Accounting for Photophysical Processes and Specific Signal Intensity Changes in Fluorescence-Detected Sedimentation Velocity

Abstract

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.

Monochromatic multicomponent fluorescence sedimentation velocity for the study of high-affinity protein interactions

“…For example, for rsEGFP with a scanning beam of 13 mW we measure a depletion rate of 5.08 [4.97–5.19; 95% CI]×10 –4 /sec, approaching a final fluorescence of 13.4 [13.0–13.9; 95% CI]% its initial value, associated with a particle sedimentation coefficient of 2.50 [2.45–2.54; 95% CI] S and apparent molar mass of 29.5 [26.5–33.0; 95% CI] kDa. By contrast, as previously established ( Zhao et al, 2014b ), no photophysical processes are detectable under this illumination for other fluorophores such as fluorescein-based DL488 ( Figure 2b ) and standard EGFP.…”

“…The psFPs make up a class of fluorescent proteins that can be actively switched between fluorescent and non-fluorescent states using different wavelengths of illumination. While they have been engineered for entirely different purposes in nanoscience and fluorescence imaging, we have previously observed that under the illumination conditions of FDS-SV they are induced to slowly switch by virtue of the excitation light when radially scanning the spinning sample ( Zhao et al, 2014b ). Even though the mechanism of photoswitching in psFPs generally may involve multiple states, in the low-power exposure that occurs during sedimentation using the FDS the process is quantitatively modeled very well as a single exponential.…”

Sedimentation velocity analytical ultracentrifugation for characterization of therapeutic antibodies

“…The above difficulties can be easily overcome by careful design and performance of the experiments and associated data analysis, leading to truly exceptional results (Zhao et al 2014a;Chaturvedi et al 2017a). It has been shown previously that, due to the large number of data points acquired during a typical SVexperiment, meaningful results can be derived even when boundary amplitudes are comparable with the stochastic noise of the data acquisition (Zhao et al 2012).…”

Sedimentation coefficient distributions of large particles

“…The approach is very general, and without any further complications refinements on χ 1, nd are possible to account for solvent compressibility, 51 time-varying centrifugal fields, 52 , the finite scanning speed of the scanning systems, 3 optical signal magnification gradients, 53 or spatiotemporal modulation of signal increments in fluorescence detection. 54 …”