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Studying High-Affinity Protein Interactions by Fluorescence Optical Analytical Ultracentrifugation

Friday, November 08, 2013 — Poster Session IV

2:00 p.m. – 4:00 p.m.

FAES Academic Center (Upper-Level Terrace)




  • H Zhao
  • S Lomash
  • M.L Mayer
  • P Schuck


Analytical ultracentrifugation (AUC) offers substantial advantages for studying protein complex formation, providing information on size, shape and binding energies for reversible systems from analysis of sedimentation profiles of molecular mixtures in free solution. By virtue of the hydrodynamic resolution achieved in sedimentation velocity, multiple co-existing complexes can be identified, even in the presence of impurities and aggregates. Due to the limited sensitivity of conventional optical detection systems (absorbance and interference), this technique has been applied predominantly to systems with a Kd in the micromolar range or weaker. We have now explored the potential of a recently introduced fluorescence optical detection system (FDS) for AUC for studying high-affinity protein interactions. After computationally accounting for specific characteristics of the optical setup of the FDS, we found the data and fit quality to be excellent, allowing for the highly quantitative interpretation of the observed sedimentation boundaries. By exploring the lower detection limit of FDS, we observed satisfactory signals with hydrodynamic resolution at low picomolar concentrations. This allows collecting isotherms of sedimentation properties of interacting systems over the picomolar to micromolar range. Using different systems with high-affinity association we demonstrate how the FDS-AUC system can be used reliably for studying high-affinity protein complex assembly.

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