circles. The median for each group is displayed as a vertical line.</p

Distributions of calculated BSA monomer signals for the different kits and the different optical systems.

<p>The box-and-whisker plots indicate the central 50% of the data as solid line and draw the smaller and larger 25% percentiles as individual circles. The median for each group is displayed as vertical line.</p