Methods for sample labeling and meniscus determination in the fluorescence-detected analytical ultracentrifuge

Bailey, Michael F.; Angley, Lauren M.; Perugini, Matthew A.

doi:10.1016/j.ab.2009.03.045

Cited by 21 publications

(13 citation statements)

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“…In order to better determine experimentally the position of the meniscus, Bailey and colleagues have suggested the use of light oil with a dissolved fluorescent dye [10]. In the present study, however, we did not find this necessary because early scans did allow us to visually recognize the approximate meniscus location, and because the inclusion of scans from the entire sedimentation process usually carries sufficient information for the meniscus position to be determined implicitly in the model of the sedimentation boundary migration.…”

Section: Discussionmentioning

confidence: 75%

“…At very low concentrations, which generate data with extremely low signal levels only on the order of the statistical noise, we have previously shown in conventional absorbance optics that the c ( s ) analysis can still produce well-determined s -values [12]. In such cases, however, the computational method for determining the meniscus will likely not be satisfactory, and an experimental configuration with suspended mineral oil [10], or a separate measurement with the absorbance optics [9], [11] (provided the radial calibration is accurate [21]), may be very useful to constrain the meniscus position. A similar methodology may be appropriate for sedimentation data with extremely broad boundaries.…”

Section: Discussionmentioning

confidence: 99%

“…This requires judgments on the fitting limits to be made, which may be particularly difficult in the presence of smaller macromolecules and under conditions of significant back-diffusion that may partially mask this effect. Even though the use of fluorinated oil has been suggested to create an artificial transparent bottom [7], [10], a different attenuation of signal, perhaps more similar to that close to the meniscus ( Figure 2A ), might be expected even if masked by accumulation of material in the back-diffusion region. Furthermore, the addition of a water/oil interface appears to bear the potential for interactions, at least for some proteins or hydrophobic fluorophores.…”

Section: Discussionmentioning

confidence: 99%

“…First applications have been reviewed recently by Kingsbury & Laue [8]. Several characteristic deviations of FDS data from the shapes of concentration sedimentation profiles have been described, including sloping sample plateaus [7], [9], dependent on the focal depth of the optics perpendicular to the plane of rotation [7], and the attenuation of the signal close to the bottom of the solution column [7], [9], [10]. Furthermore, imperfect stability of the signal intensity has been suggested to limit the quality of fit of FDS data with standard sedimentation models [11], and the potential of non-linearity in the signal response has been considered [7]–[9], [11], [12].…”

Section: Introductionmentioning

confidence: 99%

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Tools for the Quantitative Analysis of Sedimentation Boundaries Detected by Fluorescence Optical Analytical Ultracentrifugation

et al. 2013

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Fluorescence optical detection in sedimentation velocity analytical ultracentrifugation allows the study of macromolecules at nanomolar concentrations and below. This has significant promise, for example, for the study of systems of high-affinity protein interactions. Here we describe adaptations of the direct boundary modeling analysis approach implemented in the software SEDFIT that were developed to accommodate unique characteristics of the confocal fluorescence detection system. These include spatial gradients of signal intensity due to scanner movements out of the plane of rotation, temporal intensity drifts due to instability of the laser and fluorophores, and masking of the finite excitation and detection cone by the sample holder. In an extensive series of experiments with enhanced green fluorescent protein ranging from low nanomolar to low micromolar concentrations, we show that the experimental data provide sufficient information to determine the parameters required for first-order approximation of the impact of these effects on the recorded data. Systematic deviations of fluorescence optical sedimentation velocity data analyzed using conventional sedimentation models developed for absorbance and interference optics are largely removed after these adaptations, resulting in excellent fits that highlight the high precision of fluorescence sedimentation velocity data, thus allowing a more detailed quantitative interpretation of the signal boundaries that is otherwise not possible for this system.

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Section: Discussionmentioning

confidence: 75%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Tools for the Quantitative Analysis of Sedimentation Boundaries Detected by Fluorescence Optical Analytical Ultracentrifugation

et al. 2013

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“…We obtained this parameter from absorbance intensity scans taken after the fluorescence scans were completed. An alternative approach has recently been described,72 but requires the addition of a fluorescence dye suspended in light oil that may perturb the sample. A mass extinction coefficient of 3.3 fringes/ (mg/ml) was used for interference data.…”

Section: Methodsmentioning

confidence: 99%

Energetics of SecA Dimerization

Wowor

Kendall

et al. 2011

Journal of Molecular Biology

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Summary Transport of many proteins to extracytoplasmic locations occurs via the general secretion (Sec) pathway. In Escherichia coli, this pathway is comprised of the SecYEG protein conducting channel and the SecA ATPase. SecA plays a central role in binding the signal peptide region of preproteins, directing preproteins to membrane-bound SecYEG and promoting translocation coupled with ATP hydrolysis. Although it is well-established that SecA is crucial for preprotein transport and thus cell viability, its oligomeric state during different stages of transport remains ill defined. We have characterized the energetics of SecA dimerization as a function of salt concentration and temperature and defined the linkage of SecA dimerization and signal peptide binding using analytical ultracentrifugation. The use of a new fluorescence detector permitted analysis of SecA dimerization down to concentrations as low as 50 nM. The dimer dissociation constants are strongly dependent on salt. Linkage analysis indicates that SecA dimerization is coupled to the release of about five ions, demonstrating that electrostatic interactions play an important role in stabilizing the SecA dimer interface. Binding of signal peptide reduces SecA dimerization affinity such that Kd increases about 9-fold from 0.28 μM in the absence of peptide to 2.68 μM in the presence of peptide. The weakening of the SecA dimer that accompanies signal peptide binding may poise the SecA dimer to dissociate upon binding to SecYEG.

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Analytical Ultracentrifugation (AUC): An Overview of the Application of Fluorescence and Absorbance AUC to the Study of Biological Macromolecules

Edwards

Muthurajan

Bowerman

et al. 2020

CP Molecular Biology

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The biochemical and biophysical investigation of proteins, nucleic acids, and the assemblies that they form yields essential information to understand complex systems. Analytical ultracentrifugation (AUC) represents a broadly applicable and information‐rich method for investigating macromolecular characteristics such as size, shape, stoichiometry, and binding properties, all in the true solution‐state environment that is lacking in most orthogonal methods. Despite this, AUC remains underutilized relative to its capabilities and potential in the fields of biochemistry and molecular biology. Although there has been a rapid development of computing power and AUC analysis tools in this millennium, fewer advancements have occurred in development of new applications of the technique, leaving these powerful instruments underappreciated and underused in many research institutes. With AUC previously limited to absorbance and Rayleigh interference optics, the addition of fluorescence detection systems has greatly enhanced the applicability of AUC to macromolecular systems that are traditionally difficult to characterize. This overview provides a resource for novices, highlighting the potential of AUC and encouraging its use in their research, as well as for current users, who may benefit from our experience. We discuss the strengths of fluorescence‐detected AUC and demonstrate the power of even simple AUC experiments to answer practical and fundamental questions about biophysical properties of macromolecular assemblies. We address the development and utility of AUC, explore experimental design considerations, present case studies investigating properties of biological macromolecules that are of common interest to researchers, and review popular analysis approaches. © 2020 The Authors.

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Methods for sample labeling and meniscus determination in the fluorescence-detected analytical ultracentrifuge

Cited by 21 publications

References 11 publications

Tools for the Quantitative Analysis of Sedimentation Boundaries Detected by Fluorescence Optical Analytical Ultracentrifugation

Tools for the Quantitative Analysis of Sedimentation Boundaries Detected by Fluorescence Optical Analytical Ultracentrifugation

Energetics of SecA Dimerization

Analytical Ultracentrifugation (AUC): An Overview of the Application of Fluorescence and Absorbance AUC to the Study of Biological Macromolecules

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