title: Tools for the quantitative analysis of sedimentation boundaries detected by fluorescence optical analytical ultracentrifugation.
abstract: 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.
authors.affiliation: Dynamics of Macromolecular Assembly Section, Laboratory of Cellular Imaging and Macromolecular Biophysics, National Institute of Biomedical Imaging and Bioengineering, National Institute of Health, Bethesda, Maryland, United States of America.
authors.forename: Huaying
authors.initials: H
authors.lastname: Zhao
authors.forename: Ernesto
authors.initials: E
authors.lastname: Casillas
authors.forename: Hari
authors.initials: H
authors.lastname: Shroff
authors.forename: George H
authors.initials: GH
authors.lastname: Patterson
authors.forename: Peter
authors.initials: P
authors.lastname: Schuck
mesh_terms: D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000032:analysis*; D015233:Models, Statistical; D008970:Molecular Weight; D055095:Optics and Photonics / Q000295:instrumentation; D012984:Software; D014461:Ultracentrifugation / Q000295:instrumentation* / Q000379:methods
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins