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

Thursday, November 07, 2013 — Poster Session II
12:00 p.m. – 2:00 p.m.	FAES Academic Center (Upper-Level Terrace)	NIBIB	BIOENG-23

Authors

P. Schuck
E. Casillas
H. Shroff
G.H. Patterson
H. Zhao

Abstract

Fluorescence optical detected sedimentation velocity analytical ultracentrifugation (FDS-SV) is a recent technique for the study of macromolecules at nanomolar concentrations and below. This has significant promise for the study of high-affinity protein interactions. So far, however, most applications of FDS-SV were qualitative in nature, and fits to the FDS data with moderate or high signal-to-noise ratio routinely exhibited systematic deviations from the expected macromolecular sedimentation profiles. To address this problem, we have developed computational models that accommodate unique characteristics of the confocal fluorescence detection system, including 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. We show that the experimental data provide sufficient information to determine the parameters required for first-order approximation of these effects. Systematic deviations of FDS-SV 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 FDS-SV data, thus allowing a detailed and reliable quantitative interpretation that is otherwise not possible for this system.