Sedimentation velocity analysis of protein interactions in whole cell extracts using a fluorescence detection system

Authors

• H Zhao
• K Rainey
• G Patterson
• P Schuck

Abstract

Analytical ultracentrifugation (AUC) is a classical biophysical technique to characterize proteins and their interactions in solution. A recently introduced fluorescence detection system (FDS) strongly increases the sensitivity of the detection in AUC, and offers the potential to study proteins and protein interactions in biological fluids. Here, we explore the potential and challenges posed by the application of FDS-AUC to the study of protein interactions in whole cell extracts, using enhanced green fluorescent protein (EGFP) as a model system. At experimentally feasible concentrations of E.coli cell extract (3 mg/mL), no impact of this environment on the sedimentation of EGFP is discernable. Autofluorescence of the E.coli cell extract sets the detection limit of EGFP in the low nanomolar range. In comparison with E.coli, the mammalian Cos7 cell extract has less background autofluorescence, which lowers the detection limit for EGFP to the subnanomolar range. To investigate protein interactions, monoclonal anti-EGFP antibody was added to the E.coli cell extract with exogenous EGFP and the Cos7 cell extract with exogenous or endogenous EGFP. All three arrangements show shifts in the sedimentation coefficient distribution that reflect complex formation. Quantitatively, a small decrease of the complex sedimentation coefficient can be observed in cell extracts due to hydrodynamic nonideality, still allowing the determination of complex size and stoichiometry. In summary, this study demonstrates the potential for studying interactions of unpurified GFP-tagged proteins in bacterial and mammalian cell extracts using fluorescence detected AUC.

Scientific Focus Area: Biomedical Engineering and Biophysics

This page was last updated on Friday, March 26, 2021