Accounting for photophysical processes and specific signal intensity changes in fluorescence-detected sedimentation velocity.

Publisher: American Chemical Society Country of Publication: United States NLM ID: 0370536 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1520-6882 (Electronic) Linking ISSN: 00032700 NLM ISO Abbreviation: Anal Chem Subsets: MEDLINE

Original Publication: Washington, American Chemical Society.

Ultracentrifugation*; Proteins/*chemistry; Algorithms ; Animals ; Cattle ; Fluorescein-5-isothiocyanate/chemistry ; Fluorescence Recovery After Photobleaching ; Green Fluorescent Proteins/chemistry ; Hydrodynamics ; Lasers ; Serum Albumin, Bovine/chemistry

Fluorescence detected sedimentation velocity (FDS-SV) has emerged as a powerful technique for the study of high-affinity protein interactions, with hydrodynamic resolution exceeding that of diffusion-based techniques, and with sufficient sensitivity for binding studies at low picomolar concentrations. For the detailed quantitative analysis of the observed sedimentation boundaries, it is necessary to adjust the conventional sedimentation models to the FDS data structure. A key consideration is the change in the macromolecular fluorescence intensity during the course of the experiment, caused by slow drifts of the excitation laser power, and/or by photophysical processes. In the present work, we demonstrate that FDS-SV data have inherently a reference for the time-dependent macromolecular signal intensity, resting on a geometric link between radial boundary migration and plateau signal. We show how this new time-domain can be exploited to study molecules exhibiting photobleaching and photoactivation. This expands the application of FDS-SV to proteins tagged with photoswitchable fluorescent proteins, organic dyes, or nanoparticles, such as those recently introduced for subdiffraction microscopy and enables FDS-SV studies of their interactions and size distributions. At the same time, we find that conventional fluorophores undergo minimal photobleaching under standard illumination in the FDS. These findings support the application of a high laser power density for the detection, which we demonstrate can further increase the signal quality.

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United States Intramural NIH HHS

0 (Proteins)
0 (enhanced green fluorescent protein)
147336-22-9 (Green Fluorescent Proteins)
27432CM55Q (Serum Albumin, Bovine)
I223NX31W9 (Fluorescein-5-isothiocyanate)

Date Created: 20140820 Date Completed: 20150828 Latest Revision: 20211021

20250114

PMC4165462

10.1021/ac502478a

25136929