Constrained fits of the 1dPCH data to two-species allows for the examination of monomer and oligomer populations.

<p>A. A model was assumed where Ste50 could exist as either a monomer with fixed brightness, or oligomer with unconstrained brightness and number (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001931#s4" target="_blank">Materials and Methods</a>, and main text). Results are displayed to show the percentage of each component. Error bars are the standard error of the mean. B. Average brightness values for autofluorescence, GFP, GFP-GFP, and GFP-GFP-GFP from the data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001931#pone-0001931-g003" target="_blank">Figure 3</a>, fit to a line. Error bars are the standard deviation. The line represents the best fit of the data to a linear model with a slope of 1959 and intercept 1193, which was then extrapolated toward higher brightness. Average brightness observed for the Ste50 oligomer from the analysis described above are marked on the extrapolated part of the plot.</p

1dPCH analysis of Ste50-GFP and Ste11-GFP probes homo-oligomerization.

<p>A. Example curves for GFP and mCherry (mCH.) controls in live yeast. B. Notched box plots of PCH fits, ranging from 18 to 30 individual, 7 second data traces from 5 to 10 cells. For auto-fluorescence measurements, data represents 7 measurements for mCherry and 15 measurements for GFP. 50 µs bins were used to generate the PCH distributions. C. Notched box plots of 1dPCH fits of GFP tagged species, with lines (same color scheme as in B) representing average brightness values of monomer, dimer, and trimer controls for a basis of comparison.</p

Yeast strains used in this study.

<p>all strains are S288C background, his3Δ1;leu2Δ0;met15Δ0;ura3Δ0.</p

Fluctuation data can probe protein-protein interactions.

<p>A. Example traces of fluctuation data for dual-color experiments. B. Data can be analyzed by correlation analysis to examine concentration, diffusion, and co-diffusion of red and green particles. C. 1dPCH examines the distribution of photon events per time interval, and reports concentration and ‘brightness’, or oligomeric status. D. 2dPCH reports simultaneously concentration, interaction, oligomerization, and binding stoichiometry of heterogeneous complexes. An example two-dimensional PCH histogram is shown, with frequency versus number of green photons and number of red photons per time bin. E. Example, two-dimensional plot of a fit of modeled 2dPCH data. If a monomer red or green probe has a brightness of 3000 CPSM, for example, the plot demonstrates points one would expect to find values for with non-interacting monomeric species, or interacting monomeric species, or interacting dimeric species, as labeled.</p

2dPCH analysis of Ste50-mCherry and Ste11-GFP detects binding stoichiometry.

<p>50 µs bins were used. Data were fit to a one-component model or two-component model, as explained in the text. Symbols and bars represent the averages and standard deviations, respectively. Schematic representations of average stoichiometry observed; possible geometries of the interactions (see main text) are displayed next to the corresponding regions of the graph.</p