Sedimentation of Reversibly Interacting Macromolecules with Changes in Fluorescence Quantum Yield

Area of Science:

• Biophysical chemistry
• Macromolecular interactions
• Analytical ultracentrifugation

Background:

• Sedimentation velocity analytical ultracentrifugation (SV-AUC) with fluorescence detection is a sensitive method for studying macromolecular complexes.
• Current data interpretation relies on weight-average sedimentation coefficients (s_w), assuming constant signal across all species.
• This assumption fails when complex formation alters fluorescence properties, such as quantum yield.

Purpose of the Study:

• To challenge the prevailing dogma in SV-AUC data interpretation regarding signal-averaged sedimentation coefficients.
• To develop a more general binding model that accounts for coupled transport in systems with signal changes.
• To improve the accuracy of determining binding affinity and stoichiometry in complex macromolecular systems.

Main Methods:

• Utilized sedimentation velocity analytical ultracentrifugation with fluorescence detection.
• Applied effective particle theory to model reaction-coupled migration.
• Developed a generalized binding model incorporating hydrodynamic cotransport and signal changes.

Main Results:

• Demonstrated that the standard s_w model is insufficient when fluorescence signals change during complex formation.
• Showed that coupled transport significantly impacts observed migration in rapidly reversible systems.
• The new model accurately describes systems with altered fluorescence quantum yields, including quenching, enhancement, or FRET.

Conclusions:

• The developed binding model provides a more accurate framework for SV-AUC analysis of interacting systems with fluorescence signal variations.
• This advancement is crucial for studying macromolecular complexes where fluorescence properties change upon binding.
• The findings enhance the utility of SV-AUC for characterizing complex stoichiometry, affinity, and shape.