A maximum entropy analysis of protein orientations using fluorescence polarization data from multiple probes

Area of Science:

• Biophysics
• Structural Biology
• Biochemistry

Background:

• Fluorescent probes enable orientation determination of protein subunits within macromolecular assemblies.
• Quantitative interpretation of fluorescence polarization data requires known probe orientation.
• Understanding protein orientation is vital for elucidating function in complex biological systems.

Purpose of the Study:

• To present an analytical method for determining protein orientational distributions using maximum entropy.
• To enable quantitative interpretation of fluorescence polarization experiments for protein orientation and motion.
• To guide the selection of optimal probe orientations for enhanced information content.

Main Methods:

• Development of a maximum entropy analytical method to derive protein orientational distributions.
• Utilizing fluorescence polarization data from probes at predetermined orientations.
• Introducing a 'figure of merit' to assess the independence of probe orientations.

Main Results:

• The maximum entropy method yields the broadest distribution compatible with data, requiring no a priori assumptions.
• The method successfully recovers simulated orientational distributions with one and two peaks.
• Experimental data from myosin regulatory light chain (RLC) in muscle fibers revealed complex axial and azimuthal orientation relationships.

Conclusions:

• Maximum entropy analysis offers a robust, unique approach to determine protein orientational distributions.
• The choice and independence of probe orientations are critical for accurate results.
• The method highlights limitations in experimental data and guides future experimental design for detailed orientational analysis.