Fluorescence Polarization and Fluctuation Analysis: A method for studying the structure of protein complexes.

<p>(A) Six possible structures for a hypothetical protein (Blue hexagon) tagged with Venus (yellow cylinder). Example 1 depicts monomers. Examples 2 and 3 depict dimers. Attached Venus molecules are in close apposition for example 2 but not for example 3. Examples 4 and 5 depict hexamers. Pairs of attached Venus molecules are in close apposition for example 4 but not for example 5. Example 6 depicts monomers that are confined to a sub-compartment (dashed line) where they are expressed at a high concentration. (B) Schematic for microscope to measure FPFA. (C) Micro-time data measured for a homogenate prepared from cells expressing Venus monomers are used to calculate fluorescence lifetime histograms for photons detected by either the parallel or perpendicular hybrid-detectors as a function of time after the laser excitation pulse (left panel). These two decay curves are then used to calculate the time-resolved anisotropy (right panel top), or fluorescence lifetime (right panel bottom) of Venus monomers. Red dashed lines show fitting to a single exponential model. (D) Macro-time data measured for a homogenate prepared from cells expressing Venus monomers are used to calculate auto- and cross-correlation functions for photons detected in the parallel and perpendicular hybrid-detectors. A single diffusible-component 3-D Gaussian model was used to fit the cross-correlation (red dashed line), and to estimate the correlation time and the average number of molecules in the observation volume. The excitation power used was 10.2 mW.