Analytical Ultracentrifugation as a Tool for Studying Protein Interactions

Area of Science:

• Biochemistry and Biophysics
• Macromolecular Science
• Analytical Chemistry

Background:

• Analytical ultracentrifugation (AUC) has seen significant advancements in the last two decades.
• Progress is driven by improvements in instrumental, theoretical, and computational methodologies.

Purpose of the Study:

• To review key developments in sedimentation equilibrium (SE) and sedimentation velocity (SV) analysis.
• To highlight new approaches for studying macromolecular interactions using AUC.

Main Methods:

• Sedimentation Equilibrium (SE): Analysis of tracer SE at high concentrations with non-ideality, and strategies for heterogeneous interactions.
• Sedimentation Velocity (SV): Numerical solutions of the Lamm equation, noise decomposition, diffusion deconvoluted distributions, and multi-signal analysis.
• Effective particle theory for interpreting co-migration of interacting components in SV.

Main Results:

• Developed strategies for global multi-signal and multi-speed SE analysis with implicit mass conservation.
• Enabled direct boundary fitting and diffusion deconvoluted sedimentation coefficient distributions in SV.
• Effective particle theory provides robust interpretation of heterogeneous interacting systems in SV.

Conclusions:

• SE and SV techniques offer new avenues for studying macromolecular interactions across diverse affinities (attractive/repulsive).
• These methods are applicable to both dilute and concentrated solutions, including self-associating proteins and multi-protein complexes.