Global Analysis of Data from Multiple Biophysical Methods for the Analysis of Protein Complex Stoichiometry and Affinity

Monday, September 22, 2014 — Poster Session I
12:00 p.m. – 2:00 p.m.	FAES Academic Center	NIBIB	BIOENG-12

* FARE Award Winner

Authors

• H Zhao
• J Ma
• P Schuck

Abstract

Analytical ultracentrifugation (AUC) is a powerful technique for elucidating protein complex formation, providing information on size, shape and binding energies for reversible systems from the analysis of sedimentation profiles of molecular mixtures in free solution. By virtue of the hydrodynamic resolution achieved in sedimentation velocity experiments, multiple co-existing complexes can be identified, and be discriminated from impurities and aggregates. A recently introduced fluorescence optical detection system (FDS) strongly enhances the potential of AUC for quantifying high-affinity protein interactions due to the superb sensitivity of fluorescence. We have developed computational approaches for the analysis of FDS data that correct for geometric and temporal aberrations intrinsic to the use of confocal laser optics, and provide excellent fits to the observed sedimentation boundaries. This allows us to fully exploit the statistics of FDS data, which provides detection limits and hydrodynamic resolution at low pM concentrations, extending the dynamic range for measuring Kds with this technique to 9 orders of magnitude from pM to mM range. Using different interactions systems we demonstrate how the FDS-AUC system can be reliably applied to study high-affinity protein self- and hetero-associations at orders of magnitude lower concentrations and with much higher accuracy than previously possible.

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